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venous thromboembolism ( vte ) , which includes deep vein thrombosis and pulmonary embolism , is a common complication of cancer and cancer treatment.13 patients with active malignancy are at 47-fold higher risk of symptomatic vte than the general population,4,5 and this prothrombotic risk is further exacerbated by chemotherapy , hormonal therapy , and surgery.3,68 in addition , patients with cancer who develop vte are at higher risk of recurrent thrombotic complications than noncancer patients with vte.9,10 despite the strong association of vte with cancer , there is wide variation in risk , with rates ranging from 0.6% to 7.8% in population - based case - control studies.9,11,12 predisposing factors for vte in cancer patients include tumor site,1315 stage ( advanced / metastatic cancer),13,14 patient - related factors ( advanced age , comorbidity , prolonged immobilization),5,15,16 and treatment - related factors , such as specific antineoplastic agents and growth factors.5,17 few studies have assessed the economic impact of vte in cancer patients .
the limited data available suggest that management of cancer - associated vte is both resource - intensive and costly,1820 partly due to the high frequency of vte recurrence , complications of anticoagulant therapy,18 and length of hospital stay.18,21 a review of medical records of cancer patients who developed vte between 1994 and 1997 indicated that the mean cost of hospitalization for initiation of anticoagulant therapy was usd 20,065 ( 2002 usd values).18 little is known about the current real - world economic burden of vte events in ambulatory cancer patients undergoing chemotherapy or how this might differ according to the site of the cancer .
therefore , the objective of this study was to examine all - cause and vte - associated resource utilization and assess , from the us health payer s perspective , the incremental cost of vte in a large , real - world , contemporary cohort of ambulatory patients initiating chemotherapy for six selected common solid tumors ( lung , colorectal , pancreatic , gastric , bladder , and ovarian ) .
this retrospective observational cohort study was based on health care data collected from the ims / pharmetrics patient - centric database ( ims health inc ) for the period january 2004 to december 2009 .
this large database provides integrated enrolment , medical , and prescription claims information from more than 90 managed care organizations and medicare , and represents the health services of over 58 million patients across the us .
it includes both inpatient and outpatient diagnoses ( in international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] format ) , and procedures ( in current procedural terminology , fourth edition , and health care common procedure coding system formats ) , and outpatient prescription records .
additional data elements include patient demographics , health plan type , payer type , provider specialty , and health plan enrolment dates .
the ims / pharmetrics patient - centric database has been used extensively for health economics and health outcomes research in various therapy areas , including vte.22 all patient records used in the study were deidentified in compliance with the health insurance portability and accountability act of 1996 , so this study was exempt from institutional review board overview .
patients aged 18 years with an inpatient diagnosis of malignant neoplasm of the lung ( icd-9-cm code 162.0 , 162.2162.5 , 162.8 , 162.9 ) , pancreas ( 157.0157.4 , 157.8 , 157.9 ) , stomach ( 151.0151.6 , 151.8 , 151.9 ) , colon / rectum ( 153.0154.3 , 154.8 ) , bladder ( 188.x ) , or ovary ( 183.0 , 183.2183.5 , 183.8 , 183.9 ) , and who received cytotoxic chemotherapy between january 2005 and december 2008 ( index event identification period ) were selected .
use of chemotherapy was indicated by pharmacy claims with relevant national drug code or healthcare common procedure coding system drug codes . for inclusion in the study , patients were additionally required to have continuous medical and prescription drug coverage for 12 months before and 12 months after their first ( index ) cycle of chemotherapy during the index event identification period .
patients receiving biologic agents or targeted therapy alone , in the absence of chemotherapy , were excluded from the study , as were patients with a diagnosis of vte ( icd-9-cm code 451.1 , 451.11 , 451.19 , 451.2 , 451.81 , 451.83 , 451.84 , 451.9 , 453.4 , 453.41 , 453.42 , 453.8 , 453.9 , 997.2 , 415.1 , 415.11 , 415.12 , 415.19 ) , severe renal impairment , hemorrhagic stroke , or thrombocytopenia within 12 months before the initial cycle of chemotherapy , and patients with major bleeding ( including gastrointestinal or ulcer - related bleeding ) within 3 months of the initial cycle of chemotherapy .
in addition , patients receiving antithrombotic / thrombolytic treatment , as indicated by pharmacy claims with national drug code and healthcare common procedure coding system codes for anticoagulants , glycoprotein iib / iiia receptor antagonists , and thrombolytics , less than 2 weeks before the index cycle of chemotherapy were excluded .
chronic treatment with antiplatelet agents such as aspirin , clopidogrel , or ticlopidine was permitted . within the resulting dataset
, cancer patients with evidence of a vte event within 12 months following initiation of chemotherapy , as indicated by the presence of an inpatient or outpatient claim with an icd-9-cm code for deep vein thrombosis or pulmonary embolism ( as listed above for vte ) , were identified in accordance with a prior claims study of the incidence of deep vein thrombosis and pulmonary embolism.20 for the purposes of cost analysis , additional eligibility criteria were applied , with patients being required to have continuous medical and prescription drug coverage for 12 months after their frist ( index ) vte event . using propensity score methods,23 patients with vte were individually matched to those cancer patients without evidence of a vte event during this period to form two matched cancer cohorts , ie , vte and non - vte ( matched control ) .
each patient with vte was matched according to propensity score and key patient characteristics , such as cancer site , with three non - vte patients .
propensity scores were generated using a logistic regression model , with patient cohort ( vte versus non - vte ) as the dependent variable , and age , gender , geographic region , and health plan type as independent variables .
individually matched vte and non - vte patients were required to have propensity scores that differed by no more than 0.005 points from each other .
if a vte patient could not be matched with three non - vte cancer controls , then the vte patient was excluded from the analysis .
the success of propensity score matching was confirmed by the absence of statistically significant differences ( p > 0.05 ) in age , gender , geographic region , or health plan type between the resulting vte and non - vte patient cohorts .
information on patients demographics ( gender , age , us census bureau geographic region , health plan type ) and clinical characteristics ( medical conditions / comorbidities , charlson comorbidity index,24,25 initial chemotherapy ) were collected for both the vte and non - vte ( control ) cohorts over the 12-month baseline period preceding the index vte event .
for the vte cohort , the index event was defined as the first vte diagnosis occurring after the initial cycle of chemotherapy ; for the non - vte cohort , the index event was a random date occurring within the 12-month period following the initial cycle of chemotherapy .
resource utilization ( number of hospitalizations , days in hospital , outpatient medical services , outpatient prescriptions ) and costs ( inpatient , outpatient , total payments ) were determined for the vte and non - vte cancer cohorts over the 12-month period following the index vte event .
resource usage and costs were determined for all - cause claims ( ie , all claims ) and vte - related claims ( ie , claims with an associated icd-9-cm diagnostic code for deep vein thrombosis or pulmonary embolism as defined above ) .
vte - related hospitalization was defined as a hospitalization that contained a diagnostic code for deep vein thrombosis or pulmonary embolism .
the incremental impact of vte on all - cause and vte - related health care costs was determined for the entire cancer cohort and separately for patients with bladder , colorectal , lung , ovarian , pancreatic , and gastric cancer .
costs were adjusted using multivariate regression to account for demographic ( age , gender , region , health plan ) and clinical ( vte type , charlson comorbidity index ) covariates .
all costs were inflation - adjusted to 2010 levels , using the cpi medical care index .
intercohort comparisons of patient demographic , clinical , and treatment characteristics , and clinical outcomes were performed using student s t - test . a p value of , 0.05 was considered statistically significant .
resource utilization and unadjusted health care costs were summarized with descriptive statistics and compared between cohorts using student s t - test .
a generalized linear model was fitted to the cost data with a gamma probability distribution and a log link function .
statistical analyses were conducted using sas version 9.2 ( sas institute inc , cary , nc ) .
this retrospective observational cohort study was based on health care data collected from the ims / pharmetrics patient - centric database ( ims health inc ) for the period january 2004 to december 2009 .
this large database provides integrated enrolment , medical , and prescription claims information from more than 90 managed care organizations and medicare , and represents the health services of over 58 million patients across the us .
it includes both inpatient and outpatient diagnoses ( in international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] format ) , and procedures ( in current procedural terminology , fourth edition , and health care common procedure coding system formats ) , and outpatient prescription records .
additional data elements include patient demographics , health plan type , payer type , provider specialty , and health plan enrolment dates .
the ims / pharmetrics patient - centric database has been used extensively for health economics and health outcomes research in various therapy areas , including vte.22 all patient records used in the study were deidentified in compliance with the health insurance portability and accountability act of 1996 , so this study was exempt from institutional review board overview .
patients aged 18 years with an inpatient diagnosis of malignant neoplasm of the lung ( icd-9-cm code 162.0 , 162.2162.5 , 162.8 , 162.9 ) , pancreas ( 157.0157.4 , 157.8 , 157.9 ) , stomach ( 151.0151.6 , 151.8 , 151.9 ) , colon / rectum ( 153.0154.3 , 154.8 ) , bladder ( 188.x ) , or ovary ( 183.0 , 183.2183.5 , 183.8 , 183.9 ) , and who received cytotoxic chemotherapy between january 2005 and december 2008 ( index event identification period ) were selected .
use of chemotherapy was indicated by pharmacy claims with relevant national drug code or healthcare common procedure coding system drug codes . for inclusion in the study , patients were additionally required to have continuous medical and prescription drug coverage for 12 months before and 12 months after their first ( index ) cycle of chemotherapy during the index event identification period .
patients receiving biologic agents or targeted therapy alone , in the absence of chemotherapy , were excluded from the study , as were patients with a diagnosis of vte ( icd-9-cm code 451.1 , 451.11 , 451.19 , 451.2 , 451.81 , 451.83 , 451.84 , 451.9 , 453.4 , 453.41 , 453.42 , 453.8 , 453.9 , 997.2 , 415.1 , 415.11 , 415.12 , 415.19 ) , severe renal impairment , hemorrhagic stroke , or thrombocytopenia within 12 months before the initial cycle of chemotherapy , and patients with major bleeding ( including gastrointestinal or ulcer - related bleeding ) within 3 months of the initial cycle of chemotherapy .
in addition , patients receiving antithrombotic / thrombolytic treatment , as indicated by pharmacy claims with national drug code and healthcare common procedure coding system codes for anticoagulants , glycoprotein iib / iiia receptor antagonists , and thrombolytics , less than 2 weeks before the index cycle of chemotherapy were excluded .
chronic treatment with antiplatelet agents such as aspirin , clopidogrel , or ticlopidine was permitted . within the resulting dataset , cancer patients with evidence of a vte event within 12 months following initiation of chemotherapy , as indicated by the presence of an inpatient or outpatient claim with an icd-9-cm code for deep vein thrombosis or pulmonary embolism ( as listed above for vte ) ,
were identified in accordance with a prior claims study of the incidence of deep vein thrombosis and pulmonary embolism.20 for the purposes of cost analysis , additional eligibility criteria were applied , with patients being required to have continuous medical and prescription drug coverage for 12 months after their frist ( index ) vte event . using propensity score methods,23 patients with vte were individually matched to those cancer patients without evidence of a vte event during this period to form two matched cancer cohorts , ie , vte and non - vte ( matched control ) . each patient with vte was matched according to propensity score and key patient characteristics , such as cancer site , with three non - vte patients .
propensity scores were generated using a logistic regression model , with patient cohort ( vte versus non - vte ) as the dependent variable , and age , gender , geographic region , and health plan type as independent variables .
individually matched vte and non - vte patients were required to have propensity scores that differed by no more than 0.005 points from each other .
if a vte patient could not be matched with three non - vte cancer controls , then the vte patient was excluded from the analysis .
the success of propensity score matching was confirmed by the absence of statistically significant differences ( p > 0.05 ) in age , gender , geographic region , or health plan type between the resulting vte and non - vte patient cohorts .
information on patients demographics ( gender , age , us census bureau geographic region , health plan type ) and clinical characteristics ( medical conditions / comorbidities , charlson comorbidity index,24,25 initial chemotherapy ) were collected for both the vte and non - vte ( control ) cohorts over the 12-month baseline period preceding the index vte event . for the vte cohort ,
the index event was defined as the first vte diagnosis occurring after the initial cycle of chemotherapy ; for the non - vte cohort , the index event was a random date occurring within the 12-month period following the initial cycle of chemotherapy .
resource utilization ( number of hospitalizations , days in hospital , outpatient medical services , outpatient prescriptions ) and costs ( inpatient , outpatient , total payments ) were determined for the vte and non - vte cancer cohorts over the 12-month period following the index vte event . resource usage and costs were determined for all - cause claims ( ie , all claims ) and vte - related claims ( ie , claims with an associated icd-9-cm diagnostic code for deep vein thrombosis or pulmonary embolism as defined above ) .
vte - related hospitalization was defined as a hospitalization that contained a diagnostic code for deep vein thrombosis or pulmonary embolism . the incremental impact of vte on all - cause and vte - related health care costs was determined for the entire cancer cohort and separately for patients with bladder , colorectal , lung , ovarian , pancreatic , and gastric cancer .
costs were adjusted using multivariate regression to account for demographic ( age , gender , region , health plan ) and clinical ( vte type , charlson comorbidity index ) covariates .
all costs were inflation - adjusted to 2010 levels , using the cpi medical care index .
intercohort comparisons of patient demographic , clinical , and treatment characteristics , and clinical outcomes were performed using student s t - test .
resource utilization and unadjusted health care costs were summarized with descriptive statistics and compared between cohorts using student s t - test .
a generalized linear model was fitted to the cost data with a gamma probability distribution and a log link function .
statistical analyses were conducted using sas version 9.2 ( sas institute inc , cary , nc ) .
a total of 63,453 patients with a diagnosis of bladder , colorectal , lung , ovarian , pancreatic , or gastric cancer , and who were undergoing chemotherapy during the index event identification period , were identified from the ims / pharmetrics patient - centric database . of these
, 9352 cancer patients fulfilled the study inclusion criteria and provided a pool of patients for propensity score matching . within this pool ,
912 patients with vte were identified and matched with 2736 non - vte ( control ) cancer patients .
the two patient cohorts were well balanced in terms of age ( mean 63.3 versus 63.0 years ) and gender ( both 56% female ) distributions .
however , the vte cohort had more frequent comorbidity than the non - vte cohort ( overall mean cci score 6.8 versus 5.6 ; p < 0.0001 ) , including pulmonary and hepatic disease , obesity , and congestive heart failure ( table 1 ) .
for both patient cohorts , the most frequent cancer types were lung cancer ( 37.1% of patients ) and colorectal cancer ( 33.0% , table 1 ) . during the post - index follow - up period , cancer patients who experienced vte had approximately three times as many all - cause hospitalizations ( mean 1.38 versus 0.55 per patient ; p < 0.0001 ) and days in hospital ( mean 10.19 versus 3.37 per patient ; p < 0.0001 ) as cancer patients without vte .
furthermore , outpatient medical claims ( mean 291.44 versus 173.39 per patient ; p < 0.0001 ) and outpatient prescription claims ( mean 39.97 versus 33.07 per patient ; p <
cancer patients who experienced vte also incurred significantly higher ( unadjusted ) overall ( all - cause ) inpatient costs ( mean usd 21,299 versus usd 7459 per patient ; p < 0.0001 ) , outpatient medical costs ( mean usd 47,091 versus usd 29,901 per patient ; p < 0.0001 ) , outpatient prescription costs ( mean usd 6569 versus usd 4331 per patient ; p < 0.0001 ) and total health care costs ( mean usd 74,959 versus usd 41,691 per patient ; p <
0.0001 ) over the 12-month post - vte follow - up period than those without vte ( table 2 ) .
the total cost per hospitalization for deep vein thrombosis and pulmonary embolism was ( mean ) usd 6654 and usd 10,753 , respectively . as anticipated , vte - related hospitalizations ( mean 0.63 versus 0.0 per patient ; p < 0.0001 ) and vte - related days in hospital ( mean 5.8 versus 0.0 per patient ; p <
0.0001 ) were confined exclusively to cancer patients with vte rather than those without vte .
likewise , vte - related outpatient medical claims ( mean 23.3 versus 0.0 per patient ; p < 0.0001 ) and outpatient prescription claims ( mean 4.8 versus 0.0 per patient ; p < 0.0001 ) were limited to cancer patients with vte .
cancer patients who experienced vte incurred ( unadjusted ) mean vte - related inpatient costs of usd 5202 per patient , outpatient medical costs of usd 2063 per patient , outpatient prescription costs of usd 1982 per patient , and total vte - related health care costs of usd 9247 per patient over the 12-month post - vte follow - up period ( table 2 ) .
after adjusting for demographic and clinical differences between cancer patients with and without vte , the predicted incremental all - cause health care cost of vte ( compared with no vte ) was ( mean ) usd 30,538 ( standard error [ se ] usd 2349 ) per patient , while the predicted incremental vte - related health care cost of vte ( compared with no vte ) was ( mean ) usd 9202 ( se usd 378 ) per patient ( both p <
0.0001 ) over the 12-month post - index follow - up period . predicted mean incremental all - cause health care costs of vte ranged from usd 11,946 per patient for gastric cancer to usd 38,983 per patient for pancreatic cancer ( figure 1 ) . predicted mean incremental vte - related health care costs of vte ranged from usd 4524 per patient for bladder cancer to usd 17,205 per patient for pancreatic cancer ( figure 1 ) .
a total of 63,453 patients with a diagnosis of bladder , colorectal , lung , ovarian , pancreatic , or gastric cancer , and who were undergoing chemotherapy during the index event identification period , were identified from the ims / pharmetrics patient - centric database . of these
, 9352 cancer patients fulfilled the study inclusion criteria and provided a pool of patients for propensity score matching . within this pool ,
912 patients with vte were identified and matched with 2736 non - vte ( control ) cancer patients .
the two patient cohorts were well balanced in terms of age ( mean 63.3 versus 63.0 years ) and gender ( both 56% female ) distributions .
however , the vte cohort had more frequent comorbidity than the non - vte cohort ( overall mean cci score 6.8 versus 5.6 ; p < 0.0001 ) , including pulmonary and hepatic disease , obesity , and congestive heart failure ( table 1 ) .
for both patient cohorts , the most frequent cancer types were lung cancer ( 37.1% of patients ) and colorectal cancer ( 33.0% , table 1 ) .
during the post - index follow - up period , cancer patients who experienced vte had approximately three times as many all - cause hospitalizations ( mean 1.38 versus 0.55 per patient ; p < 0.0001 ) and days in hospital ( mean 10.19 versus 3.37 per patient ; p < 0.0001 ) as cancer patients without vte .
furthermore , outpatient medical claims ( mean 291.44 versus 173.39 per patient ; p < 0.0001 ) and outpatient prescription claims ( mean 39.97 versus 33.07 per patient ; p < 0.0001 ) were also higher in cancer patients with vte versus those without vte .
cancer patients who experienced vte also incurred significantly higher ( unadjusted ) overall ( all - cause ) inpatient costs ( mean usd 21,299 versus usd 7459 per patient ; p < 0.0001 ) , outpatient medical costs ( mean usd 47,091 versus usd 29,901 per patient ; p < 0.0001 ) , outpatient prescription costs ( mean usd 6569 versus usd 4331 per patient ; p < 0.0001 ) and total health care costs ( mean usd 74,959 versus usd 41,691 per patient ; p <
0.0001 ) over the 12-month post - vte follow - up period than those without vte ( table 2 ) .
the total cost per hospitalization for deep vein thrombosis and pulmonary embolism was ( mean ) usd 6654 and usd 10,753 , respectively .
as anticipated , vte - related hospitalizations ( mean 0.63 versus 0.0 per patient ; p < 0.0001 ) and vte - related days in hospital ( mean 5.8 versus 0.0 per patient ;
p < 0.0001 ) were confined exclusively to cancer patients with vte rather than those without vte . likewise , vte - related outpatient medical claims ( mean 23.3 versus 0.0 per patient ; p < 0.0001 ) and outpatient prescription claims ( mean 4.8 versus 0.0 per patient ; p < 0.0001 ) were limited to cancer patients with vte .
cancer patients who experienced vte incurred ( unadjusted ) mean vte - related inpatient costs of usd 5202 per patient , outpatient medical costs of usd 2063 per patient , outpatient prescription costs of usd 1982 per patient , and total vte - related health care costs of usd 9247 per patient over the 12-month post - vte follow - up period ( table 2 ) .
after adjusting for demographic and clinical differences between cancer patients with and without vte , the predicted incremental all - cause health care cost of vte ( compared with no vte ) was ( mean ) usd 30,538 ( standard error [ se ] usd 2349 ) per patient , while the predicted incremental vte - related health care cost of vte ( compared with no vte ) was ( mean ) usd 9202 ( se usd 378 ) per patient ( both p < 0.0001 ) over the 12-month post - index follow - up period . predicted mean incremental all - cause health care costs of vte ranged from usd 11,946 per patient for gastric cancer to usd 38,983 per patient for pancreatic cancer ( figure 1 ) .
predicted mean incremental vte - related health care costs of vte ranged from usd 4524 per patient for bladder cancer to usd 17,205 per patient for pancreatic cancer ( figure 1 ) .
this population database study demonstrated that high - risk cancer patients initiating contemporary chemotherapy regimens for common solid tumors who experienced vte had approximately three times as many hospitalizations and days in hospital , and one - third more outpatient claims , than propensity - matched cancer patients without vte over the 12-month post - vte follow - up period .
total ( all - cause ) health care costs were about 80% higher in cancer patients with vte ( mean unadjusted cost usd 74,959 per patient ) in comparison with matched cancer patients without vte ( usd 41,691 ) .
however , it should be emphasized that this vte cost differential could be partly due to clinical differences between the two groups .
patients with vte had more frequent comorbidity than those without vte , although some of this might have been due to the complications of vte itself .
the risk of vte is influenced by factors such as disease stage , treatment modality , and primary cancer site.9,11,15,17,26 pancreatic and gastric cancers are associated with the highest rates of vte , whereas breast cancers and melanomas show the lowest rates.3,17 however , we used propensity matching techniques23 to ensure that the vte and non - vte cohorts were matched with regard to their type of cancer .
furthermore , generalized linear models were used to adjust for demographic and clinical confounders and thereby avoid this potential bias . after adjusting for these and other covariates through multivariate regression ,
vte accounted for 30% of the overall difference in health care costs between cancer patients with and without vte .
this figure varied widely between the various cancer types , ranging from a low of 13.5% in bladder cancer to a high of 91.1% in gastric cancer .
it should be noted that the estimates of the overall incremental cost of vte are likely to be inflated through inclusion of the costs of treating vte - related complications as well as the costs of vte itself .
the economic burden of vte is primarily driven by the development of complications ( notably bleeding , and , in the longer term , vte recurrence and post - thrombotic syndrome ) , and by the length of hospitalization.18 information from the general medical population suggests that the direct medical costs of vte are large,19,27,28 with the annual overall cost of vte to the health care system in the us estimated to exceed usd 1.5 billion.28 a large retrospective us database analysis ( 19972004 ) reported that the median annualized direct medical costs of a primary deep vein thrombosis or pulmonary embolism event in the general population were usd 17,512 and usd 18,901 , respectively , rising to usd 20,569 ( 2004 usd values ) when complicated by post - thrombotic syndrome.27 the high rates of bleeding complications ( about 12% ) and vte recurrence ( about 17%21% ) among cancer patients with vte10,18 suggest that cancer - associated vte is likely to be more costly than vte in the general medical population , although available information is sparse . a retrospective analysis ( 19921994 )
attributed about 6% of oncology bed occupancy directly to vte and vte - associated complications.21 a review of medical records of cancer patients ( n = 529 ) presenting with deep vein thrombosis during the 19941997 period indicates that the mean length of hospital stay for initiation of anticoagulation was 11 days , and that the mean cost of the index hospitalization was usd 20,065 ( 2002 usd values).18 our findings suggest that direct medical costs amount , on average , to about usd 75,000 per patient over the first 12 months following a vte event , and that the adjusted incremental cost associated with vte in cancer ranges from approximately usd 12,00039,000 per patient per year , depending on the tumor site .
this study provides a contemporary estimate of the economic burden of cancer - associated vte in the ambulatory setting , where most cancer care occurs .
multiple randomized studies have shown the benefit of thromboprophylaxis both in hospitalized patients with acute medical illness , including cancer , and in the surgical setting.29,30 however , most cancer care , and therefore most episodes of vte , occur in the outpatient setting.31 risk factors identified for development of symptomatic chemotherapy - associated vte in cancer outpatients include : cancer site , prechemotherapy platelet count 350 109/l , hemoglobin level , 100 g / l or use of erythropoiesis - stimulating agents , prechemotherapy leukocyte count .
35 kg / m,2,26 and these factors collectively form the basis of a validated vte risk scoring system for cancer patients receiving chemotherapy.26,32 a number of candidate biomarkers of thrombotic risk in cancer have been proposed , including d - dimer , soluble p - selectin , c - reactive protein , and tissue factor.3336 addition of d - dimer and soluble p - selectin to the vte risk scoring system increases its predictive accuracy.32 however , d - dimer and p - selectin assays are not routinely performed in cancer patients .
the current american society of clinical oncology and national comprehensive cancer network guidelines recommend thromboprophylaxis in the outpatient setting only for highly selected cancer patients , particularly multiple myeloma patients receiving thalidomide - based or lenalidomide - based combination regimens.8,37 recent controlled studies demonstrate that pharmacologic anticoagulation is effective and safe in reducing vte in selected outpatients receiving chemotherapy .
these include protecht , a study of nadroparin in patients with solid tumors,38 conko 004 , a study of enoxaparin in pancreatic cancer,39 and fragem , a study of dalteparin in pancreatic cancer.40 the largest study of thromboprophylaxis , save - onco , was a randomized , placebo - controlled trial of the ultralow - molecular - weight heparin , semuloparin , in patients initiating chemotherapy for solid tumors ( lung , pancreas , stomach , colon / rectum , bladder , and ovary).41 the six common solid tumors included in our analysis were also investigated in the protecht and save - onco studies .
our findings suggest that thromboprophylaxis targeted at cancer patients at high risk of vte can potentially reduce the economic burden of vte and , hence , overall cancer costs . as an administrative claims database investigation ,
patient - centric database represents a commercially insured population and may contain biases or have limited applicability to other populations .
information on tumor stage , histologic subtype , and leukocyte and platelet counts , which may influence the risk of bleeding and thrombosis , was not included in the database .
use of claims data also precludes verification of diagnoses and raises the possibility of coding error , given that it was not possible to verify whether diagnoses and procedures were accurately coded
. finally , the predicted incremental health care costs of vte are likely to be conservative estimates , because they do not take into account the cost of vte complications or the medical costs arising as a result of vte - imposed interruption of cancer therapy .
the high numbers of all - cause hospitalizations , days in hospital , and outpatient claims associated with cancer patients with vte , in comparison with matched cancer patients without vte , indicate that vte imposes a substantial clinical and economic burden in the ambulatory oncology setting . in the first 12 months following the index vte event , all - cause health care costs were , on average , usd 30,538 per patient higher in cancer patients with vte than in those without vte , with vte - related health care costs accounting for approximately one - third of this difference .
measures to prevent cancer - associated vte in the outpatient setting offer the potential to reduce the high health care utilization and costs of high - risk cancer patients .
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backgroundthis study examines venous thromboembolism ( vte)-associated resource utilization and real - world costs in ambulatory patients initiating chemotherapy for selected common high - risk solid tumors.methodshealth care claims data ( 20042009 ) from the ims / pharmetrics patient - centric database were collected for propensity score - matched adult cancer ( lung , colorectal , pancreatic , gastric , bladder , or ovarian ) patients initiating chemotherapy with vte ( n = 912 ) and without vte ( n = 2736 ) .
health care resource utilization ( inpatient , outpatient , and outpatient prescription drug claims ) and costs were compared between the two cohorts during the 12-month follow - up period after the index vte event .
incremental costs were adjusted for demographic and clinical covariates.resultscancer patients with vte had approximately three times as many all - cause hospitalizations ( mean 1.38 versus 0.55 per patient ) and days in hospital ( 10.19 versus 3.37 ) , and more outpatient claims ( 331 versus 206 ) than cancer patients without vte ( all p < 0.0001 ) . cancer patients with vte incurred higher overall all - cause inpatient costs ( mean usd 21,299 versus usd 7459 per patient ) , outpatient costs ( usd 53,660 versus usd 34,232 per patient ) , and total health care costs ( usd 74,959 versus usd 41,691 per patient ) than cancer patients without vte ( all p < 0.0001 ) .
total mean vte - related health care costs were usd 9247 per patient over 12 months .
adjusted mean incremental all - cause health care costs of vte were usd 30,538 per patient for cancer overall , ranging from usd 11,946 for gastric to usd 38,983 for pancreatic cancer.conclusion:vte is associated with significant inpatient and outpatient resource utilization , and increased all - cause ( in addition to vte - related ) health care costs among ambulatory cancer patients .
measures to prevent outpatient cancer - associated vte may reduce health care utilization and costs in this population .
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clinic of obstetrics , clinical center university of sarajevo is a tertiary level of health care with more than 3,000 births per year . during the 1992 - 1995 war the old building of the clinic of obstetrics and gynecology was the target of destruction , so the clinic was relocated into another building within the clinical center university of sarajevo . during 2010 the original building of the clinic of obstetrics and gynecology at the location
jezero in sarajevo was rebuild , officially opened and put into operation on november 25 , 2010 .
two years later , there was the reorganization of the clinic operation , so in the framework of the existing building was organized a discipline that consists of two clinics or the clinic of obstetrics and gynecology clinic .
clinic of obstetrics currently has about 15 doctors , specialist in obstetrics and gynecology , more than 10 doctors on residency and about 20 nurses and other supporting staff .
birth room of the clinic of obstetrics is organized to provide services 24 hours a day throughout the year . in the delivery rooms
are located the most modern equipment for vaginal delivery , with a total of 10 beds for delivery , with room for delivery in the presence of a spouse ( partner ) . as part of
the clinic of obstetrics , there are operating rooms with direct access from the delivery room in case of emergencies .
the goal is to show the operation at the birth room of clinic for gynecology and obstetrics for the year 2012 , frequency of completed deliveries by caesarian section , also describe manners of delivery and to provide a description of other techniques and manual interventions that have been applied during childbirth .
the study was conducted at the clinic of obstetrics , clinical center university of sarajevo .
a database based on the data from the delivery room protocol is made in which are entered all details regarding delivery ( parity , mode of delivery , interventions during birth , information on newborn ) .
data were entered in ms excel , imported into the statistical program spps v.20.0 and then statistically analyzed . for the statistical analysis of qualitative data we used chi - square tests , and for quantifying data anova .
in the study period of one year at the clinic of obstetrics there were 3216 ( 100% ) deliveries .
of the total number of births vaginally completed delivery was in 2101 ( 65% ) cases and cesarean section in 1115 ( 35% ) cases ( figure 3 ) .
the analysis of the frequency of the number of births per month reveals that the lowest number of births per month was during february with a total of 234 ( 7.27% ) and the highest percentage in september ( 9.48% ) .
the average number of births per day in the 2012 amounted to 8.81 ( figure 4 ) .
anova test showed a statistically significant difference in the incidence of cesarean sections on a monthly basis ( p<0.05 ) . in the 2012 the total of 48 pregnant women completed delivery by caesarean section after a failed trial of vaginal delivery ( figure 5 ) .
of the total number of pregnant women delivered during the study period in 1603 ( 49.84% ) cases they were nulliparous , second child had 1186 ( 36.87% ) , while third or more delivery was in 427 ( 13.27% ) cases .
chi - square test showed a statistically significant difference ( =663.02 , df=2 , p 0.05 ) in the parity of pregnant women who gave birth in the 2012 ( figure 6 ) .
the number of births completed by cesarean section in 2012 amounted to 1115 ( 100% ) . in 731 ( 65.56% ) cases
it was the first cesarean section , repeated - iterative caesarean section was performed in 353 ( 31.66% ) cases , while reiterative caesarean section was performed in 31 ( 2.78% ) cases with statistically significant difference in the incidence of cesarean section ( =660.02 , df=2 , p<0.05 ) .
table 1 shows the number of births in relation to the number of babies . of the total number of births 3156 ( 98.13% )
were singleton pregnancies , twin pregnancy and childbirth was in 59 ( 1.83% ) cases and the triplets were only 1 ( 0.03% ) case .
of the total number of births in the 2012 in term deliveries had a total of 2907 ( 89% ) , while premature births had 370 ( 11% ) pregnant women .
analysis of infants weight has led to the fact that in 2012 was born 3006 ( 91.73% ) infants with birth weight over 2500 g , with a body weight of 1000 - 2500 g was born 240 ( 7.32% ) infants , while the body weight of 500 - 1000 g had a total of 31 ( 0.95% ) infants .
of the total number of deaths , 15 had birth weight from 10002500 g , while 8 had a body weight over 2500 g .
of all obstetric surgeries in 2012 , the majority was manual explorations of the uterus ( 197 ) , followed by manual placental lysis ( 49 ) . among other surgeries
was performed 21 vacuum extractions , 15 hand assisted births in case of breech births and 3 births with use of forceps .
for centuries the pregnancy caused fear and caution of poor outcome for the mother or child .
all of us , who have ever attended childbirth , are aware of the fact , that there is nothing more natural and normal than normal birth and that there is nothing abnormally as abnormal childbirth and nothing
so astonishingly quickly yarn from one to another ( 1 ) . work of birth room at the clinic of obstetrics is based on a 24-hour working time and involvement of experts in the field of obstetrics . in the 2012 year at the clinic of obstetrics , 3216 woman gave birth .
abadzic in his study showed that the percentage of caesarean sections at the clinic of obstetrics , clinical center of sarajevo university is in relation to the total number of births and in the 1996 it was 8.57% while the percentage of 2007 was 27.75% .
for a 12 years period the number of caesarean sections is tripled compared to the total number of births ( 2 ) . the upward trend in cesarean sections
obstetric practice is witness to the world trend of increasing rates of caesarean sections in the past few years .
australia and the united states have the highest rate of caesarean sections in the developed world of 28.5% and 29.1% ( 3 ) .
similar trends of increasing rates of caesarean sections occur in latin america , especially in mexico and brazil 25.7% and 27.9% , as well as in other developing countries , such as india ( kerala state ) , 21.4% ( 5 ) . although optimal rate of cesarean sections remains point of many debates .
fatusic and colleagues in a retrospective study at clinic of gynecology and obstetrics in tuzla , processed medical data on cesarean sections completed in five years period , from 1984 to 1988
. period which they processed was not affected by the epidemic of cesarean sections . in the study period
was performed 6.47% ( 1819 ) caesarean section in relation to the total number of births . of the total percentage of performed caesarean sections 90.22% ( 1641 )
was an unplanned cesarean section while 9.78% ( 178 ) was elective caesarean sections ( 6 ) .
the largest number of births in the 2012 year was by primiparous women ( 1603 ) , then the second child ( 1186 ) .
perinatal mortality in this period amounted to 7.12 . abadzic in his study showed that the rate of perinatal mortality in 1996 amounted to 16.65 whereas in 2007 it amounted to 9.60 ( 2 ) .
the study , the rate of cesarean delivery and perinatal mortality in infants at maichin dom
( 7 ) is performed in bulgaria in the period from 1976 to 2000 and showed that the increase in the cesarean section rate of 4.8% to 24.4% lead to the reduction of perinatal mortality from 27.7 to 11.4 . in the group of women with normal pregnancy , perinatal mortality is 8 - 11 at the frequencies of performed cesarean sections of 15 - 16% , while in high - risk pregnancies perinatal mortality is the same with the rate of performed cesarean sections of 24 - 26% .
no correlation between cesarean section rates and perinatal mortality of singleton infants over 2500 g is a study conducted in iceland which provided new data when obstetric trends are in question ( 8) .
information is gathered through icelandic birth registry for a period of 20 years from 1987 to 2006 .
perinatal mortality for this period amounts to an average of 2 annually and the range is from 0.8 to 4.1 . cesarean section rate varied between 11.9% and 16.7% and did not correlate with perinatal mortality . among primiparas ,
the rate of cesarean section was increased from 13.1 to 17.8% , without correlation with perinatal mortality , which averaged to 1.7 .
the number of births at the clinic of obstetrics , clinical center university of sarajevo has gradually decreased , while the number of caesarean sections is constantly increasing . during the 2012 perinatal mortality
has decreased and the percentage of infants with low birth weight ( < 2500 g ) is in limits of the birth of such children in the european union .
a small number of completed vaginal deliveries after previous cesarean section warn that we should instead prefer instead of elective cesarean section first a test delivery .
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clinic for gynecology and obstetrics , clinical center university of sarajevo represents a tertiary level of health care with more than 3,000 births during one year .
the aim of this article is to present the operation of the birth room at the clinic for gynecology and obstetrics in 2012 .
data were obtained on the basis of protocol of deliveries from the birth room .
material and methods : analyzed are the total number of births , the incidence of obstetric surgeries and other manual interventions . results : over the study period , there were a total of 3216 births , of which by caesarean section was , completed 1115 .
the highest number of births was in september ( n=305 ) .
the largest number of women who gave birth was nulliparous .
also among primiparous is performed the majority of cesarean sections ( n=731 ) . in the study period ,
multiple births were recorded in a total of 60 ( 59 twins , 1triplets ) .
number of premature births was 370 , and the perinatal mortality was 7.12 . number of newborn with birth weight below 2500 g was 271 . among manual interventions in the delivery room
mostly was used manual exploration of the uterus followed by the manual lysis of the placenta .
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cystinuria is an autosomal recessive disorder of amino acids transport affecting the epithelial cells of the renal tubules and gastrointestinal tract .
it is characterized by increased urinary excretion of cystine and the dibasic amino acids ( ornithine , lysine and arginine ) , and manifested by recurrent renal stones due to the poor solubility of cystine in the urine .
there are three different types of cystinuria according to the urine phenotype in heterozygotes : type i ( mim220100 ) , non - type i ( mim600918 ) , and mixed . in type i cystinuria , heterozygotes have a normal pattern of amino acid excretion in the urine ( 1 ) . in non - type i
cystinuria , heterozygotes have a variable degree of hyperexcretion of cystine and diabasic amino acids . patients with mixed type cystinuria inherit type i and non - type i alleles from either parent .
two genes responsible for cystinuria have been identified . the first locus gene , slc3a1 , located on chromosome 2p-16.3 - 21 encoding rbat ( related to b amino acid transporter ) ,
was identified to be the cause of type i cystinuria in 1994 ( 2 ) .
a second locus gene that is responsible for non - type i cystinuria was mapped on chromosome 19q12 - 13.1 .
encoding for b at ( b amino acid transporter ) by linkage analysis and later identified as slc7a9 by international cystinuria consortium ( 3 ) . at present ,
79 mutations in slc3a1 were reported and over 50 mutations in slc7a9 have been described so far ( 1 ) .
there have been several reports of cystinuria associated with neurological manifestations ; mental retardation , muscular dystrophy , hypotonia and dwarfism , paroxysmal dyskinesia , migraine , subacute combined degeneration of the spinal cord , and cerebellar atrophy ( 4 - 6 ) . in terms of pathogenetic correlation between cystinuria and various neurological conditions , it is still undetermined whether these two conditions share a common pathogenesis or are coincidental .
we report a case of non - type i cystinuria associated with neurologic symptoms and identified new missence mutation g173r in slc7a9 gene .
a 13-yr - old boy was admitted due to increasing difficulty in walking and clumsiness of the movement , noticed at about 5 yr of age by his mother .
he held his head at 3 months , turned over at 5 months , and sat alone at 10 months of age .
his neurodevelopments were delayed since then ; standing with support at 16 months , walk alone at 21 months of age .
he used his first meaningful words at 2 yr of age , make phrases at 4 yr of age , and simple sentences at 5 yr of age . at the age of 5
yr , his mother noticed that he walked with a slow and broad - based gait , and all his movements were weak .
however , he did not sought medical advice . his difficulty in walking was slowly aggravated , and he also had a difficulty in academic achievement recently .
physical examination revealed minor dysmorphic features : long face , macrognathia , high arched palate , and large ear .
neurologic examination disclosed mild higher cortical dysfunction , dysarthria , upper limb and gait ataxia , hyperreflexia with bilateral extensor toe signs .
the sensory examination disclosed no abnormalities except that vibration and position sense were impaired in lower extremities .
neuropsychological evaluation revealed mild mental retardation on the k - wisc - iii : full - scale intelligence quotient , 62 ; verbal scale intelligence quotient , 79 ; performance scale intelligence quotient ; 47 .
somatosensory evoked potential of posterior tibial nerve stimulation revealed no cortical waves ( p1 ) suggestive of central conduction dysfunction through the posterior column of the spinal cord .
however , spine magnetic resonance imaging ( mri ) did not disclosed structural lesions in the spinal cord .
chromosome analysis and fragile x mental retardation 1 ( fmr1 ) gene studies are normal .
cerebrospinal fluid examination , serum and cerebrospinal fluid lactate / pyruvate level , urine organic acid analysis , and plasma amino acid analysis were normal .
quantitative amino acid analysis of the urine disclosed increased excretion of cystine ( 1,345 m / g cr ) and an excess of the dibasic amino acids ; ornithine , lysine , and arginine ( fig .
his mother and sister were screened by quantitative amino acid analysis of the urine and showed variable degree of cystine and dibasic amino acids excretion ( fig .
mutation analysis of slc3a1 and slc7a9 genes was performed on genomic dna samples , which were extracted from peripheral blood lymphocytes of the patient , his mother , and his sister .
amplification of individual exons of the two genes was performed using intronic primers obtained from the literature ( 7 ) .
direct sequencing of entire coding region of slc3a1 and slc7a9 genes of the patient revealed both a g ( normal ) and a ( mutant ) at nucleotide 535 in exon 5 , resulting in the presence of a gly ( codon gga ) at amino acid position 173 and a arg ( codon aga ) in heterozygote state ( fig .
we identified the novel mutation , g173r and 6 known polymorphisms ( table 1 ) . to confirm that g173r is not a polymorphism which could be found in normal control , we screened this mutation in 50 unrelated healthy korean .
the patients has only one mutation , g173r in slc7a9 gene , therefore we screened for slc3a1 mutations to ensure that no other mutations were missed .
g173r was also detected in the genomic dna of the patient 's mother and sister .
cystinuria is an inherited disorder of cystine and dibasic amino acids ; ornithine , lysine , and arginine .
this disorder is mainly expressed by the formation of cystine stones in the urinary tract .
more than 80% of patients develop their first stone within the first 2 decades . on the other hand cystinuira
has been occasionally described in association with a various neurological manifestations ; mental retardation , muscular dystrophy , hypotonia and dwarfism , paroxysmal dyskinesia , migraine , subacute combined degeneration of the spinal cord , and cerebellar atrophy ( 4 - 6 ) . our patient is similar to these in that there is clinical evidence of posterior column , corticospinal tract involvement , and mental retardation .
pathogenetic correlation between cystinuria and various neurological conditions is still undetermined , and a relevant explanation must be needed .
if the amino acid transport systems of the brain closely resemble those of the kidney and gastrointestinal tract , one might expect that cystinuria could impaired brain functions ( 8 , 9 ) .
experimental results have demonstrated that transport systems for entry of cystine and cysteine into brain differ from those of kidney and intestine ( 10 ) .
it is unlikely that cystinuric patients are at risk of impaired cerebral functions because of difference in the amino acid transport systems of the nervous system ( 9 ) .
however , scriver et al . ( 4 ) reported that homozygous cystinuria is statistically more prevalent in patients in mental hospitals than in the general population .
this finding suggest that cystinuric patients are still at higher risk for impaired cerebral function .
cystinuria is currently classified into three types according to the urinary phenotype in heterozygotes : type i ( mim220100 ) , non - type i ( mim600918 ) , and mixed . in non - type i heterozygotes showed a variable degree of urinary hyperexcretion of cystine and dibasic amino acids .
probands excreting more than 1,300 m / g cr are considered as phenotypically homozygote ( 11 ) .
i cystinuria , there is no uptake of cystine by intestinal mucosal cells and there is no rise in serum cystine following an oral cystine load . in non - type i
cystinuria , the intestinal transport of dibasic amino acids is disturbed , but not abrogated .
there is some increment in plasma cystine following oral cystine load especially in patients which were previously classified as type iii cystinuria by old classification scheme ( 9 ) . in this patient ,
quantitative amino acid analysis disclosed increased urinary cystine excretion ( 1,345 m / g cr ) within the range of phenotypic homozygote , and an excess of the dibasic amino acids ; ornithine , lysine , and arginine ( fig .
his mother and sister also showed increased excretion of cystine and diabasic amino acids in urine ( fig .
. therefore , patient was diagnosed as non - type i cystinuria . despite the classification of the disease described above
, a new scheme based only on genetic aspects has recently been proposed : type a , caused by mutations in both alleles of slc3a1 ; type b , caused by mutations in both alleles of slc7a9 ; the possibility of type ab , with one mutation on each of the above genes ( 1 ) .
we screened the family with patient for gene mutation by sequencing of the slc7a9 and slc3a1 gene , and identified a new missence mutation g173r in slc7a9 gene in heterozygote state .
the present search for mutations covered the whole coding region and neighboring intron / exon boundaries . despite careful analysis of the slc3a1 and slc7a9 genes ,
there are still a number of patients that lack mutations or display only heterozygosity ( 1 ) .
recently , two distinct recessive contiguous gene deletion syndrome , hypotonia - cystinuria syndrome and 2p21 deletion syndrome , associated with type i cystinuria have been described ( 12 , 13 ) . in these contiguous gene deletion syndromes , various neurologic manifesations such as mental retardation , hypotonia , developmental delay , neonatal seizure , facial dysmorphism , and mitochondrial respiratory chain deficiencies
this contiguous gene deletion is already known to disrupt the coding region of at least 4 genes ( slc3a1 , prepl , ppm1b and c2orf34 ) .
deletion of slc3a1 gene causes cytinuria , and loss of the other 3 genes are elucidated to contribute neurologic manifestations in these contiguous gene deletion syndromes . in this pedigree ,
patient 's mother and sister did not showed clinical symptoms of cystinuria , but biochemical and molecular genetic data disclosed them as heterozygote of non - type i cystinuria .
they also did not showed neurologic symptoms similar to those of the patient even though they had same g173r mutation in slc7a9 gene .
although the neurologic manifestations of the patient probably are not to be caused by g173r mutation itself , it may be explained by unidentified other genes associated with slc7a9 gene mutation including large gene deletion such as a contiguous gene deletion syndrome which may be transmitted to the patient from paternal side .
extensive genetic evaluation must be needed in the patient and his father to answer this question .
, we identified a new mutation , g173r , in the exon 5 of slc7a9 gene , in the patient with non - type i cystinuria associated with neurologic manifestations which presented as mental retardation and ataxia .
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cystinuria is an inherited renal and intestinal disease characterized by defective amino acids reabsorption and cystine urolithiasis .
it is unusually associated with neurologic symptoms .
mutations in two genes , slc3a1 and slc7a9 , have been identified in cystinuric patients .
this report presents a 13-yr - old boy with cystinuria who manifested difficulty in walking , ataxia , and mental retardation .
somatosensory evoked potential of posterior tibial nerve stimulation showed the central conduction dysfunction through the posterior column of spinal cord .
he was diagnosed non - type i cystinuria by urinary amino acid analysis and oral cystine loading test .
we screened him and his family for gene mutation by direct sequencing of slc3a1 and slc7a9 genes . in this patient , we identified new missence mutation g173r in slc7a9 gene .
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chorea can be caused by a variety of diseases , including neurodegenerative disorders , vascular events , toxic - metabolic states , and immunologic and infectious diseases .
we describe a patient who presented with hemichorea as the initial manifestation of diabetes mellitus ( dm ) and responded partially to the glycemic control .
a 63-year - old , healthy hispanic man with no prior history of medical illness presented with subacute onset , gradually progressive hemichorea of 6 weeks duration . on evaluation , he was found to have non - ketotic hyperglycemia with high serum glucose ( 328 mg / dl ) , elevated hemoglobin a1c ( 9.9% ) , and absent ketones .
magnetic resonance imaging of the brain demonstrated hyper intense signals in bilateral basal ganglia on t1w images .
the patient continued to have hemichorea at 3 months follow - up and required haloperidol for control of the involuntary movements .
involuntary movements , particularly hemichorea , can be a manifestation and rarely be a presenting sign of dm .
a 63-year - old , right - handed hispanic man was brought to the emergency room with the complaint of abnormal movements of right arm and right leg for about 6 weeks .
the patient initially developed intermittent twitches in the right shoulder and arm as noticed by his daughter , which the patient was unaware of .
two weeks later , he was briefly admitted to an outside hospital for dizziness , dehydration , and high serum glucose levels up to 1,100 mg / dl .
after 6 days of discharge from the hospital , he experienced worsening of involuntary movements which now involved whole right upper extremity and progressed in 2 weeks to right leg and foot .
these movements were interfering with his daily activities prompting him to come to our hospital .
he did not have abnormal behavior , headache , and weakness on any side or sensory disturbances .
he was a reformed smoker and alcoholic . on examination , he was alert , awake , and oriented
motor examination revealed normal strength , but hypotonia , hyporeflexia , and rapid , jerky , continuous non - stereotyped involuntary choreiform movements involving right upper and lower extremities .
he could not walk without support due to abnormal movements in right leg and foot .
his serum glucose was 328 mg / dl , hba1c level 9.9% , and serum ketones were negative .
1a ) revealed a 11.7 cm area of subtle increased density in left lentiform nucleus with surrounding decreased attenuation .
magnetic resonance imaging ( mri ) brain with gadolinium contrast showed non - enhancing hyper intense lesions in bilateral lentiform and caudate nuclei on t1w images and old stable infarcts ( figs .
( a ) non - contrast ct scan showing ill - defined hyperdensity in left putamen .
( d ) axial t1-w with contrast showing no enhancement of abnormal signals after gadolinium .
despite adequate glycemic control using insulin , he continued to have hc and responded partially to haloperidol . at 3-month follow - up ,
polyphagia , polydipsia , weight loss , fatigue , and weakness are common presenting symptoms in patients with dm .
chorea is a rare manifestation of nkh in dm and was described first in 1960 ( 1 ) . it has rarely been described as the presenting sign of new onset diabetes ( 2 , 3 ) .
hemichorea has been more commonly described to be associated with nkh than generalized chorea . in a meta - analysis of 53 patients by oh et al . , mean age was 71 years and male : female ratio was 1:1.8 ( 4 ) . in a study of 35 patients ,
patients typically present with subacute onset , gradually progressive involuntary movements over days to weeks associated with high glucose levels , absent ketones , and high serum osmolality .
however , chorea starting few days after the hyperglycemic crisis and even after adequate sugar control has been reported ( 6 ) .
the movement disorder in nkh range from mild choreoathetoid movements to hemiballism with violent flinging movements .
associated features such as personality changes , seizures , painful sensory symptoms , and weakness have been described as well ( 7 , 8) .
usually , movements resolve within 2448 h of aggressive sugar control ( 9 , 10 ) .
however , prolonged hc and relapse after the initial response has also been described ( 11 , 12 ) .
reported five female patients who developed chorea concurrently or shortly after hyperglycemic episode and all patients continued to have persistent chorea despite sugar control for 6 months to 5 years of follow - up ( 6 ) .
patients with hc with incomplete recovery from glycemic control may respond well to the conventional neuroleptics , such as haloperidol , perphenazine , and chlorpromazine .
also , risperidone and an anticonvulsant such as topiramate may be useful ( 1113 ) .
the prognosis of chorea associated with nkh has been reported as good ( 10 ) .
striatum may be directly susceptible to alterations of blood glucose because even patients with hypoglycemia can develop choreoathetoid movements ( 14 ) .
ischemia secondary to hyperviscosity or neural injury due to hyperglycemia could be the possible cause for abnormal movements ( 15 ) .
reported two cases of dm and chorea where hemorrhagic infarction was caused by diapedesis from damaged but not ruptured capillaries ( 17 ) .
it is also possible that chorea in nkh is due to a functional abnormality rather than structural disease .
putamen may have inhibitory influence on globus pallidus and lesions of putamen cause uninhibited activity of globus pallidus leading to choreiform movements .
neurotransmitter dysfunction mainly hyperactivity of dopaminergic neurons is thought to be a prominent cause . in nkh , brain metabolism shifts to anaerobic pathway and brain utilizes gaba as energy source . unlike in diabetic ketoacidosis , gaba is not resynthesized and rapid depletion of gaba leads to decreased acetyl choline synthesis .
reduced gaba , acetyl choline , decreased energy , and regional metabolic failure may cause basal ganglia dysfunction leading to chorea ( 4 ) .
majority have hyperintense signals in contralateral putamen on t1-weighted images . in few case series , patients also had hyperintensities in contralateral caudate ( 19 ) .
patients can also have bilateral basal ganglia hyperintense signals on t1w images . in the meta - analysis of 53 cases by oh et al .
, all patients had hyperintense signals in contralateral putamen on t1w images and six ( 11% ) patients had bilateral basal ganglia lesions ( 4 ) . usually , there is hypointensity or no significant alternation in t2-weighted images and on contrast mri lesions are mostly non - enhancing .
another characteristic finding is the resolution of abnormal signals in follow - up imaging although it may be seen well ahead of clinical improvement ( 4 ) .
we suggest that detailed neuroimaging should be performed including dwi and swi to exclude infarction , hidden ischemia ( perfusion deficit ) , and hemorrhagic processes , which may affect treatment strategies .
pet scan in three patients with chorea and nkh showed markedly reduced rates of cerebral glucose metabolism in the corresponding lesions on t1-weighted images on mri suggesting evidence of regional metabolic failure ( 13 ) . as dm is a widely prevalent disease and hemichorea due
to nkh is amenable to rapid sugar control , it is important for physicians to be aware of this condition and consider it in appropriate clinical settings .
why chorea persists in some patients of nkh despite optimal glycemic control is not entirely clear though neuronal loss from ischemia and putaminal microhemorrhage are possible causes
involuntary movements , particularly hemichorea , can be a manifestation and rarely be a presenting sign of dm . usually , hemichorea is associated with nkh .
exact pathogenesis is unclear ; direct affection of striatum by altered glucose levels is possible .
all the listed authors have participated actively in the study , have met the requirements for the authorship , and have read and approved the submitted manuscript .
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backgroundchorea can be caused by a variety of diseases , including neurodegenerative disorders , vascular events , toxic - metabolic states , and immunologic and infectious diseases .
we describe a patient who presented with hemichorea as the initial manifestation of diabetes mellitus ( dm ) and responded partially to the glycemic control.case reporta 63-year - old , healthy hispanic man with no prior history of medical illness presented with subacute onset , gradually progressive hemichorea of 6 weeks duration . on evaluation
, he was found to have non - ketotic hyperglycemia with high serum glucose ( 328 mg / dl ) , elevated hemoglobin a1c ( 9.9% ) , and absent ketones .
magnetic resonance imaging of the brain demonstrated hyper intense signals in bilateral basal ganglia on t1w images .
he was diagnosed to have dm . despite optimal glycemic control with insulin ,
the patient continued to have hemichorea at 3 months follow - up and required haloperidol for control of the involuntary movements.significanceinvoluntary movements , particularly hemichorea , can be a manifestation and rarely be a presenting sign of dm .
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alpha-1 antitrypsin deficiency ( aatd ) is a congenital autosomical codominant condition characterized by low plasma levels of alpha-1 antitrypsin ( aat ) in the blood and tissues .
more than 120 genetic variants of the aat gene have been identified and classified into three major categories : normal , with genotype m , characterized by aat within normal ranges ; deficient , characterized by reduced but detectable aat plasma levels with genotypes z , s , and m - like ; and null , currently designated as q0 , with no detectable plasma levels.1 aatd is one of the most common congenital disorders with an estimated prevalence between one in 2,857 and one in 5,097 in usa1 and between one in 2,175 and one in 5,164 in spain.2 aatd predisposes the development of certain diseases , especially copd in adults and liver disease , which is more frequent in children .
other less frequent conditions associated with aatd are panniculitis , vasculitis , and fibromyalgia.1 the world health organization recommends the testing of all copd patients,3 and the european respiratory society and american thoracic society guidelines recommend the testing of all symptomatic adults with persistent airway obstruction , such as copd , emphysema , and asthma with incompletely reversible airflow obstruction , individuals with unexplained liver disease , and adults with necrotizing panniculitis or multisystemic vasculitis.1 similarly , the spanish society of pneumology and thoracic surgery ( separ ) recommends that all copd patients should be tested at least once in their lives.4 despite these recommendations , aatd is significantly underdiagnosed , and most of the patients are detected long after the onset of pulmonary or liver disease .
another implication of this late diagnosis is the delay in the detection of affected relatives , which hinders the implementation of measures , such as abstaining from tobacco exposure.5,6 underdiagnosis of aatd is a challenge , particularly , for primary care ( pc ) physicians who attend most of the copd patients , and this is usually the first point of contact of patients with health care providers . computerized databases of medical records are increasingly used in clinical research to enhance the knowledge about the management and progression of this disease based on real - life data.7 database studies help to understand real clinical practice and to design public health strategies to improve the quality of care .
the objective of this study was to describe the patterns of diagnosis of aatd in pc in catalonia , spain .
this was an epidemiological , population - based , observational study aimed to quantify and compare the number of aat determinations performed in the pc in catalonia during two 2-year periods ( 20072008 and 20102011 ) and to describe the characteristics of the individuals tested and the management of those with deficient values .
data for this study were obtained from the system for the development of research in primary care ( sidiap ) database , a computerized database containing anonymized patient records for the 5.8 million people registered in the 279 pc centers of the catalan health institute ( > 80% of catalonia s population ) .
all general practitioners in the catalan health institute use the same specific software called ecap to record the clinical information of their patients .
health professionals gather this information using codes of international statistical classification of diseases and related health problems , tenth revision , and structured forms designed for the collection of variables .
sidiap combines information from the electronic medical records with data from other databases and registers , such as laboratory test results ( from the laboratory databases ) , the pharmacy register , and the national mortality register.8,9 for the purpose of the study , we checked the quality of the sidiap database .
high - quality data were obtained from 2007 onward ; however , data from 2012 were not available at the time of the initiation of the study .
therefore , to compare two periods of the same length , we used data from 2007 to 2008 and 2010 to 2011 .
the study was approved by idiap jordi gol ethics committee ( barcelona , spain ) .
this was a retrospective study with data from an anonymized database , so it was not necessary to request patient consent .
based on the levels obtained in the determination , individuals were classified as follows : no deficiency : aat > 100 mg / dl ; intermediate deficiency : aat between 50 mg / dl and 100 mg / dl ; and severe deficiency : aat < 50 mg / dl.10 since indications for aat testing differ by age group , we classified individuals younger than 15 years as children and analyzed them separately .
demographic and clinical characteristics were recorded for all the study populations . for individuals with intermediate and severe deficiencies , we collected data on referrals to a specialist , complementary tests ( spirometry and computerized tomography scans ) , pharmacologic treatment , and number of respiratory infections during the 6 months following the determination .
a descriptive analysis of each period ( 20072008 and 20102011 ) and of the totality of the sample was performed separately for children and adults . for qualitative variables , absolute frequencies and corresponding percentages
quantitative variables following a normal distribution were described by mean and standard deviation , while those not following a normal distribution were described using the median and 2575 percentiles .
differences between groups were performed using the chi - square test for categorical variables , while continuous variables were tested using the student s t - test ( or the mann
all statistical analyses were performed using a statistical software package ( spss version 20.0 ; ibm corporation , armonk , ny , usa ) .
all the individuals with an aat determination during the study period were included . based on the levels obtained in the determination
, individuals were classified as follows : no deficiency : aat > 100 mg / dl ; intermediate deficiency : aat between 50 mg / dl and 100 mg / dl ; and severe deficiency : aat < 50 mg / dl.10 since indications for aat testing differ by age group , we classified individuals younger than 15 years as children and analyzed them separately .
demographic and clinical characteristics were recorded for all the study populations . for individuals with intermediate and severe deficiencies
, we collected data on referrals to a specialist , complementary tests ( spirometry and computerized tomography scans ) , pharmacologic treatment , and number of respiratory infections during the 6 months following the determination .
a descriptive analysis of each period ( 20072008 and 20102011 ) and of the totality of the sample was performed separately for children and adults . for qualitative variables , absolute frequencies and corresponding percentages
quantitative variables following a normal distribution were described by mean and standard deviation , while those not following a normal distribution were described using the median and 2575 percentiles .
differences between groups were performed using the chi - square test for categorical variables , while continuous variables were tested using the student s t - test ( or the mann
all statistical analyses were performed using a statistical software package ( spss version 20.0 ; ibm corporation , armonk , ny , usa ) .
in total , 12,409 determinations of serum aat were performed during the 4 years of the study , of which 1,335 ( 10.7% ) were children .
the number of determinations was higher in the second period ( 5,559 determinations in 20072008 and 6,850 determinations in 20112011 ) due to the low number of individuals tested in 2007 .
nonetheless , the rate of individuals tested per year did not increase significantly after 2008 ( table 1 ) .
the mean age of the individuals tested was 52.6 ( sd 16.3 ) years in adults and 4.6 ( sd 4.1 ) years in children , with an equal distribution between sexes . among adults ,
the most frequent comorbidities in adults were dyslipidemia ( 27.6% ) , hypertension ( 27.4% ) , diabetes mellitus ( 11.7% ) , depression ( 10.1% ) , and ischemic heart disease ( 4% ) . up to 41% of children and 18.5% of adults were receiving treatment for a respiratory disease at the time of the determination
demographic characteristics are shown in tables 2 and 3 . as a possible indication for aat determination ,
3,195 ( 28.9% ) adults and 393 ( 29.4% ) children had a previous diagnosis of a disease related to aatd . up to 17.7% of children and
nine percent of children were between the age 0 year and 1 year , suggesting neonatal jaundice as the most likely indication . during the previous year
, 31.3% of individuals had had at least one respiratory infection and 1.3% had had pneumonia .
the mean aat plasma level was 147.2 ( 36.7 ) mg / dl in adults and 154.1 ( 37.2 ) mg / dl in children . in total , 663 ( 5.3% ) individuals ( 56 children ) had an intermediate aat deficiency , while 24 ( 0.2% ) individuals ( two children ) had a severe deficiency , with a prevalence of 0.19 cases of severe deficiency per 100 determinations .
patients with severe deficiency were younger than individuals with normal aat levels ( 42.5 years vs 52.9 years , p=0.003 ) and were more likely to have a previous diagnosis of copd or emphysema ( 45.5% vs 10.8% , p<0.05 ) ( table 3 ) . during the 6-month follow - up ,
four of the patients with severe deficiency ( 18.1% ) were newly diagnosed with copd or emphysema , two ( 9.1% ) following diagnostic spirometry and one ( 4.2% ) after a computerized tomography scan .
only three patients ( 13.6% ) were referred to a pneumologist and another patient was referred to internal medicine ( table 4 ) .
in total , 12,409 determinations of serum aat were performed during the 4 years of the study , of which 1,335 ( 10.7% ) were children .
the number of determinations was higher in the second period ( 5,559 determinations in 20072008 and 6,850 determinations in 20112011 ) due to the low number of individuals tested in 2007 .
nonetheless , the rate of individuals tested per year did not increase significantly after 2008 ( table 1 ) .
the mean age of the individuals tested was 52.6 ( sd 16.3 ) years in adults and 4.6 ( sd 4.1 ) years in children , with an equal distribution between sexes . among adults ,
the most frequent comorbidities in adults were dyslipidemia ( 27.6% ) , hypertension ( 27.4% ) , diabetes mellitus ( 11.7% ) , depression ( 10.1% ) , and ischemic heart disease ( 4% ) . up to 41% of children and 18.5% of adults were receiving treatment for a respiratory disease at the time of the determination
as a possible indication for aat determination , 3,195 ( 28.9% ) adults and 393 ( 29.4% ) children had a previous diagnosis of a disease related to aatd . up to 17.7% of children and
nine percent of children were between the age 0 year and 1 year , suggesting neonatal jaundice as the most likely indication . during the previous year , 31.3% of individuals had had at least one respiratory infection and 1.3% had had pneumonia .
the mean aat plasma level was 147.2 ( 36.7 ) mg / dl in adults and 154.1 ( 37.2 ) mg / dl in children . in total , 663 ( 5.3% ) individuals ( 56 children ) had an intermediate aat deficiency , while 24 ( 0.2% ) individuals ( two children ) had a severe deficiency , with a prevalence of 0.19 cases of severe deficiency per 100 determinations .
patients with severe deficiency were younger than individuals with normal aat levels ( 42.5 years vs 52.9 years , p=0.003 ) and were more likely to have a previous diagnosis of copd or emphysema ( 45.5% vs 10.8% , p<0.05 ) ( table 3 ) . during the 6-month follow - up ,
four of the patients with severe deficiency ( 18.1% ) were newly diagnosed with copd or emphysema , two ( 9.1% ) following diagnostic spirometry and one ( 4.2% ) after a computerized tomography scan .
only three patients ( 13.6% ) were referred to a pneumologist and another patient was referred to internal medicine ( table 4 ) .
the results of this study show that the number of aat determinations performed in the pc in catalonia , spain , is low and has not increased after 2008 . in addition , in most cases , we could not identify the reason for requesting the test , and after detection of a severe deficiency , some individuals were not tested further or referred to a specialist .
aatd is one of the most common congenital disorders but remains significantly underdiagnosed despite the recommendations of national and international guidelines.1,2 moreover , there is still a large delay between the onset of symptoms and diagnosis,11 with no significant improvement in this delay during the last decades.12 in spain , it is estimated that 12,026 patients have a severe deficiency,13 wit1,700 of these cases corresponding to catalonia alone.14 nonetheless , only 511 cases from all over the country are diagnosed and included in the spanish registry for aatd.15 possible explanations for the underdiagnosis of aatd have been addressed in previous studies .
knowledge of aatd is generally poor even for trainees who declared a special interest in respiratory medicine.16 among nonpulmonologists , awareness of aatd is low in comparison with other respiratory diseases,17 with the consequent low rate of testing for aatd.11,12,18 a survey carried out in spain and portugal showed that the main reasons for not testing for aatd are the referral of patients to other specialists for testing or the erroneous perception of the high cost of the test.19 despite the current recommendations of testing symptomatic adults with persistent airway obstruction and individuals with unexplained liver disease,1,2 the rate of aat determinations observed in our study along the years varied from 4.33 determinations per 10,000 inhabitants in 2007 to 6.85 determinations per 10,000 inhabitants in 2008 , with intermediate values for 2010 and 2011 .
data from a recent study performed with data from sidiap indicated that a mean of 6,932 new patients were diagnosed with copd per year between 2007 and 2012 , a figure well above the 375 mean number of copd patients tested yearly for aatd during the 4 years of our study.9 although underdiagnosis of the deficiency has been reported in many countries , to our knowledge , this low rate of aat determinations in the general population and in copd patients has not been previously reported , thereby not allowing comparison of our findings with data from other countries or geographical areas . regarding the reasons for requesting aat determination , we observed that only 13% of the adults tested had copd , chronic bronchitis , or emphysema and half had high transaminase levels , which could justify the request for aat determination .
these results concur with the observation that > 70% of pc physicians in spain were aware of liver complications of aatd , but very few decided to test all copd patients.19 similarly , only 0.5% of children had a codified liver disease at the time of aat determination , and interestingly , the number of children tested for transaminitis and asthma was similar , although aatd is not a recognized cause of respiratory diseases in childhood.20 in an attempt to improve the rate of diagnosis of aatd in copd , several screening initiatives or case findings have been developed,2125 some being carried out in the pc.22,23 in the iddea project of case finding of aatd in copd patients , volunteer pc physicians were provided with filter paper to collect dried blood spots , together with information about aatd and a web tool .
the ratio of recruitment only reached 6.6 patients per participant over the 9-month collection period , being somewhat low considering that the estimated prevalence of copd in spain is 10.2% of adults older than 40 years.26 however , among the individuals tested , 4% were carriers of the severe deficient allele z , and 0.34% were diagnosed with severe homozygous pizz deficiency.23 jain et al24 implemented an electronic alert to encourage guideline - based testing for aatd .
this alert was displayed for patients with obstructive spirometry results , and this tool was associated with an increase in the frequency of testing .
other strategies , such as programs to educate respiratory physicians27 and the combination of an awareness program with the offer of free diagnostic testing,28 resulted in high rates of detection of individuals with severe aatd .
population screening programs in areas of high prevalence or protocols to measure and phenotype aat in selected patients were found to be effective at detecting aatd patients.2931 our study has some limitations .
first , the reason to request a complementary test is not recorded in the sidiap database , and we can not be completely certain of the indication leading to the aat determination . we can only assume the reason based on the codified diagnosis or the results of liver function tests .
second , databases are also subject to possible diagnostic and miscoding biases.32 however , considering that our main objective was to quantify the number of aat determinations performed , we believe that this possible bias had little impact , if any , on the main objective of the study . on the other hand , the sidiap database includes data from > 80% of the population of our area , thereby ensuring the representativeness of the results for the whole population of catalonia .
our study shows that the rate of testing for aat in pc is still low , and the reasons for requesting the determination often remain unclear .
these results should help to design interventions to increase the awareness and the diagnosis of aat in selected individuals or populations according to the current guidelines for the diagnosis and management of aatd .
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introductionalpha-1 antitrypsin deficiency ( aatd ) remains an underdiagnosed condition despite initiatives developed to increase awareness .
the objective was to describe the current situation of the diagnosis of aatd in primary care ( pc ) in catalonia , spain.methodswe performed a population - based study with data from the information system for development in research in primary care , a population database that contains information of 5.8 million inhabitants ( 80% of the population of catalonia ) .
we collected the number of alpha-1 antitrypsin ( aat ) determinations performed in the pc in two periods ( 20072008 and 20102011 ) and described the characteristics of the individuals tested.resultsa total of 12,409 aat determinations were performed ( 5,559 in 20072008 and 6,850 in 20102011 ) , with 10.7% of them in children . as a possible indication for aat determination ,
28.9% adults and 29.4% children had a previous diagnosis of a disease related to aatd ; transaminase levels were above normal in 17.7% of children and 47.1% of adults . in total , 663
( 5.3% ) individuals had intermediate aatd ( 50100 mg / dl ) , 24 ( 0.2% ) individuals had a severe deficiency ( < 50 mg / dl ) , with a prevalence of 0.19 cases of severe deficiency per 100 determinations .
nine ( 41% ) of the adults with severe deficiency had a previous diagnosis of copd / emphysema , and four ( 16.7% ) were diagnosed with copd within 6 months.conclusionthe number of aat determinations in the pc is low in relation to the prevalence of copd but increased slightly along the study period .
the indication to perform the test is not always clear , and patients detected with deficiency are not always referred to a specialist .
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the microrna- ( mirna- ) guided rna silencing pathway is a recently discovered process that is able to regulate gene expression by acting on messenger rna ( mrna ) at posttranscriptional level .
mirna biogenesis is mediated by dicer which catalyzes the processing of double - stranded rnas ( dsrnas ) into 22 nt - long small mirnas . the initial transcript , or primary mirna ( pri - mirna ) , can be hundreds to thousands nucleotides long and , like any other pol ii transcript , undergoes capping and polyadenylation .
the mature mirna is part of a 60 to 80-nucleotide stem - loop structure contained within the pri - mirna .
the first step in mirna biogenesis occurs in the nucleus and requires the excision of this hairpin structure .
the excised hairpin , called pre - mirna , is exported to the cytoplasm , and the pre - mirna is then processed by another rnase iii enzyme called dicer .
this endonuclease removes the loop region of the hairpin , releasing the mature mirna : mirna * duplex . during the assembly of
the rna - induced silencing complex ( risc ) with the mirna , only one strand of the duplex is loaded , whereas the complementary mirna * strand is removed and degraded .
the mature mirna is now ready to direct its activity on a target mrna by binding mirna responsive elements usually located in the 3untranslated region ( 3utr ) of the transcript .
this association may result in either cleavage or translational repression of the target mrna , depending on the degree of base - pairing between the mirna and the responsive element .
perfect complementarity generally results in cleavage , whereas imperfect base - pairing leads to translational repression .
these alternative effects might also reflect differences in the biochemical composition of the risc complex associated to each specific mirna : mrna duplex .
the proteins in the argonaute ( ago ) family are very tightly bound to small single - stranded rnas within risc , as the rna - protein interaction persists even under high - salt conditions .
the paz domain of ago has been implicated in rna binding , and the piwi domain seems to furnish risc with effector - nuclease function .
the wide range of molecular weights reported for risc complex ( between 140 and 500 kda ) represents several different versions of the complex that contain other factors in addition to ago . because the other components of risc are not required for slicing
, they may have a role in other aspects of risc activity , for example , substrate turnover and/or risc subcellular localization .
this variation may also represent species differences or may reflect developmental- or tissue - specific variations in risc composition .
mirna genes represent about 1%-2% of the known eukaryotic genomes and constitute an important class of fine - tuning regulators that are involved in several physiological or disease - associated cellular processes .
mirnas are conserved throughout the evolution , and their expression may be constitutive or spatially and temporally regulated . even in viral infections these small non - coding rnas can contribute to the repertoire of host - pathogen interactions .
the resources needed to study in details such interactions or to investigate their therapeutic implications have been recently reviewed .
increasing efforts have been made to identify the specific targets of mirnas , leading to speculation that mirnas may regulate at least 30% of human genes .
computational predictions suggest that each mirna can target more than 200 transcripts and that a single mrna may be regulated by multiple mirnas .
this entails that mirnas and their targets are part of complex regulatory network and outline the widespread impact of mirnas on both the expression and evolution of protein - coding genes .
however , artificial tethering of ago proteins to the 3utr of a reporter mrna is sufficient to induce its translational repression .
this evidence suggests that mirnas may act to guide the deposition of the risc complex onto a specific site of the target mrna . to date , the computational identification of mirna targets and the validation of mirna - target interactions represent fundamental steps in disclosing the contribution of mirnas toward cell functions .
the prediction of mirna targets by computational approaches is based mainly on mirnas complementarity to their target mrnas , and several web - based or stand - alone computer softwares are used to predict mirna targets . among them , targetscans , pictar , and miranda are the most common target prediction programs while mirbase , argonaute , mirnamap , and mirgen are databases combining the compilation of mirnas with target prediction modules . here ,
we summarize and discuss the most recent in silico and biological approaches aimed to unravelling the functional interactions between mirnas and their targets with a special emphasis to combined methods for more accurate mirna target gene prediction .
it is now well established that the formation of a double - stranded rna duplex through the binding of mirna to mrna in the rna - induced silencing complex ( risc ) triggers either the degradation of the mrna transcript or the inhibition of protein translation .
however , experimental identification of mirna targets is not straightforward , and in the last few years , many computational methods and algorithms have been developed to predict mirna targets . even though target prediction criteria may vary widely ,
most often they include : ( 1 ) strong watson - crick basepairing of the 5 seed ( i.e. , positions 28 ) of the mirna to a complementary site in the 3utr of the mrna , ( 2 ) conservation of the mirna binding site , and ( 3 ) a local mirna - mrna interaction with a positive balance of minimum free energy ( mfe ) .
these requirements should be accompanied by a good structural accessibility of the surrounding mrna sequence . however
, it is likely that other important parameters for functional mirna - target interactions remain to be identified .
the first step in the prediction procedure requires the identification of potential mirna binding sites in the mrna 3utr according to specific base - pairing rules .
the second step involves the implementation of cross - species conservation requirements . among the most popular prediction algorithms , we recall pictar , targetscan , and miranda .
each algorithm has a definite rate of both false positive and false negative predictions . in common practice ,
more than one algorithm is used to make reliable predictions about a particular gene or a specific mirna .
surprisingly , different algorithms provide different predictions , and the degree of overlap between different lists of predicted targets is sometimes poor or null .
it has been predicted that up to 30% of mammalian genes are regulated by mirnas [ 1113 ] , and many regulatory patterns are likely to be regulated by them .
however , when the number of genes under study is on the order of several hundreds or thousands ( like in microarray experiments ) , a gene - by - gene search of mirna targets of interest becomes impractical .
furthermore , when dealing with such a number of genes that may be coregulated , the evaluation of groups of genes with common binding sites for one or specific mirnas or families of mirnas is surely more informative .
this goal may be reached using classical enrichment statistics , testing over - representation of the mirna target predictions within the selected set of genes ( see also next paragraph ) : the statistical methods are similar to those used for the gene ontology annotation ( http://www.geneontology.org/go.tools.html ) .
however , few prediction algorithms able to clarify mirna function or integrate data coming from different experimental high - throughput techniques are currently available .
therefore , there is the need to develop accurate computational methods for the identification of functional mirna - target interactions . undoubtedly , a computational method able to efficiently combine gene expression studies ( mrna profiles ) with mirnas expression profiles for a reliable prediction of mirna target is essential .
in fact , using the results of both mirna and gene expression profiling , the prediction of mirna - mrna associations through the identification of anticorrelated pairs should be refined ; based on the well - established knowledge of mirna function , an upregulation of a specific mirna will lead to lower expression of its mrna targets , and a downregulation of a specific mirna will lead to higher levels of its target genes .
this effect is more clearly visible from in vitro studies where the system is perturbed either by the over - expression or by the silencing of a specific mirna [ 15 , 16 ] .
therefore , a ranking of downregulated ( or upregulated ) genes coupled to several mrna predictions should allow the researcher to obtain a more reliable estimate of the real mirna targets and finally their function [ 12 , 13 ] .
unfortunately , so far this approach led to few examples , and the available software and algorithms will be briefly commented here .
in contrast , a biological approach has led to the development of several techniques that appear to be efficient alternatives to computational methods .
these applications , briefly reviewed in this paper , are able to solve , at least in part , the problem of high - throughput validation of mirna targets in vivo .
-omics data are commercially available or free for nonprofit organizations ( table 1 ) .
these systems are usually general purpose environments in which small databases of experimental samples can be built ; the data can be filtered and normalized and also analyzed in depth using a number of statistical techniques such as analysis of variance ( anova ) , hierarchical clustering , principal component analysis ( pca ) , among others .
the same systems also offer annotation instruments such as enrichment statistics for a set of reference databases , including lists of mirnas targeting all the known genes .
the predictions come usually from the most popular computational predictors ( targetscan , pictar , miranda ) and are not validated by databases of experimental mirna - mrna interactions .
given any mrna expression profile and a selected gene list , this approach allows a first investigation of the mirnas likely to directly modulate , at least partially , the mrna degradation rate or indirectly modulate the mrna transcription and translation rates .
these techniques are not specifically tailored to the problem of integrating parallel mirna and mrna gene profiles obtained within the same experiment but are useful in combining data within the same analytical environment . of these tools ,
algorithms for functional annotation , such as fatigo , have been integrated into a single and user friendly interface .
the software genespring is a commercial package that offers , together with a wide range of standard and advanced statistical analysis methods , other enrichment statistics for functional annotations .
this last feature is further developed in the ingenuity pathway analysis system , specifically designed for functional and pathway analysis .
other analysis software such as the popular bioconductor package and the mev from the tigr institute , are open source projects that undergo constant updates .
bionconductor works within the r language environment , which enables it to be directly integrated with several other r libraries such as the topkcemc reported in table 2 .
the usefulness of bioinformatic integration of mrna and mirna expression data into an interaction database ( transcriptome interaction database ) was emphasized by chen et al . .
however , the functional significance of many mirnas is still largely unknown due to the difficulty in identifying target genes and the lack of genome wide expression data combining mirna results . in table 2
there is a list of some recent algorithms or tools developed to investigate the effect of mirnas on mrna expression profiles , to better predict mirna targets and to integrate different data sources .
sigterms is a novel software package ( a set of microsoft excel macros ) that has been recently developed : for a given target prediction database , it retrieves all mirna - mrna functional pairs represented by an input set of genes . for each mirna
, the software computes an enrichment statistic for over - representation of predicted targets within the gene set .
this could help to define roles of specific mirnas and mirna - regulated genes in the system under study .
in the hands of researchers , sigterms is a powerful tool that allows rates of false positive and false negative responses to be minimized .
one method to decrease the incidence of false positive predictions and to narrow down the list of putative mirna targets is to compare the in silico target predictions to the genes that are differentially expressed in the biological system of interest .
sigterms can support this type of analytical approach allowing the user to manipulate , filter , and extract different output from mirna - mrna sets .
another recently reported application is mirgator that integrates target predictions , functional analyses , gene expression data and genome annotations .
since the function of mirna is mostly unknown , diverse experimental and computational approaches have been applied to elucidate their role [ 24 , 25 ] . in this context
, mirgator provides a utility for statistical enrichment tests of target genes , performed for gene ontology ( go ) function , genmapp and kegg pathways , and for various diseases .
expression correlation between mirna and target mrna / proteins is evaluated , and their expression patterns can be readily compared with a user friendly interface . at present , mirgator supports only human and mouse genomes .
another major task facing researchers studying complex biological systems is the integration of data from high - throughput
recently , some mirna - bioinformatic aspects like the biological and therapeutic repertoire of mirnas , the in silico prediction of mirna genes and their targets , and the bioinformatic challenges lying ahead have been reviewed .
combined modeling of multiple raw datasets can be extremely challenging due to their enormous differences , while rankings from each dataset might provide a common base for integration .
aggregation of mirna targets , predicted from different computational algorithms is one of these problems .
another challenging issue is the integration of results from multiple mrna studies based on different platforms .
however , one of the methods recently proposed in the literature makes use of a global optimization technique , the so - called cross entropy monte carlo ( cemc ) .
this algorithm , called topkcemc , searches iteratively for the optimal list that minimizes the sum of weighted distances between the candidate ( aggregate ) list and each of the input - ranked lists .
the distance between two ranked lists is measured using both the modified kendall 's tau measure and the spearman 's footrule .
the application of this technique in the field of mirna seems appropriate when the diverse predicted targets from different computational algorithms are combined together to give an aggregate list that is more informative for downstream experiments [ 12 , 13 ] .
this algorithm is a clear example of what we think may be well suited for combining mrna and mirna data to furnish a list of more reliable mirna targets . in fact
, the comparison should be made combining the classical list of mirna targets ( obtained from different prediction softwares ) and a list of ranked downregulated ( or upregulated ) mrnas .
another proposed method of inferring the effective regulatory activities of mirnas requires integrating microarray expression data with mirna target predictions .
as previously mentioned , the method is based on the idea that regulatory activity changes of mirnas could be reflected by the expression changes of their target transcripts ( measured by microarray techniques ) . to verify the hypothesis ,
this method has been applied to selected microarray data sets measuring gene expression changes in cell lines after transfection or inhibition of specific mirnas .
results indicate that this method can detect activity enhancement of the transfected mirnas as well as activity reduction of the inhibited mirnas with high sensitivity and specificity .
furthermore , this inference is robust with respect to false positive predictions ( i.e. , nonspecific interactions when silencing a mirna or when the gene downregulation is erroneously associated to a direct mirna targeting ) .
this method is a generalization of the gene set enrichment analysis ( gsea ) , which was proposed to identify gene sets associated with expression change profiles .
the first example of a direct correlation between mrna expression levels and the 3utr motif composition has been recently reported .
this algorithm , a novel application of reduce , has also led to the hypothesis that the number of vertebrate mirna could be larger than previously estimated .
the algorithm 's rationale is based on the assumption that motifs within 3utrs make a linear contribution to enhancing or inhibiting mrna levels .
the significant motifs are chosen by iteratively looking at the individual contribution that brings the greatest reduction in the difference between the model and the expression data .
motifs with a p - value lower than a defined threshold are retained and listed .
this method was ultimately demonstrated to be more sensitive than the current target prediction algorithms not relying on cross - species comparisons .
the same approach has been followed in another recent paper . here , the authors demonstrated that the effect of a mirna on its target mrna levels can be measured within a single gene expression profile .
this method , however , used a known public dataset of expression both for mirna and mrna , limiting the usefulness of the conclusions .
however , the success of this approach has revealed the vast potential for extracting information about mirna function from other gene expression profiles . a novel bayesian model and learning algorithm , genmir++ ( generative model for mirna regulation ) , has also been proposed .
genmir++ accounts for patterns of gene expression using mirna expression data and a set of candidate mirna targets .
a set of high - confidence functional mirna targets is obtained from the data using a bayesian learning algorithm . with this model , the expression of a targeted mrna transcript can be explained through the regulatory action of multiple mirnas .
genmir++ allows accurate identification of mirna targets from both sequence and expression data and allows the recovery of a significant number of experimentally verified targets , many of which provide insight into mirna regulation . in table 3
we summarize some research articles where the authors have combined expression data for mirna and mrna , using standard analytical techniques but without the use of specifically designed algorithms . in a recent approach aimed at identifying mirna targets ,
an experimental and analysis workflow was used to find a set of genes whose expression is modulated by mir-140 .
this method is based on the manipulation of a mirna activity in mouse cell lines , where mir-140 is expressed at a moderate level , thus making it easier both to repress or enhance its activity .
expression of mrnas repressed or enhanced upon mirna overexpression and silencing , respectively , was profiled . within the set obtained by the intersection of the up- and down regulated mrnas measured by microarrays ,
the authors searched for complementary seed sequences in the 3utr section of transcripts : 21 out of 49 mrnas were identified as candidate direct targets , while the others as potential indirect ones .
interestingly , none of the 21 identified candidates were computed by popular predictors such as targetscan , mirbase , and pictar , though one of these targets , cxcl12 , was validated by northern blot and luciferase assay .
this method suggests that the use of more cell lines would certainly increase the set of experimentally identified targets .
in fact , since some of them were already found to have escaped the analysis , they were unaffected by the type of cell manipulation chosen in this approach .
this method appears to be conservative and tends to find false negative targets especially if they are not affected at the mrna level .
a different type of combined analysis of mrna and mirna profiles is often used in the field of tumors : cancers may be classified into various subclasses or may respond differently to various chemotherapeutic procedures . to correctly distinguish two subtypes of carcinomas (
i.e. , the colorectal cancer that can be characterized by microsatellite pathway either stability or instability ) , the authors have identified two different gene signatures from the mrna and mirna expression profiles .
the two signatures were extracted by standard statistical techniques such as correct t - test , pam ( prediction analysis of microarray ) and svm ( support vector machine , provided by gene spring software , see table 1 ) .
then , their ability to classify the samples was tested through a hierarchical clustering , both separately and together .
results showed that the better performance was obtained when the two signatures were combined together in a single clustering tree , proving once more the well - assessed crucial role played by mirnas in the genesis of cancers .
both mrna and mirna gene profiles coupled to hierarchical clustering techniques were recently used in obtaining a deeper understanding of the cancer biology of the wilm 's tumor .
a serious problem that affects the results of antineoplastic treatments is , together with a correct diagnosis and classification , the choice of the right chemotherapeutic agent .
again , both mrna and mirna expression signatures of sensitive and resistant cell lines were used to predict patient response to a panel of commonly used chemotherapy agents .
the signatures were first used to cluster analyze samples from real breast cancer patients , then also as predictors to separate patients into nonresponders / responders to each treatment .
the mirna profiles were also finally analyzed to investigate the biological mechanisms underlying the resistance / response to the agents used in the study , making use of the prior knowledge about the experimentally validated targets of the selected mirnas .
finally , we would like to report a few examples that show how a biochemical approach may overcome all the difficulties encountered with the computational approach .
so far , the small number of available validated mirna targets has hindered the evaluation of the accuracy of mirna - target prediction software .
method has been proposed for the capture of all known conserved mirna - mrna target relationships in caenorhabditis elegans , with a lower false positive rate than other standard methods .
this quantitative mirna target prediction method allows an accurate weighting of some immunoprecipitation - enriched parameters , finally optimizing sensitivity to verified mirna - target interactions and specificity . as indicative examples , two recent studies on c. elegans used immunoprecipitation of mirna - containing ribonucleoprotein complexes and evaluated that only 30%45% of mirnas associated with these complexes contain perfectly matched , conserved seed elements in their 3utrs [ 36 , 37 ] .
although these datasets have provided important insights into parameters associated with functional interactions , this approach is limited to the detection of mirna - target interactions that result in transcript destabilization and does not identify stable , translationally repressed target mrnas .
recently , immunoprecipitation of the risc has been used to identify mrnas that stably associate with the endogenous risc .
this study recovered 3404 mrna transcripts that specifically coprecipitate with the mirna - induced silencing complex ( mirisc ) proteins ain-1 and ain-2 .
ain - ip set of mrna transcripts provided a biologically derived estimate of how many genes are targeted by mirnas : in this case , at least one - sixth of c. elegans genes .
the authors used these features to develop the prediction algorithm mirwip , which scores mirna target sites by weighting site characteristics in proportion to their enrichment in the experimental ain - ip set .
mirwip has improved overall performance compared to previous algorithms , in both recovery of the ain - ip transcripts and correct identification of genetically verified mirna - target relationships without a requirement for alignment of target sequences .
mirwip in its current form is supported by immunoprecipitation experiments that identify transcripts by their probable association with mirnas , even if these experiments do not directly provide information about what particular mirna ( or set of mirnas ) is responsible for mirisc association . finally ,
because the mirisc immunoprecipitation approach may be biased toward the identification of stable mirna - target complexes , mirna - induced target destabilization can be screened using complementary datasets , such as microarray assays to identify mrna transcripts that change in response to mirna activity . to overcome the above mentioned difficulties and
since the identification of the downstream targets of mirnas is essential to understand cellular regulatory networks , a direct biochemical method for mirna target discovery has been proposed that combines risc purification with microarray analysis of bound mrnas .
a biochemical method of identifying mirna targets holds the promise of deepening the understanding of the determinants of mirna - mediated regulation , particularly by revealing targets that are repressed without changes in mrna levels .
identification of this class of targets will provide an opportunity to study sequences or structural features determining mirnas regulatory fate .
as a model , mir-124a has been used because its targets are well known and studied .
this method consisted in the ago2 co - immunoprecipitation of mrna targets followed by microarray profiling of mrnas .
as a result , it has been proven that not only most of the immunoprecipitated mrnas analyzed were direct mir-124a targets but also a significant subset was downregulated .
a novel sequencing era is going to dramatically change our view of studying gene expression , posttranscriptional modifications , dna copy number variations , and snps .
novel high - throughput sequencing techniques are emerging at an impressive speed on the market and on the scientific community . in the near future
, these novel approaches will surely help to elucidate the function of mirnas and their role as fine regulators .
one of the most important recently reported work is based on this approach . whereas conventional methods rely on computational prediction and subsequent experimental validation of target rnas , the proposed method consists in the direct sequencing of more than 28 000 000 signatures from the 5 ends of polyadenylated products of mirna - mediated mrna decay .
briefly , by matching millions of 5 end sequences of rna cleavage products back to their corresponding sequences in the genome , additional sequences flanking the potential cleavage sites were identified .
these were used to identify matches to known or new potential mirnas that could direct their cleavage .
even though this study was conducted on arabidopsis thaliana , we expect that the proposed method will also be rapidly applied to other genomes for the understanding of the role and functions of mirnas . in summary
, we have addressed the issue of combining mrna and mirna expression data from different points of view . while biological validation of a predicted target is critical , failure to biologically validate the expression of a certain mirna
it is possible that the mirna is not expressed in the examined tissues , the mirna is expressed only in specific phase of cell cycle , or that the mirna is expressed in low abundance , which escapes detection by the technique used .
this latter cause is especially problematic for mirna that shares a high degree of sequence homology with another mirna .
expression of an abundant mirna may therefore mask the expression of a rare one that is very similar in sequence , especially when using polymerase chain reaction amplification . while several methods already exist to predict mirna targets , albeit with a heterogeneous and wide range of results , there are few tools and algorithms or even only analysis workflow capable of elucidating the functional role of mirnas .
the wider availability of experimentally validated mirna targets and their action mechanisms will certainly permit in the near future more reliable computational predictions .
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all microrna ( mirna ) target finder algorithms return lists of candidate target genes .
how valid is that output in a biological setting ?
transcriptome analysis has proven to be a useful approach to determine mrna targets .
time course mrna microarray experiments may reliably identify downregulated genes in response to overexpression of specific mirna .
the approach may miss some mirna targets that are principally downregulated at the protein level .
however , the high - throughput capacity of the assay makes it an effective tool to rapidly identify a large number of promising mirna targets .
finally , loss and gain of function mirna genetics have the clear potential of being critical in evaluating the biological relevance of thousands of target genes predicted by bioinformatic studies and to test the degree to which mirna - mediated regulation of any validated target functionally matters to the animal or plant .
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stress urinary incontinence ( sui ) is a social disease caused by numerous contributing factors such as natural childbirth obesity , hormonal deficiencies and changes in collagen fibers , to name a few . as a result of these causes , the renal pelvis and urethra sag .
there are various methods of treatment for sui depending on how advanced the condition is . at present , the implantation of mini slings is gaining recognition as a method of treating sui [ 3 , 4 ] .
the objective of this study was to assess the effectiveness of this method using specific materials in the treatment of sui .
in the years 2007 to 2011 , there were 160 women surgically treated for sui , and 140 ( 87.5% ) of them remained under observation .
the average age of the patients was 62 years ( 3685 ) ; the average body mass index was 28 ( 2533 ) ; the average amount of births was of 2.5 per patient ( 04 ) , and 75% of them were post menopausal .
surgical prep stage , various physical tests were done , such as the urinary stress test , and abdominal usg to determine the residual urine after urination ; in addition to a urodynamic exam and urine culture tests .
surgical usg did not reveal any residual urine after urination , and excluded other bladder conditions while the urodynamic exam excluded an overactive bladder condition . in 40 cases ,
first degree sui was diagnosed ; in 70 cases , second degree ; in 30 cases , third degree , and in 30 cases there were mixed symptoms of sui . in 20 patients
it was noted that second degree sui had returned after earlier surgical treatment : six patients had undergone the burch colposuspension ; seven , the pereyra needle suspension procedure ; seven , intravaginal slingplasty ( ivs ) .
those patients having mixed symptoms had been treated with 5 or 10 mg of solifenacin for 4 weeks before qualifying for surgical treatment . in the case of first degree sui ,
all patients were first referred to rehab and physical therapy ; however , without satisfying effect . among patients treated with mini - slings johnson & johnson 's tvt
secur tape was implanted in 65 cases ; bard 's adjust mini sling in 70 cases ; and ams 's mini arc in just 5 cases .
secur is a mesh tape secured to tissue on the rear surface of the descending branch of the pubic bone ( figure 1 ) . when adjust tape is implanted , the tape is anchored in the obturator foramen .
patients assessed their improvement on a vas scale , in addition to gynecological and urinary stress tests .
all patients reported to the 2 department of urology in d for 1 month , 3 month , 6 month and 1 year check ups after the surgical procedure .
the effectiveness of the procedure was assessed on the basis of the urinary stress test and the vas .
after the removal of the catheter , 115 patients were able to urinate properly and were fully able to retain their urine during a gynecological examination and urinary stress test . in the case of 25 patients
, there was a major improvement with significant reduction of sui symptoms rated on the basis of interviews and the vas . in 9 cases , temporary urine retention occurred and confirmed by usg examination ( 5 with tvt secur tape , 3 with adjust tape , 1 with mini arc tape ) .
for these cases , foley catheters were inserted into the bladder for a period of 5 to 7 days . in one case , as a result of chronic urine retention , the patient was discharged home with the catheter in place ( a tvt secur recipient ) .
operative vaginal bleeding was observed , but surgical intervention or post operative revision was unnecessary ( 2 with adjust tape , 2 with tvt secur tape ) . assessing the improvement of life quality was done with the use of the vas .
the visual analogue scale ( vas ) is a common form of response option in health outcome studies and is generally presented as a single line of 100 mm with anchor words at either end .
the average rating before the surgical procedure was 3 , whereas the rating increased to an average of 8.5 after the implantation of the mini slings . during the following 12 months of monitoring
, the vas ratings were not changed by the patients , nor were there any recurrences of the condition ( table 1 ) .
cumulative results of mini sling procedures for sui of different authors compared to own results
in our study 3 out of 5 ( 60% ) fully recovered when treated with the mini arc sling .
arc implantation in 91.4% of patients suffering from sui : there were no recorded intra or postoperative complications , nor complaints of strong pain or observed urinary retention .
full recovery was observed in 69.1% , while partial recovery was observed in 20.6% of patients who underwent the procedure . for 10.3% of the patients ,
the authors noted one incident of urinary infection after the procedure and one incident of urinary incontinence de novo . in operations with the usage of tvt secur there was shown that 56 out of 65 ( 86.15% ) patients gained full recovery . in the work of meschia and associates , full recovery
was noted in 78% of cases after surgical treatment using the tvt secur system .
the postoperative rating was a subjective patient assessment , which noted minor complications after surgery including disorders in urination , urinary infection and urinary incontinence de novo .
in khandwala and associates study , 141 women suffering with sui took part in the study where tvt secur tape was the chosen material for implantation .
full recovery was noted in 82% of the cases , while partial recovery in 11% , and no improvement in 7% .
results of our study show that 65 out of 70 ( 92.86% ) treated with adjust tape gained full recovery . according to m. abdel fattah and associates , in their prospective multicenter study there was determined the effectiveness and safety of the adjust method was determined . in the post
the authors also undertook further study to determine results concerning the use of local anesthesia vs. general anesthesia for the procedure .
they found that there was less blood loss and fewer problems with urination , percentage wise , when the procedure was performed under local anesthesia .
the percentage difference was statistically significant . among early complications noted were vaginal bleeding ( 4 patients ) which did not require surgical intervention or blood transfusions ; and temporary urine retention in 10 patients . during the procedures , there was no damage done to the bladder , urethra , large blood vessels or nerves .
slings is an effective , minimally invasive and relatively safe method of sui treatment . in almost all cases full
recovery was noted , especially in the group where sui had reoccurred in patients having previous procedures once rated as
slings may become an important fixture in modern urogynecology and may possibly be named the gold standard in treating sui .
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introductionstress urinary incontinence ( sui ) is a social disease caused by numerous contributing factors such as natural childbirth , obesity , hormonal deficiencies and changes in collagen fibers , to name a few .
currently , mini slings , among all the surgical treatment methods , have gained significance .
the aim of this study was to establish the effectiveness of this method.material and methodsfrom 2008 to 2012 , one hundred sixty women suffering from sui underwent surgical procedures to implant mini
slings under the middle part of the urethra and 140 ( 87.5% ) of them remained under observation . in 65 cases , johnson & johnson 's tvt secur was used ; in 70 cases , bard 's adjust mini
sling was used ; and in 5 cases , ams mini arc mini
sling was used .
the average period of hospitalization was 3 days per admission , operation and discharge day.resultsin 82 cases , patients urinated well after the removal of the catheter , and had full urine continence .
fourteen patients showed great improvement , and in four cases temporary urine retention was observed . in two cases vaginal bleeding
was observed , yet there was no need for wound revision .
evaluation of the quality of life improvement was done using the visual analog scale ( vas).conclusionsthe obtained results allowed the conclusion that the implantation of mini slings is a low invasive , relatively safe and effective procedure for the treatment of sui even in cases of recurrence .
almost full recovery was achieved in all the cases of this study . the mini
sling has become an important element in modern urogynecology .
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the estimation of 24-hour urine protein excretion or 24-hour urine albumin excretion using urine protein to creatinine ratio ( upcr ) and urine albumin to creatinine ratio ( uacr ) , respectively , is well established in clinical practice and promulgated by practice guidelines .
the original studies were , however , performed prior to the standardization of creatinine assays [ 24 ] .
the standardized creatinine values may differ by 520% from values obtained by methods not calibrated or traceable to the isotope dilution mass spectrometry ( idms ) standard . as an example , if standardized creatinine is 20% higher than previously measured , upcr or uacr may be 17% lower on estimation .
conversely if standardized creatinine is 10% lower , then upcr or uacr may be 11% higher on estimation .
therefore , it is clear that creatinine standardization has important implications for the proteinuria estimation equations and their application in clinical practice .
research studies and clinical practice may favor upcr over uacr for reason of cost , and there are no reliable methods to convert ratios .
however , the new iteration of the clinical practice guidelines for the identification and classification of chronic kidney disease incorporated urine albumin to creatinine ratio as part of risk stratification [ 8 , 9 ] . in this study , we assessed the correlation of early morning spot urine tests to 24-hour urine protein and albumin excretion , provided conversion equations developed from a population of patients with a variety of chronic kidney disease , and compared their predictive effects for gfr decline , end - stage renal disease ( esrd ) , and mortality .
, stable ckd patients were recruited from outpatient clinics for a study on glomerular filtration rates ( gfr ) .
they collected 24 hr urine collections and presented the next day for a gfr measurement and also provided early morning urine and blood samples .
the 24 hr urine collection and early morning spot urine were tested for protein , albumin , and creatinine concentrations .
urine protein was measured using a pyrogallol - based assay calibrated to the manufacturer 's internal standard .
urine albumin was measured using a peg - enhanced immunoturbidimetric assay and was also calibrated to an internal standard .
creatinine was measured by an enzymatic method ( creatininase ) in a central laboratory accredited by the college of american pathologists and the assay was calibrated with manufacturer - provided materials traceable to standardized creatinine ( national institute for standards and technology standard reference material 967 ) measured by isotope dilution mass spectrometry ( as recommended by the national kidney disease education program , http://www.nkdep.nih.gov/ ) . to extract the last known serum creatinine , the hospital clinical laboratory database was reviewed .
all participants were cross - referenced with our esrd database for date of dialysis initiation or death .
all longitudinal follow - up data for this study were correct till january 15 , 2012 .
this study was approved by the national healthcare group , domain - specific review board ( d/07/524 and 2007/00225 ) .
where appropriate , variables were naturally log - transformed before linear regression to correct for nonnormal distribution and nonconstant variability of observed points around the regression line .
we used bland - altman analysis of agreement to assess uacr and upcr in predicting 24 hr urine albumin excretion and 24 hr urine protein excretion , respectively . for comparisons with earlier studies , we used non - si units in calculating the urine protein or albumin to creatinine ratios [ 3 , 4 ] .
conversion equations between uacr , upcr , 24 hr urine protein excretion , and 24 hr urine albumin excretion were developed . to assess these equations , an external dataset of 45 participants with similarly collected 24 hr urine sample followed by an early morning spot urine sample the next day was used as a validation test dataset . the predicted variable
was compared to the measured values by pearson correlation r and wilcoxon signed rank test . because clinical practice and research analysts commonly use uacr and upcr to estimate the respective 24 hr urine excretion rates above the clinically significant thresholds of albumin > 300 mg / day and protein > 0.5 g / day , we also analyzed the measured ( main derivation dataset , n = 232 ) and equation - predicted uacr and upcr ( validation dataset , n = 45 ) for their predictive abilities in terms of sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) [ 11 , 12 ] .
we estimated gfr using the ckd - epi equation . to compare the predictive performance of each method of urine protein or albumin assessment with gfr decline
we used stepwise linear regression by p value threshold for entry or exit from the models in a mixed direction with no rules to select the variables for models predicting gfr decline .
the initial variables screened were age , gender , ethnicity , initial serum creatinine , initial serum urea , smoking history , medical history of diabetes , hypertension , coronary artery disease , peripheral artery disease , systolic blood pressure , diastolic blood pressure , body mass index , serum albumin , uacr , upcr , 24 hr urine protein excretion , and 24 hr urine albumin excretion .
the urine estimation or measurement method in the best model was substituted in turn for three additional models for comparisons . variables with p values 0.05 and
we assessed the linear regression models by reviewing the r , aic ( akaike information criterion ) , and bic ( bayesian information criterion ) [ 14 , 15 ] . since r always will increase with an increasing number of predictor variables in a model , we select our model using the aic and bic which penalizes models for having large number of predictor variables .
aic might favor more complex models and overfit , while bic may select a parsimonious model and underfit .
for our model assessments , the lowest aic and bic criteria indicate the best model .
similarly , we also assessed the predictive performance of urine protein estimations for the combined end - point of esrd and death using cox - proportional hazards modeling .
analyses were performed on jmp 10 ( cary , nc , usa ) and r ( http://www.r-project.org/ ) .
four patients had 24-hour urine protein excretion > 3.5 g. the correlation of spot urine estimation ratios ( upcr and uacr to 24 hr urine protein and albumin excretion , resp . ) is shown in figure 1 .
uacr appears to predict 24 hr urine albumin excretion better as the slope is closer to 1 .
the prediction equations are
( 1)log 24 hr urine protein excretiong = 0.617019 + 0.7150918log upcrmg / mg;hhhhhhhhhhhhhhhhhhhhhhhhr2=0.64,p<0.001,log 24 hr urine albumin excretiong = 0.800153 + 0.8257142log uacrmg / mg;hhhhhhhhhhhhhhhhhhhhhhhh(r2=0.74,p<0.001 ) .
upcr has poorer correlation to 24 hr urine protein excretion ( r = 0.80 , 95% ci 0.750.84 ) than uacr has to 24 hr urine albumin excretion ( r = 0.86 , 95% ci 0.820.89 )
. uacr is highly correlated to upcr ( r = 0.99 , 95% ci 0.99 - 0.99 ) ( figure 2 ) .
24 hr urine albumin excretion is also highly correlated to 24 hr urine protein excretion ( r = 0.99 , 95% ci 0.99 - 0.99 ) .
uacr is less correlated with 24 hr urine protein excretion ( r = 0.78 , 95% ci 0.720.83 ) than upcr is with 24 hr urine albumin excretion ( r = 0.81 , 95% ci 0.760.85 ) .
the sensitivity and specificity of uacr of between 30 and 300 mg / mg for predicting 24 hr urine albumin excretion of between 30 and 300 mg ( microalbuminuria ) are 0.69 and 0.77 , respectively .
the sensitivity and specificity of uacr of > 300 mg / mg for predicting 24 hr urine albumin excretion of > 300 mg ( macroalbuminuria ) are 0.94 and 0.84 , respectively .
the sensitivity and specificity of upcr of > 0.5 mg / mg for predicting 24 hr urine protein excretion of > 0.5 g / day are 0.90 and 0.81 , respectively .
the various conversion equations derived from the main study data ( n = 232 ) are in table 2 . using the external validation dataset ( n = 45 ) for assessing the performance of the conversion equations , predicted spot urine ratios were highly correlated but significantly differed from measured spot urine values .
( r = 0.63 , 95% ci 0.410.78 ) better than uacr with predicted 24 hr urine albumin excretion
( r = 0.55 , 95% ci 0.310.73 ) . the sensitivity and specificity of predicted - uacr , calculated from upcr , of > 300 mg / mg for predicting 24 hr urine albumin excretion of > 300 mg ( macroalbuminuria ) are 0.059 and 1.0 , respectively .
the sensitivity and specificity of predicted - uacr , calculated from 24 hr urine protein excretion , of > 300 mg / mg for predicting 24 hr urine albumin excretion of > 300 mg ( macroalbuminuria ) are 1.0 and 0.89 , respectively .
the sensitivity and specificity of predicted - upcr , calculated from uacr , of > 0.5 mg / mg for predicting 24 hr urine protein excretion of > 0.5 g / day are 0.1 and 1.0 , respectively .
there were 225 patients with available follow - up serum creatinine to determine estimated gfr decline .
by stepwise linear regression , we developed 4 final models to compare the performance of uacr , upcr , and 24 hr urine protein or albumin excretion ( table 3 ) .
in all models , the method of urine protein or albumin assessment and the initial serum urea were significant .
all the models had good predictive performance for gfr decline , with the best performance in the model that included 24 hr urine protein excretion , followed in order by 24 hr urine albumin excretion , upcr , and uacr .
there were 19 patients who reached esrd ( 9/19 , 47% women ) and 9 ( 3/9 , 33% women ) who died during the follow - up period ( 4 had esrd before dying ) .
patients who reached esrd were of similar age ( 59.8 10.3 versus 58.8 13.0 years ) but had a higher serum creatinine ( 311 113 versus 138 75 mol / l ) and 24 hr urine albumin ( 1515 1251 versus 283 504 mg ) .
we created 4 cox proportional hazard models to compare the performance of the various proteinuria assessments for predicting the combined end - point of esrd and death ( table 4 ) .
all models , which included the standard adjusters ( age , gender , and ethnicity ) , were significant for all methods of proteinuria assessment ( all p < 0.001 ) and log- serum urea ( all p < 0.001 ) .
this is the first prospective study of a multiethnic asian population with a wide variety of ckd ( diabetic and nondiabetic ) patients that simultaneously evaluates early morning spot urine prediction ratios to 24 hr urine collections , while accounting for the standardization of the creatinine assay .
previous studies were retrospective and did not have simultaneous collection of spot urine and 24 hr urine collections . clinical practice and research involving ckd patients are highly dependent on the use of urine protein or albumin to creatinine ratios as estimates of their respective 24 hr urine excretions . yet , many are unaware of the implications of the use of urine ratios [ 17 , 18 ] .
( 1 ) does uacr or upcr predict 24 hr urine albumin or protein excretion ?
( 2 ) which spot urine ratio predicts 24 hr urine excretion better ? ( 3 ) in our setting , how do the ratios relate to the 24 hr urine excretions ? especially , now that we have standardized creatinine assays but not for albumin and protein assays . and
, of course , ( 4 ) which parameter ( ratios or 24 hr urine excretions ) predicts longitudinal outcomes data better ( gfr declines , esrd , or mortality in ckd patients ) ?
our study shows that uacr is correlated to 24 hr urine albumin excretion better than upcr .
we did not find as high a correlation for upcr as the earlier studies [ 3 , 4 ] .
this may be partly due to creatinine calibration which results in a systematically larger ratio .
the clinical practice guideline for the identification and classification of ckd incorporates uacr in addition to estimated gfr for staging ckd .
our study derived the conversion equations for clinical research or practice needing spot or 24 hr urine albumin or protein excretions , specific to the assay methods . on average , predicted spot urine ratios were reasonably correlated but were significantly different from measured values .
many clinical and research databases contain both uacr and upcr in the same patients ; analysts often apply conversion equations for the purposes of analysis .
but the sensitivity of predicted uacr or predicted upcr for identifying clinically significant 24 hr urine excretion rates is poor .
therefore , we do not recommend using the conversion equations of uacr to upcr , and vice versa .
however , the performance of predicted - uacr calculated from 24 hr urine protein excretion appears to be acceptable for identifying clinically significant proteinuria of > 0.5 g / day .
nonetheless , in clinical practice , it is currently not recommended to interchangeably convert urine albumin and urine protein concentrations .
contrary to the findings by ruggenenti et al . in their cohort of only nondiabetic patients
, we did not find that upcr predicted gfr decline better than 24 hr urine protein excretion .
but , in all our models , all methods of assessing urine protein excretion rates were significant for predicting gfr decline , esrd , and mortality . in our opinion
, this supports the current practice of using spot urine tests as estimates of assessing 24 hr urine protein or albumin excretion [ 11 , 16 ] . and
all of the methods of assessments are significantly associated with predicting clinical outcomes in a multiethnic asian population with different types of ckd , making our results more generalizable and supportive of clinical practice and research .
the strengths of our study include a fairly large multiethnic asian population comprising of both diabetic and nondiabetic ckd , with systematically collected spot and 24 hr urine when compared to previous studies [ 3 , 4 ] .
we also used turbidimetry , a robust method for determining albumin concentrations , although some others advocate using nephelometry as the preferred albumin assay method .
our study is also limited by fewer ckd patients with nephrotic - range proteinuria ( 24 hr urine protein excretion > 3.5 g ) .
however , it had been shown that spot urine estimates were less accurate at higher levels of proteinuria [ 3 , 4 ] .
the urine estimation to 24 hr measurements may be less accurate since the urine collections are self - directed .
conversely , others would also argue that the derived equations are more reflective of actual practice , and , therefore , prediction equations and longitudinal analyses will be more valid and generalizable to clinical practice .
moreover , in practice , we are generally interested in categories of proteinuria excretion , namely , < 1 g / day , 1 to 3 g / day , and > 3 g / day , and that , at higher levels , one should obtain a 24 hr urine collection to ascertain the parameters for initiating treatment of ckd .
the sample size limits the accuracy of the multivariate regression models , and further definitive studies are required .
in summary , we appraised the use of urine spot ratios for assessing urine protein excretion rates and developed helpful conversion equations for both clinical research and practice .
we showed that all methods of urine protein assessment were comparable for clinical end - points , and any method can be used in clinical practice or research .
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background . the use of spot urine protein to creatinine ratios in estimating 24 hr urine protein excretion rates for diagnosing and managing chronic kidney disease ( ckd ) predated the standardization of creatinine assays .
the comparative predictive performance of spot urine ratios and 24 hr urine collections ( of albumin or protein ) for the clinical outcomes of ckd progression , end - stage renal disease ( esrd ) , and mortality in asians is unclear .
we compared 4 methods of assessing urine protein excretion in a multiethnic population of ckd patients .
methods . patients with ckd ( n = 232 ) provided 24 hr urine collections followed by spot urine samples the next morning .
we created multiple linear regression models to assess the factors associated with gfr decline ( median follow - up : 37 months , iqr 2641 ) and constructed cox proportional - hazards models for predicting the combined outcome of esrd and death .
results .
the linear regression models showed that 24 hr urine protein excretion was most predictive of gfr decline but all other methods were similar .
for the combined outcomes of esrd and death , the proportional hazards models had similar predictive performance . conclusions .
we showed that all methods of assessments were comparable for clinical end - points , and any method can be used in clinical practice or research .
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the possibility of repeated thrombolysis has increased because of the widespread use of thrombolytic treatments , an improved chance of survival after thrombolytic treatment , and an increase in life expectancy.1,2 in myocardial infarction and pulmonary embolism , repeated thrombolytic therapy using intravenous recombinant tissue plasminogen activator ( rt - pa ) has been reported to be safe and effective.3 - 5 however , repeated thrombolytic therapy in patients with acute ischemic stroke is rarely reported , and almost all previous reports were of patients who received intravenous rt - pa.2,6 - 8 only 1 case report detailed a successful endovascular treatment for recurrent basilar artery occlusions.9 recently , multimodal thrombolytic therapy , which includes intravenous and intra - arterial thrombolytic drugs and mechanical thrombectomy , has been introduced and is fre quently used by stroke teams.10 - 13 the goals of this study are to 1 ) identify how frequently repeated thrombolytic treatments are performed since the existence of multimodal thrombolytic treatments , and 2 ) characterize the safety and outcome of repeated thrombolytic therapy in patients with acute ischemic stroke .
we drew subjects for this study from the yonsei stroke registry.14 we selected patients with acute ischemic stroke who had received thrombolytic treatments within a 10-year period ( from august 2001 to july 2011 ) .
frequency of thrombolysis was determined in each patient , and initial stroke severity was assessed by national institutes of health stroke scale ( nihss ) scores .
potential cardiac sources of embolism were defined according to the trial of org 10172 in the acute stroke treatment classification.15 this study was approved by the severance hospital institutional review board of yonsei university health system ( 4 - 2012 - 0553 ) .
the detailed protocol for thrombolytic treatment has been previously reported.16 - 19 thrombolytic treatment was performed using intravenous rt - pa ( actilyse , boehringer ingelheim , germany ) , intra - arterial urokinase ( urokinase , yuhan , seoul , korea ) , or intra - arterial mechanical devices ( microwire , agility 10 , cordis , miami , fla . , usa ; penumbra , alameda , ca , usa ; solitaire , ev3 inc , irvine , ca , usa ) .
inclusion and exclusion criteria for thrombolytic treatments were based on previous trials.20,21 patients who could be treated within 3 hours after the onset of symptoms received intravenous rt - pa ( 0.9 mg / kg with 10% bolus injection , followed by continuous infusion of the remainder over 60 minutes ) .
after intravenous rt - pa infusion , further treatment with intra - arterial urokinase or mechanical thrombectomy was permitted for patients who showed an unsatisfactory clinical response ( improvement on the nihss score < 50%).19,22 patients who could be treated within 3 - 6 hours after symptom onset were considered for intra - arterial urokinase ( up to 1 million units ) or mechanical thrombectomy .
abciximab was also allowed in patients with reocclusion.16 recanalization was evaluated at 244 hours after thrombolysis using magnetic resonance angiography or computed tomography angiography .
the thrombolysis in cerebral infarction ( tici ) grading system ( grade 0 : no perfusion ; grade 1 : penetration with minimal perfusion ; grade 2a : partial filling 2/3 of the entire vascular territory ; grade 2b : complete filling , but the filling is slower than normal ; grade 3 : complete perfusion ) was used to assess the success or failure of recanalization.23 successful recanalization was defined as tici 2a .
hemorrhagic transformations were assessed by computed tomography or magnetic resonance imaging at 244 hours after thrombolysis , or whenever clinical deterioration was suspected .
symptomatic hemorrhagic transformation was defined as any increase in the nihss score that could be attributed to intracerebral hemorrhage ( ich ) on brain imaging studies.20 independent neurologists determined functional outcomes at 3 months with the modified rankin scale ( mrs ) .
data were analyzed using spss 18.0 ( spss , chicago , il , usa ) for windows .
the pearson chi - square test or fisher 's exact test was used to compare frequencies . for continuous variables ,
data distributions were examined for normality using the kolmogorov - smirnov test . provided that the data did not deviate from normal distribution ,
the mean and standard deviation were calculated and parametric tests ( independent sample t - tests ) were applied . for data that were not normally distributed ,
we report descriptive statistics as the median and interquartile range ; these were compared using non - parametric mann - whitney u tests .
we drew subjects for this study from the yonsei stroke registry.14 we selected patients with acute ischemic stroke who had received thrombolytic treatments within a 10-year period ( from august 2001 to july 2011 ) .
frequency of thrombolysis was determined in each patient , and initial stroke severity was assessed by national institutes of health stroke scale ( nihss ) scores .
potential cardiac sources of embolism were defined according to the trial of org 10172 in the acute stroke treatment classification.15 this study was approved by the severance hospital institutional review board of yonsei university health system ( 4 - 2012 - 0553 ) .
the detailed protocol for thrombolytic treatment has been previously reported.16 - 19 thrombolytic treatment was performed using intravenous rt - pa ( actilyse , boehringer ingelheim , germany ) , intra - arterial urokinase ( urokinase , yuhan , seoul , korea ) , or intra - arterial mechanical devices ( microwire , agility 10 , cordis , miami , fla .
, usa ; penumbra , alameda , ca , usa ; solitaire , ev3 inc , irvine , ca , usa ) .
inclusion and exclusion criteria for thrombolytic treatments were based on previous trials.20,21 patients who could be treated within 3 hours after the onset of symptoms received intravenous rt - pa ( 0.9 mg / kg with 10% bolus injection , followed by continuous infusion of the remainder over 60 minutes ) .
after intravenous rt - pa infusion , further treatment with intra - arterial urokinase or mechanical thrombectomy was permitted for patients who showed an unsatisfactory clinical response ( improvement on the nihss score < 50%).19,22 patients who could be treated within 3 - 6 hours after symptom onset were considered for intra - arterial urokinase ( up to 1 million units ) or mechanical thrombectomy .
recanalization was evaluated at 244 hours after thrombolysis using magnetic resonance angiography or computed tomography angiography .
the thrombolysis in cerebral infarction ( tici ) grading system ( grade 0 : no perfusion ; grade 1 : penetration with minimal perfusion ; grade 2a : partial filling 2/3 of the entire vascular territory ; grade 2b : complete filling , but the filling is slower than normal ; grade 3 : complete perfusion ) was used to assess the success or failure of recanalization.23 successful recanalization was defined as tici 2a .
hemorrhagic transformations were assessed by computed tomography or magnetic resonance imaging at 244 hours after thrombolysis , or whenever clinical deterioration was suspected .
symptomatic hemorrhagic transformation was defined as any increase in the nihss score that could be attributed to intracerebral hemorrhage ( ich ) on brain imaging studies.20 independent neurologists determined functional outcomes at 3 months with the modified rankin scale ( mrs ) . a good outcome was defined as an mrs score 2 .
data were analyzed using spss 18.0 ( spss , chicago , il , usa ) for windows .
the pearson chi - square test or fisher 's exact test was used to compare frequencies . for continuous variables ,
data distributions were examined for normality using the kolmogorov - smirnov test . provided that the data did not deviate from normal distribution ,
the mean and standard deviation were calculated and parametric tests ( independent sample t - tests ) were applied . for data that were not normally distributed ,
we report descriptive statistics as the median and interquartile range ; these were compared using non - parametric mann - whitney u tests .
during the 10-year study period , a total of 437 patients with acute ischemic stroke underwent thrombolytic therapy . of these ,
a total of 7 patients ( 1.6% ) received thrombolytic therapy twice in chronologically separate events .
the median age for patients who received repeated thrombolytic therapy was 71 years old , and 4 patients were female .
the median time interval between the first and second thrombolytic treatment was 16 months ( ranging from 6 days to 76 months ) .
the median time from the onset of symptoms to thrombolytic therapy was 120 hours for the first thrombolytic treatment , and 125 hours for the repeated treatment .
initial median nihss scores were 12 for the first treatment and 15 for the repeated treatment .
the occluded cerebral arteries were the middle cerebral arteries in 6 patients and the internal carotid artery in 1 patient . in the second attack ,
all of the patients experienced the occlusion in the same cerebral arteries as the first ischemic strokes .
however , the right and left sides of the particular arteries were reversed for 4 patients in the second attack . in the first thrombolytic treatment , the treatment modalities were intravenous rt - pa in 4 patients , intra - arterial urokinase in 3 patients , and mechanical treatments in 3 patients .
5 patients were treated with intravenous rt - pa , 3 with intra - arterial urokinase , and 5 with mechanical treatments .
all patients who received repeated thrombolytic treatments had 1 or more potential sources of cardiac embolism .
after the first thrombolysis , recanalization was achieved in all patients ( tici 3 in 2 patients and 2b in 4 patients ) with no symptomatic ichs .
five ( 71.4% ) out of 7 patients showed good outcome after 3 months ( mrs 0 in 4 patients , mrs 1 in 1 , and mrs 4 in 2 ) .
similar to the first thrombolysis , after the second thrombolysis , recanalization was achieved in all patients and no symptomatic ich occurred .
all patients recovered to the baseline mrs score for the first thrombolytic treatment . to examine safety and outcome of patients with repeated thrombolysis ,
age , sex , initial stroke severity , door to admission time , initial systolic blood pressure and glucose level , and risk factors including hypertension , diabetes mellitus , hypercholesterolemia , and smoking , were not different between patients with single and repeated treatments .
the frequency of potential sources of cardiac embolism , however , was different ( 100% in the repeated thrombolytic treatment vs. 59.6% in the single treatment , p=0.045 ) . among the thrombolytic modalities ,
mechanical thrombolysis was more commonly performed in patients with repeated treatments ( 71.4% vs. 38.4% , p=0.027 ) .
outcome variables , including recanalization , symptomatic ich , and good outcome were similar ( table 2 ) .
during the 10-year study period , a total of 437 patients with acute ischemic stroke underwent thrombolytic therapy . of these ,
a total of 7 patients ( 1.6% ) received thrombolytic therapy twice in chronologically separate events .
the median age for patients who received repeated thrombolytic therapy was 71 years old , and 4 patients were female .
the median time interval between the first and second thrombolytic treatment was 16 months ( ranging from 6 days to 76 months ) .
the median time from the onset of symptoms to thrombolytic therapy was 120 hours for the first thrombolytic treatment , and 125 hours for the repeated treatment .
initial median nihss scores were 12 for the first treatment and 15 for the repeated treatment .
the occluded cerebral arteries were the middle cerebral arteries in 6 patients and the internal carotid artery in 1 patient . in the second attack ,
all of the patients experienced the occlusion in the same cerebral arteries as the first ischemic strokes .
however , the right and left sides of the particular arteries were reversed for 4 patients in the second attack . in the first thrombolytic treatment , the treatment modalities were intravenous rt - pa in 4 patients , intra - arterial urokinase in 3 patients , and mechanical treatments in 3 patients .
5 patients were treated with intravenous rt - pa , 3 with intra - arterial urokinase , and 5 with mechanical treatments .
all patients who received repeated thrombolytic treatments had 1 or more potential sources of cardiac embolism .
after the first thrombolysis , recanalization was achieved in all patients ( tici 3 in 2 patients and 2b in 4 patients ) with no symptomatic ichs .
five ( 71.4% ) out of 7 patients showed good outcome after 3 months ( mrs 0 in 4 patients , mrs 1 in 1 , and mrs 4 in 2 ) .
similar to the first thrombolysis , after the second thrombolysis , recanalization was achieved in all patients and no symptomatic ich occurred .
to examine safety and outcome of patients with repeated thrombolysis , the data of 430 patients who were given a single thrombolytic treatment were compared .
age , sex , initial stroke severity , door to admission time , initial systolic blood pressure and glucose level , and risk factors including hypertension , diabetes mellitus , hypercholesterolemia , and smoking , were not different between patients with single and repeated treatments .
the frequency of potential sources of cardiac embolism , however , was different ( 100% in the repeated thrombolytic treatment vs. 59.6% in the single treatment , p=0.045 ) . among the thrombolytic modalities ,
mechanical thrombolysis was more commonly performed in patients with repeated treatments ( 71.4% vs. 38.4% , p=0.027 ) .
outcome variables , including recanalization , symptomatic ich , and good outcome were similar ( table 2 ) .
this study demonstrates that repeated thrombolytic treatments for recurrent ischemic strokes is safe and feasible . in 10 years
all repeatedly treated patients had 1 or more sources of cardiac embolism and achieved recanalization without symptomatic ich in the first thrombolytic treatment . on repeated thrombolysis
, these patients were recanalized again without symptomatic ich , and all recovered to the baseline neurological state of the first thrombolytic treatment .
compared to the patients who had a single treatment , the rate of mechanical thrombectomy was twice as high in patients with repeated treatments .
the outcome of patients with repeated thrombolysis was similar that of patients with only one thrombolysis .
in acute myocardial infarction or pulmonary embolism , repeated thrombolysis has been reported to be safe and feasible.3 - 5 in the case of acute ischemic stroke , however , its effectiveness is largely unknown .
the major risk of thrombolytic treatment in ischemic stroke is ich.24 because the brain - blood barrier is disrupted in previously infarcted tissue , repeated thrombolytic treatments may pose a greater possibility for ich .
few investigators have reported multiple administrations of thrombolytic drugs for recurrent ischemic strokes.2,6 - 8 a case series of repeated thrombolytic therapy from a german stroke database showed that 9 ( 1.8% ) out of 496 thrombolyzed patients were repeatedly treated with intravenous rt - pa for chronologically separate acute ischemic strokes over a 4-year period.2 in that study , the median time span between the first and second thrombolysis was 10 months ( range , 3 to 48 ) , 67% of patients had favorable outcomes , and no symptomatic ich occurred .
current guidelines recommend that a stroke in the prior 3 months is considered an exclusionary criterion for intravenous rt - pa.24 this recommendation , however , is derived from clinical trials for acute myocardial infarction , and the information on acute ischemic stroke is limited.25 repeated use of rt - pa after 1 day theoretically will not lead to toxic plasma levels because the half - life of rt - pa is 6 min , and more than 80% of rt - pa is eliminated via urine within 18 h after administration.26,27 there are only 4 case reports of repeated thrombolytic therapy within 3 months , where the time interval between the first and second thrombolysis was 24 to 90 hours.7 - 9,28 three of those patients received intravenous rt - pa , and the remaining patient underwent intra - arterial chemical and mechanical thrombolysis .
all patients showed good outcomes without symptomatic ich . in our data , 1 patient received the second thrombolysis within 6 days ( patient 1 ) .
cardioembolic occlusion occurred on the right internal carotid artery in the first stroke , and recurrent occlusion on the left internal carotid artery in the second stroke .
both thrombi were completely removed using the solitaire devices , and no chemical thrombolytic agents were used .
therefore , further study of patients with prior stroke in the previous 3 months is needed and may increase the number of eligible patients .
immunologic reactions following repeated thrombolysis are another concern . in patients with myocardial infarction , repeat ed administration of streptokinase is not recommended because of its antigenicity.29 although rt - pa and urokinase are endogenous proteins and are considered non - antigenic , several studies have reported the development of antibodies against rt - pa and allergic reactions after rt - pa administrations.30 - 32 furthermore , activation of fibrinolysis may facilitate anaphylactoid reactions.33 previous reports did not show any immunological reactions in patients who were given repeated treatments.2 although we did not check titers of antibodies , our 5 patients who received repeated treatments with rt - pa or urokinase did not show any symptoms or signs of an immunological reaction .
it is noteworthy that all patients who received repeated thrombolytic treatments had 1 or more sources of cardiac embolism , most of which were atrial fibrillation .
recurrent ischemic stroke after intravenous rt - pa in patients with sources of cardiac embolism is common.28,34,35 higher recurrence of ischemic stroke in patients with cardiac embolic sources may be connected to the increased likelihood of receiving repeated thrombolysis for those patients .
currently , clinical trials of new anticoagulants yield favorable results and have been adopted for patients with non - valvular atrial fibrillation.36 recent guidelines for thrombolytic treatments also allow intravenous rt - pa use in patients who are currently taking a direct thrombin inhibitor or factor xa inhibitors with normal coagulation laboratory tests.24 further study is needed to investigate proper treatments for recurrent stroke in patients with new anticoagulants .
first , although our data were collected from consecutive patients of a single stroke team over 10 years , the number of patients with repeated thrombolytic treatment were small . considering the similar frequency of repeated thrombolysis in a different stroke database,2 it can be assumed that repeated thrombolysis is rarely performed .
second , our data do not enable us to draw conclusions about the optimal thrombolytic modality in repeated cases . in this study ,
thrombolytic modality in each case was decided according to predefined protocol ; however , conditions including age , stroke severity , occluded cerebral arteries , underlying medical conditions , and medication may influence the selection of thrombolytic modality for repeated events .
the advancement of mechanical devices for acute ischemic stroke may allow for more safe and favorable outcomes in repeated thrombolytic treatments.10,12 third , we examined the safety and outcome of patients with repeated thrombolysis through comparison to those with single thrombolytic treatment .
caution must be taken in interpreting these results . because the number of cases was small , the number of severe strokes was not large , and the thrombolysis modalities were heterogeneous , the negative results from this comparison can not be regarded as evidence for the safety and feasibility of repeated thrombolytic treatments . further prospective study is warranted .
our study investigated the safety and outcome of patients with repeated thrombolysis in the era of multimodal thrombolytic treatments .
although it is rarely performed , repeated thrombolysis appears to be safe and feasible for patients with recurrent acute ischemic stroke .
repeated thrombolytic therapy could be considered , even if patients have had previous history of thrombolytic treatments .
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background and purposewidespread use of thrombolytic treatments , along with improved chances of survival after an initial ischemic stroke , increases the possibility of repeated thrombolysis .
there are few reports , however , regarding repeated thrombolysis in patients who have suffered acute ischemic stroke .
we explored the number and outcome of patients with repeated thrombolytic therapy in the era of multimodal thrombolytic treatments.methodswe investigated patients with acute ischemic stroke who had received thrombolytic treatments for a period of 10 years .
number of thrombolysis was determined in each patient .
recanalization was defined as thrombolysis in cerebral infarction grading 2a .
symptomatic hemorrhagic transformation was defined as any increase in the national institutes of health stroke scale score that could be attributed to intracerebral hemorrhage .
a good outcome was defined as a modified rankin scale score 2.resultsof the 437 patients who received thrombolytic treatments , only 7 underwent repeated thrombolysis ( 1.6% ) .
the median age at the time of repeated thrombolytic therapy was 71 years old ; 4 of the patients were female .
all patients had 1 or more potential sources of cardiac embolism .
recanalization was achieved in all patients , in both the first and the second thrombolysis .
no symptomatic intracranial hemorrhage occurred after repeated thrombolytic treatments .
five patients ( 71.4% ) showed good outcomes at 3 months.conclusionsrepeated thrombolysis for recurrent acute ischemic stroke appears to be safe and feasible . among patients who experience recurrent acute ischemic stroke ,
thrombolytic therapy could be considered even if the patient has had previous thrombolytic treatments .
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medical charts of all persons who were hospitalized and reported with confirmed cases of e. coli o157:h7 to the new york state department of health 's communicable disease surveillance system ( cdss ) in 1998 and 1999 were reviewed according to a standardized survey form .
a hus case was defined as occurring in a patient with acute diarrhea who was hospitalized with e. coli o157:h7 infection and in whom confirmed or probable postdiarrheal hus developed .
a confirmed hus case was defined as occurring in a patient with a clear history of acute diarrhea who showed the following signs : hemolytic anemia with microangiopathic changes , renal insufficiency ( creatinine level > 1.0 mg / dl in a child < 13 years of age or > 1.5 mg / dl in an adult , or > 50% increase over baseline ) , and thrombocytopenia ( platelet count < 150,000/l ) .
a probable hus case was defined as occurring in a patient with acute diarrhea with all the above signs except microangiopathic changes in the blood smear .
statistical analysis was performed by using sas software ( sas institute , cary , nc , usa ) .
a multiple logistic regression analysis was performed to identify factors associated with development of hus . in 1998 and 1999 ,
of these , 255 patients ( 21% ) were hospitalized and 238 ( 93% ) had medical charts available for review .
thirty - six ( 15% ) patients were confirmed ( n = 29 ) or probable ( n = 7 ) hus case - patients , and 202 e. coli o157:h7infected patients without hus were identified as controls .
the risk of hus was highest among children < 5 years of age , compared with patients > 65 years ( odds ratio [ or ] 4.9 , 95% confidence interval [ ci ] 2.211.8 ) .
sixty - nine percent of hus patients were female compared with 61% of controls ( or 1.5 , 95% ci 0.83.4 ) .
the hospital stay was significantly longer for hus patients than controls ( median hospital stay 13 vs. 3 days ) .
five hus patients ( 14% ) died , including 2 children < 5 years of age , compared with 2 controls ( 1% ) .
eleven ( 31% ) case - patients and 78 ( 38% ) controls were treated with antimicrobial drugs ( not significant ) .
antimicrobial treatment was reported in 11 patients before the diagnosis of hus : 6 received antimicrobial drugs primarily for other conditions ( e.g. , urinary tract infection , otitis media , venous line sepsis ) , 1 had treatment stopped once e. coli o157:h7 was diagnosed , and we could not tell whether drug regimens were completed or discontinued in 4 patients .
hus patients were more likely than non - hus controls to have fever ( or 3.2 , 95% ci 1.66.5 ) .
the duration of diarrhea before hospitalization was significantly longer for hus patients than for non - hus controls ( median 4 vs. 2 days ) .
twenty - three ( 64% ) patients had proteinuria at admission , whereas 37 ( 18% ) controls were admitted with proteinuria ( or 7.8 , 95% ci 3.617 ) .
hematuria at admission was reported in 23 ( 64% ) patients and 57 ( 28% ) controls ( or 4.5 , 95% ci 2.19.4 ) .
twenty - nine ( 81% ) hus patients vs. 90 ( 44% ) controls had leukocyte counts > 13,000/l ( or 5.2 , 95% ci 2.212.3 ) at admission ( table 1 ) .
factors associated with hus in univariate analysis ( age < 5 years , outbreak case , fever , hematuria , proteinuria , leukocytosis at admission , and duration of diarrhea before hospitalization > 3 days ) were included in the multivariate analysis .
the following variables were associated with hus development in the multivariate analysis : proteinuria ( or 6.7 , 95% ci 1.924.1 ) , duration of diarrhea before hospitalization > 3 days ( or 6.2 , 95% ci 2.217.4 ) , age < 5 years ( or 5.9 , 95% ci 1.917.6 ) , and leukocyte count > 13,000/l ( or 4.4 , 95% ci 1.612.6 ) .
factors such as outbreak involvement , hematuria and fever were not associated with hus development ( table 2 ) .
* hus , hemolytic uremic syndrome ; or , odds ratio ; ci , confidence interval . *
hus , hemolytic uremic syndrome ; or , odds ratio ; ci , confidence interval .
this study provides additional information on potential risk factors for progression of e. coli o157:h7 infection to hus , but unlike other studies , this study used hospitalized rather than outpatient controls .
our data confirmed previous differences in risk for hus development by age group ( 35 ) .
women and girls have been reported to be at increased risk for hus development in several studies ( 10,11 ) , but our study showed no significant increased risk .
several studies have suggested that administration of antimicrobial agents increases risk for hus development ( 5,6,9,13 ) , but no significant relationship was observed between hus and the use of antimicrobial drugs in our sample .
although reports ( 5,7 ) have demonstrated a higher incidence of hus among patients with bloody diarrhea , fever , or vomiting , our multivariate analysis did not show a significant association between these characteristics and hus .
since only hospitalized patients with severe diarrhea were studied , some symptoms ( bloody stool , fever , or vomiting ) might have been reported more often than in the general population with e. coli o157:h7 infection . as a result , some significant associations might have been missed .
buteau et al . ( 14 ) reported that a diarrheal prodrome <3 days is an independent predictor of hus development in children with e. coli o157:h7 infection ; however , our study suggested that prolonged diarrhea ( > 3 days ) may increase the risk of hus .
our analysis was consistent with results of other studies that found patients with elevated leukocyte counts to be at higher risk for developing hus ( 58,14 ) .
patients with leukocytes > 13,000/l at admission in our study had 5 times the risk of hus .
protein and occult blood in urine were described as risk factors for hus in a study in japan ( 15 ) . in the current study ,
however , hus had already developed in most of these patients by the time of hospitalization , and we could not determine whether these factors preceded hus development . in summary ,
patients hospitalized for e. coli o157:h7 infection , those < 5 years of age with > 3 days of diarrhea , leukocytes > 13,000/l , and proteinuria should be monitored closely for further complications .
nine ( 25% ) of the hus patients had 4 risk factors , 11 ( 31% ) patients had 3 risk factors , and 10 ( 28% ) had 2 risk factors . in comparison ,
none of the controls had these 4 risk factors , 4 ( 2% ) had 3 risk factors , and 47 ( 23% ) had 2 risk factors .
identifying potential risk factors may allow clinicians to develop treatment interventions to prevent progression to hus .
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we reviewed medical records of 238 hospitalized patients with escherichia coli o157:h7 diarrhea to identify risk factors for progression to diarrhea - associated hemolytic uremic syndrome ( hus ) .
data indicated that young age , long duration of diarrhea , elevated leukocyte count , and proteinuria were associated with hus .
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strachan in an article that claimed an inverse relationship between the occurrence of hay fever and numbers of siblings .
according to the hypothesis , atopic disorders are due to reduced exposure to microorganisms in childhood . nowadays ,
the concept is becoming more accepted with accumulating evidence not only in atopic diseases but also in autoimmune inflammatory diseases . for instance , the incidence of multiple sclerosis ( ms ) is higher in high latitude countries (= westernized developed countries ) than in equatorial areas .
not only residents of western countries but also immigrants from developing countries are at high risk of developing inflammatory bowel diseases ( ibds ) and asthma . in the case of type 1 diabetes ( t1d ) , a similar geographical distribution to the diseases above and an inverse correlation to hygiene conditions are observed .
a population - based ecologic study in canada showed that ibd , including ulcerative colitis ( uc ) and crohn 's disease ( cd ) , correlated with a high socioeconomic status , low rate of enteric infection , and high rate of ms .
for instance , an inverse correlation between autoimmune liver diseases and strongyloides stercoralis infection was demonstrated in okinawa , japan .
cross - sectional studies on the relationship between skin prick tests and helminth infections suggested a general protective effect on the atopic reaction .
the authors summarized effects of geohelminths on the risk of asthma according to previous studies ; that is , hookworm lowered but ascaris increased the risk of asthma and trichuris had no effect . collectively , it is concluded that at least some helminths seem to have anti - allergic or anti - inflammatory effects in humans .
experimental studies have also shown protective effects of helminth infections in animal models of autoimmunity ( e.g. , colitis , arthritis , and diabetes ) and allergy ( e.g. , airway hypersensitivity ) [ 3 , 810 ] . in this review ,
we discuss possible mechanisms of anti - allergic / anti - inflammatory effects of helminths in animal models including autoimmune arthritis .
based on t cell skewing patterns and their relative importance in the pathogenesis , disorders with excessive immune responses had been briefly classified as th1 diseases and th2 diseases according to the th1/th2 paradigm . however , the recent discovery of a novel pathogenic t cell subset ( th17 ) led investigators to the concept of th17 disease .
while most atopic immune disorders ( e.g. , hay fever and bronchial asthma ) can be classified as th2 diseases , the classification of autoimmune diseases is relatively difficult . experimental autoimmune encephalomyelitis ( eae ) as a model of ms
was long thought to be a th1 disease ; however , the recent studies using il-12/23 subunit ( p35 , p19 , or p40 ) deficient mice revealed the progression of the disease to be dependent on the il-23/il-17 axis (= th17 response ) rather than il-12/ifn axis (= th1 response ) .
the pathogenic role of il-17 was shown directly by the finding that eae development was significantly suppressed in il-17-deficient mice .
regarding t1d , the diabetes observed in nod mice ( a model of t1d ) has been classified as a th1 disease despite the presence of some controversial study results [ 1416 ] .
recent reports demonstrated that th17 cells could also cause diabetes , but only after their conversion to th1-type cells [ 17 , 18 ] .
regarding ibd , the pathogenic roles of both th1 and th17 responses in tnbs - induced colitis ( a model of ibd ) are still controversial [ 1923 ] .
collectively , some experimental autoimmune disorders can not yet be distinctly classified as either th1 or th17 disease .
the effects of schistosomes and other helminths on experimental autoimmunity / allergy are summarized in table 1 .
surprisingly , helminths have been shown to suppress all types ( th1 , th2 , and th17 ) of disease in the models described above [ 2445 ] .
considering classical th1/th2 paradigm , it is reasonable to speculate that helminth - induced th2-skewing with downregulation of th1 immune responses results in an amelioration of th1 diseases .
as il-4 is known to suppress th17 development , th17 response could also be suppressed as well as th1 response in helminth - infected or helminth antigen - treated animals .
in fact , stat6-dependent il-4/il-13 signaling was shown to be essential in the suppression of tnbs - induced colitis and eae by schistosome eggs , although the authors did not measure changes of il-17 .
given that the involvement of th1 and th17 in some forms of autoimmunity is still controversial , downregulation of both t helper responses may be beneficial for the amelioration of various kinds of autoimmunity .
along with other investigators , we recently found that schistosome - infected mice became resistant to experimental arthritis accompanying down - regulation of both th1 and th17 responses of splenocytes .
reported suppression of tnbs - induced colitis by schistosome antigens , accompanying down - regulation of il-17 gene expression in the colon and mesenteric lymph node ( mln ) .
an intestinal nematode ( heligmosomoides polygyrus ) infection was also reported to suppress il-17 production in mln cells and lamina propria mononuclear cells .
the authors showed that the blocking of both il-4 and il-10 restored il-17 production in vitro .
another study revealed that fasciola hepatica - induced down - regulation of autoantigen - specific th1 and th17 responses ( and protection from eae ) was dependent on tgf- , not il-10 .
although the mechanisms of th17 's down - regulation by helminths are not yet established , some of the mechanisms might be common to those of th1 's down - regulation ( e.g. , through induction of il-4 and il-10 , down - regulation of il-12p40 ) and others might be distinct . in our study on experimental arthritis in mice , we found no increase of treg - related gene expression ( foxp3 , tgf- and il-10 ) in the paws of s. mansoni - infected mice compared to the paws of uninfected control mice .
as treg cell population was known to expand in schistosome - infected or egg - treated mice [ 31 , 32 , 35 , 50 ] , the cells might participate in the regulation of the disease systemically rather than locally . to confirm the essential involvement of treg cells in the antiarthritic effects of schistosome , further studies ( e.g. , persistent treg depletion experiments ) are necessary .
in contrast to our result , in the case of diabetes in nod mice , schistosome egg antigens induce infiltration of treg cells at a local inflamed site ( pancreas ) .
likewise , filarial nematode ( litomosoides sigmodontis ) infection induced treg cells and protected mice from the diabetes .
h. polygyrus also protected mice from the diabetes ; however , the protection was not dependent on treg cells .
in addition to schistosome and nematodes , tapeworm ( taenia crassiceps ) infection also has anti - diabetic effects in multiple low - dose streptozotocin - induced diabetes ( mlds ) in mice [ 45 , in this issue ] . in the study ,
taken together , these studies suggest that there may be various mechanisms in anti - diabetic effects of helminths .
we also found that schistosome - induced down - regulation of th1 and th17 occurred in the same period after the infection , corresponding to the beginning of egg - laying ( unpublished observation ) .
this result suggests that egg deposition is the major stimulus to lower th17 responses ( as well as th1 response ) in murine experimental schistosomiasis .
some epidemiological studies support that helminth infections are protective against atopic reactions and/or symptoms [ 5153 ] . the helminth - induced suppression of th2 diseases ( atopic disorders etc .
) is difficult to explain in terms of the th1/th2 paradigm . in the paradigm ,
theoretically , helminth infections are expected to cause ige overproduction and hypereosinophilia , followed by exacerbation of allergic reactions .
indeed , persistent bronchoalveolar eosinophilia , airway hyperresponsiveness , and exacerbation of allergic airway inflammation were observed in toxocara - infected mice .
one interesting explanation of anti - allergic effects of helminths is introduced in a review by fallon and mangan , in which th2 responses are subdivided to
allergic and modified , with helminth - induced th2 responses corresponding to the latter type characterized by predominant treg and il-10 responses and a relatively weak il-5 response .
( the authors , however , describe that such modifications are localized in the lungs and different from systemic responses . ) along with this hypothesis , administration of ascaris extract was shown to inhibit not only il-5 production but also eosinophilic inflammation in a murine asthma model .
the suppressive effect of the filarial worms on hyperreactivity was dependent on treg cells and tgf-. as helminths seem to have both allergenic and immunomodulatory components in their bodies , the balance of them may determine the outcomes ( i.e. , exacerbation or amelioration ) of allergic disorders .
helminthic infections often result in expansion and/or activation of treg cells [ 31 , 32 , 35 , 36 , 50 ] .
however , the importance of il-10 in helminth - induced suppression of atopic diseases is not fully established ; that is , mesenteric lymph node cells from heligmosomoides - infected il-10-deficient mice could confer protection against allergic airway inflammation . likewise , in humans , anti - allergic effect of ascaris lumbricoides is associated with treg , but not with il-10 . in the case of mice ,
il-35 , a recently identified effecter molecule of treg cells , might be involved in the suppression .
other than the regulation of lymphocytes , helminth - derived products have potential anti - allergic effects on various kinds of cells . for instance , the filarial product es-62 was shown to suppress the release of mediators from bone marrow - derived mast cells .
such macrophages not only suppress the pathogenesis by the parasites themselves but also may suppress pathological immune responses to autoantigens or allergens .
the involvement of aam in anti - diabetic effects was suggested in experimental tapeworm infection . on the other hand , schistosome - modulated macrophages ( but not aam )
immune cells and mediators possibly involved in the helminth - induced immunomodulation are illustrated in figure 1 .
collagen - induced arthritis ( cia ) is an autoimmune arthritis in mice and rats widely used as a model of rheumatoid arthritis ( ra ) .
although the role of the il-12/ifn axis in cia has been controversial [ 66 , 67 ] , recent findings suggest that ifn has ameliorating rather than exacerbating effects [ 6871 ] . instead
, the il-23/il-17 axis was recently shown to be important in the pathogenesis of cia [ 7176 ] as well as ra in humans . regarding anti - arthritic effects of helminths , a filarial es product es-62 and porcine roundworm ascaris suum extract were shown to suppress cia in mice . as described , we examined effects of s. mansoni infection on cia in mice . in humans ,
schistosomiasis has been reported as arthritogenic rather than anti - arthritic [ 79 , 80 ] . in our experiments , however , s. mansoni infection lowered arthritis scores and numbers of arthritic paws .
histopathological examination revealed that cell infiltration and bone / cartilage destruction were diminished in the infected mice . in cia and ra ,
we observed that the marked augmentation of il-1 and il-6 gene expression in the paws was almost completely abrogated by s. mansoni infection .
it was especially noteworthy that receptor activator of nfb ligand ( rankl ) gene expression in the inflamed paws was also abrogated by s. mansoni infection .
as rankl expression is induced by proinflammatory cytokines including il-17 , tnf- , and il-1 and essential to osteoclast development followed by bone destruction [ 8688 ] , this result suggests that schistosome infection has anti - arthritic effects preventing bone destruction .
interestingly , we also found that intraperitoneally administered schistosome worm antigens ( swap ) or egg antigens ( sea ) did not affect the progress of cia ( unpublished observation ) . in the antigen - administered mice , levels of il-10 , tnf , and il-17 produced by splenocytes were comparable to those in antigen nonadministered control mice .
thus , regarding schistosome , there is a considerable difference in immunomodulating effects between infection and antigen administration .
as described above , our experiments with schistosome showed that anti - arthritic effects were observed only in the viable worm infected mice . likewise , hunter et al .
showed that anticolitic effects of hymenolepis diminuta were dependent on a viable infection , and surprisingly , melon et al .
showed that therapeutic efficacy of the viable tapeworm was superior to dexamethazone treatment [ 44 , in this issue ] . because of these observations in experimental models , attenuated or non / lowly - pathogenic helminths are worth testing directly for therapeutic effects
in fact , porcine whipworm ( trichuris suis ) eggs and necator americanus infective larvae have been clinically tested for the treatment of chronic inflammatory or allergic diseases .
reddy and fried summarized the recent progress in clinical trials using these two intestinal nematodes .
summers et al . reported that the administration of t. suis eggs effectively ameliorated both uc and cd [ 90 , 91 ] without adverse effects .
this worm is considered superior to porcine whipworm in that repeated administration is not needed .
although it is not permissible to directly apply highly pathogenic helminths ( e.g. , schistosome ) to clinical use , purified or synthetic immunomodulatory products from such worms can be considered for clinical purposes .
various immunomodulatory molecules ( carbohydrates , proteins , and lipids ) have been identified ; for example , lacto - n - fucopentaose iii ( lnfp iii ) contained in schsitosome eggs is an oligosaccalide as the molecule affecting b cells ( especially b-1 cells ) to induce il-10 production .
a chemokine - binding protein ( cbp ) from schsitosome eggs inhibited the recruitment of neutrophils to inflammatory foci .
peroxiredoxin ( prx ) is an antioxidant protein found in various species including helminths [ 96 , 97 ] .
the molecules from s. mansoni and f. hepatica alternatively activate macrophages and are involved in the induction of a th2-type immune response .
the il-4-inducing principle of s. mansoni eggs ( ipse ) is the molecule that induces primary il-4 production from basophils . regarding anti - arthritic effects , a glycoprotein from spirometra erinaceieuropaei
was shown to suppress rankl - induced osteoclastogenesis , suggesting that there may be more anti - arthritic substances in various helminths .
it should be pointed out that helminths do not always suppress autoimmune / allergic disorders .
apart from th2-biasing abilities of helminths , allergens contained in the worms may partially explain the mechanisms of exacerbation .
mice infected with toxocara canis showed exacerbation of allergic airway inflammation whereas hookworm ( nippostrongylus brasiliensis ) infection persistently reduced airway responsiveness in mice , suggesting that there are considerable differences of outcomes even among lung - migratory nematodes . thus
the roles of th1/th17/treg - related regulatory cytokines ( il-25 , il-27 , il-35 , etc . ) in the helminth - induced suppression of allergy / autoimmunity have not been sufficiently studied .
it is well known that the roles of cytokines in human th17 differentiation are very different from those in mice [ 12 , 102 ] . for clinical applications in the future
, we should ascertain changes in th17-related cytokine patterns not only in animal models but also in patients with helminthiasis .
it was reported that concurrent filarial infection suppressed both th1 and th17 responses to mycobacterium tuberculosis .
this implies that helminths have down - regulating activity of both th1 and th17 in humans as well as in mice . in human ra ,
the relative importance of th1 and th17 is still unclear , and therefore , suppression of both t helper responses is recommended .
thus , at present we can conclude that regulatory effects on both th1 and th17 are promising characteristics of helminths as anti - inflammatory agents .
however , the interpretation of experimental results of helminth - induced immunomodulation may change depending on changes in basic immunological knowledge .
indeed , it was recently reported that t - bet expression was more important in the pathogenesis of eae than the th1/th17 balance .
therefore , further studies of helminth - induced amelioration of immune disorders should strictly follow the studies of the diseases ' pathogenesis . as noted , in experimental animal studies ,
viable helminth infections seem to be superior to the administration of worm antigens or killed worms in the therapeutic effects
. this might be due to that viable helminths can regulate secretion of immunomodulatory molecules in the most appropriate conditions for their survival .
taken together with successful treatment of uc and cd with viable porcine whipworms [ 90 , 91 ] , the optimal attenuation of human parasites by gene manipulation may be useful for the clinical application of parasitic helminths in the future .
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the prevalence of allergic and autoimmune diseases is increasing in developed countries , possibly due to reduced exposure to microorganisms in childhood ( hygiene hypothesis ) . epidemiological and experimental evidence in support of this hypothesis
is accumulating . in this
context , parasitic helminths are now important candidates for antiallergic / anti - inflammatory agents . here
we summarize antiallergic / anti - inflammatory effects of helminths together along with our own study of the effects of schistosoma mansoni on th17-dependent experimental arthritis .
we also discuss possible mechanisms of helminth - induced suppression according to the recent advances of immunology .
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low - dimension structures such as nanowires / dots / rods have attracted considerable interest to the scientific community in the past decade both for their size - dependent physical and chemical properties and their potential in the research and development of new optoelectronic and microelectronic devices .
micro / nanodevices need conductors to provide power as well as electrical / magnetic signals to make them functional .
gold and silver are among the most useful metals for many of the micro / nanodevice applications .
in contrast , indium is widely used metal in devices such as contacts , thermal interface applications , solder and cold - welding applications , because of its excellent malleability , high thermal and electrical conductivities , compressibility , and ease of application .
indium nanowires have peculiar temperature - dependent electrical properties ; which make them attractive for various applications . in particular , under the superconducting temperature , the electrical resistance of indium nanowires rapidly decreases , which is expected to play an important role in making magnetic field generators or superconducting quantum - interference devices .
synthesis and the temperature - induced metal insulator transitions of indium nanowire arrays on silicon substrate have been reported earlier [ 5 - 9 ] .
in addition , indium nanoparticles have also attracted intensive interest because they can be used as lubricants , single - electron transistors and tags for the detection of dna hybridization [ 10 - 13 ] .
although indium nanowires have been successfully synthesized by various techniques such as porous alumina template by hydraulic pressure injection technique for luminescence applications , potentiostatic stationary electrodeposition from an incl3-based solution , single crystal indium nanowires using ultrafast rate by focused ion beam ( fib ) , their mechanical properties have not been discussed much .
mechanical and structural aspects are of great importance in developing nanoscale building blocks into functional nano / microdevices in determining the long - term stability and reliability .
most mechanical properties are known to exhibit a dependence on specimen size [ 17 - 19 ] .
currently , many potential applications for these micro / nanoscale structures are not really practical because their mechanical properties have not been established [ 17 - 21 ] . a promising approach for measuring the mechanical properties of such nanostructures is direct nanoindentation [ 22 - 27 ] .
current author has employed trench template assisted vapour condensation technique to fabricate long nanowires inside the trench [ 28 - 30 ] .
such a technique is extremely useful in the sense ; it yields nanowire growth at a desired position and eliminates the need of any post - fabrication manipulation .
we report , for the first time to our knowledge , the hardness and elastic modulus of indium nanowires ( different diameters ) fabricated by trench template method using nanoindentation technique .
indium nano / microwires were fabricated inside the v - trench ( 220 m diameter , 100200 nm depth ) templates scribed by electronically controlled diamond scriber with the provision of load selection on thoroughly cleaned quartz substrate by vapour condensation ( thermal evaporation ) at high vacuum ( 1 10 mbar ) .
the process of formation of nanowire inside the trench template has been described in detail elsewhere [ 28 - 30 ] .
the morphology of the features grown inside the trenches was determined using atomic force microscope ( spa 400 of sii inc .
, japan ) operating in the non - contact dynamic force microscopy ( dfm ) mode .
the hardness and elastic modulus of indium films and nanowires were measured using a nanoindenter ( triboindenter of hysitron , usa , minneapolis ) with in situ afm imaging capability .
the machine monitors continuously and records the load and displacement the diamond indenter during indentation with a force resolution of about 1 nn and a displacement resolution of about 0.2 nm . the indenter tip was used to image and locate indium nanowires and then in situ indent on the wire with the same tip .
post - test imaging provides the ability to verify that the test was performed in the desired location , which improves the reliability of the data and aids in the explanation of unexpected test results . to avoid thermal drift effects , sufficient time ( 34 h )
has been provided to system after switching on the machine to achieve thermal equilibrium conditions .
the machine allows shallow penetration depths ( ~5 nm ) because it is inherently a load control instrument with its extremely low noise floor .
values of hardness and elastic modulus were obtained using the oliver and pharr method of fitting to the initial part of the unloading curve .
a 3-sided pyramidal diamond berkovich tip with a tip radius of 50 nm was used for indentation on the films .
first one , a triangular loading function ( 10 s load-10 s hold- 10 s unload)used to observe whether any pop - in or pop - out effects occur . at the peak loads , the hold time was kept at 10 s to determine creep effects in the films .
second one , a partial unloading function ( 50% unloading at the peak load ) was used to obtain depth - dependent hardness of the film / wire between the loading ranges from 0 to 500 n .
trench - templated deposition was carried out without any external heating and at very slow rate of deposition ( 14 a/s ) .
summarizing , the parameters which decide [ 35 - 37 ] the diameter of the nanowire so formed are trench width , trench depth , evaporation source substrate distance , tungsten filament length apart form the rate of deposition , substrate temperature , material of deposition , substrate material and its surface roughness
. lower rate of deposition is desired where material transfer to the substrate will not cause any agglomeration .
lower substrate temperature is desired for avoiding surface diffusion of adatoms while first few layers are formed .
total surface energy available when condensate atoms land on the substrate surface should be favourably minimum for the formation of nanowires . for all other parameters fixed ,
trench width / trench depth ratio ( w / h ) is the determining factor for the extent of shadowing .
there exists a window of w / h which favours organized assembled growth in the form of organized nanodots or nanowires . shadowed area has restricted vapour supply which gives rise to dewetting . dewetting along with the directionality imposed by the geometry of trench results in the formation of nanowire or organized nanodots inside the trench template .
it has been reported earlier that indium nanowire sample shows nanocrystalline peak corresponding to and crystallographic planes and do belong to cubic lattice system .
the absence of any known peaks corresponding to indium oxide in x - ray diffractogram reveals that the film / wire was not oxidized immediately . for the 200 s indium coating ( with film thickness of 100 nm as calculated from time and rate of deposition ) , nanoparticulated growth features with average grain size in the range of 130250 nm were observed onto 30-nm continuous thin film layer as shown in fig .
, nanoparticles organize by self - assembly to an organized manner in the form of nanowires , shown in fig .
nanowires form on the sidewalls of the trench template as shown in the inset of fig .
straightness is a concern for the indium metal when compared to that for the nickel and gold .
this kind of behaviour for indium especially may be due to its adhesion coefficient and its low melting point .
the difference in the vapour temperature ( as it depends on the heat transferred by the tungsten filament to the indium material while heating and vaporizing ) and the solidification temperature will decide the resulting surface feature apart from the other factors ( substrate material , surface roughness , etc . ) . from fig .
1b , it is clear that there are three different diameters of ( i.e. 500 , 1200 and 2000 nm ) wires grown in the trench .
the diameters of the wires are larger in the present study because trenches were made on quartz substrates by usual diamond scriber which had higher diamond tip diameter .
experiments are underway to controllably fabricate nanowires of diminished diameter to exploit its size - dependent behaviour .
atomic force microscope ( operated in intermittent non - contact mode ) image of a continuous indium film on quartz substrate b indium nanowires grown inside the trench template ( shown in red coloured arrows ) [ up right inset in b shows the schematic diagram of the technique and down right inset shows the growth of nanowires in a trench ] , c residual indent impression of indium nanowires ( contrast of the image was improved using microsoft office picture manager software in order to show a clear indent impression on wire kind of structure ) , d the line profile of the indent , e coloured 3-d image of indented impression figure 1c shows the afm image of the indent on the flat wire of diameter 2000 nm and the total height of 100180 nm .
figure 1e is a coloured 3d image for the residual indent impression on the same nanowire shown in fig .
1c . the peak nanoindentation depth can be as low as 50 nm ( for 500 nm dia wire ) , which is about 10% of the wire diameter . in our case of experimentation , height of nanowire / microwire
, the depth of indentation should not cross more than 10% of the film / wire thickness to avoid substrate effects in the hardness / modulus data .
the same berkovich tip was used to perform indentation test as well as for imaging after indentation .
indenting on a wire kind of structures is a bit difficult because under high load , wire can move / roll on the substrate surface hence indenter slips .
slipping of the indentation tip onto the wire top surface has been tried to avoid by placing indenter tip just midway on the top of the nano / microwire . in the present case ,
the adhesion forces between the wires and quartz substrate are strong enough to avoid wire from moving / rolling on the substrate .
interestingly , no cracking or chipping was observed and only some pile - up observed on either side of the residual indent in the nanowires case .
but under the same load , indium film did not show any pile - up effects .
pile - up correction has been done to the hardness and modulus data obtained from oliver pharr method . depending upon strain hardening of the tested material ,
the surface deformation mode of an indent can be pile - up or sink - in . in low strain - hardening materials , pile - up tends to occur due to incompressible plastic deformation .
additionally , the indenter has penetrated up to the substrate surface at higher loads , but there was no sign of radial crack formation of the film / wire .
it means that indium wires are in good adhesion towards the substrate . to confirm the values of indentation hardness calculated from oliver pharr method , we have calculated hardness using indentation projected area obtained from residual indent in the afm image .
displacement ( l - d ) curves for nanowire sample and untemplated continuous thin film are as shown in fig .
2 . figure 2a shows the l - d curve of indium thin film . while indenting smaller structures at nanoscale ( particularly nanowires kinds of structures ) , it is very difficult to avoid hitting the smaller particles on the wire surface .
figure 2 depicts how load displacement curve will be affected by hitting the particles available on the surface . in fig .
2a , indenter did not encounter any particles , and the response is that of the indium films .
figure 2b , however , the indenter tip pushes first through a particle approximately 2025 nm thick before encountering the hard surface .
the very low slope of these initial loading curves indicates that the particles are much softer than the indium nanowires beneath .
when we perform displacement off - set to 25 nm , then the loading part exactly fits to hertzian contact equation .
the behaviour of load displacement curves for a indium film , b indium wire . [ inset : plot between ln(strain rate ) and ln(stress ) , to find out the stress exponent of the indium wire ] this phenomenon of pop - in ( displacement burst or strain burst ) was attributed to very poor defect density prior to nanoindentation test so that the onset of plasticity requires load sufficient for dislocation nucleation and propagation . in indium nanowires ,
the pop - in occurred at about 35 n load and at 40 nm depth , as shown in fig .
shortly , after the first displacement burst , the load displacement curve becomes serrated as shown in fig .
2b , indicating series of small displacement bursts followed by the main one . at larger loads , these serrations disappears . the small amount of pile - up around the indenter could be because of these many number of small displacement bursts
no crack formation at the indenter edges was observed in the present study . in the previous studies , the displacement burst in indium thin films observed at 120 n .
but in the present case , pop - in occurred at very low load which might be because of the existence of poor defect density of mobile dislocations and also they require higher amount of stress to start dislocation nucleation and propagation in the nanowires .
this pop - in behaviour in metals and ceramics is often correlated to either the nucleation of dislocations in a perfect crystal or the rapid multiplication of dislocations from a limited source population .
the shear modulus of indium nanowire is 4.5 gpa calculated from effective elastic modulus , eeff , of indium films ( ~50 gpa ) , obtained from oliver pharr method and poison s ratio f(~0.3 ) .
the theoretical shear strength t for the indium nanowires is 1.0 gpa . using the above equations , the maximum shear stress calculated at which first pop - in occurred was 930 mpa for indium wire sample .
almost theoretical shear strength and maximum shear stress at which pop - in occurred are almost matching , and the very small difference between m and t is due to the experimental scatter , residual stresses in wire , defects and surface roughness .
occurrence of several discontinues in l - d curve indicates that the dislocation sources generated during the displacement burst are not very stable and unable to operate continuously at relatively low stress levels , throughout the entire - elastic plastic loading . to understand the time - dependent plastic deformation ( creep ) of the indium film and wire , a hold time of 10 s kept at a peak load of 100 n .
it can be seen from fig . 2a that the displacement was also creeping during the hold time , even though no strain burst had taken place .
the stress exponent obtained for the nanowires sample was 5.5 calculated from the slope of the curve . according to feng et al . , the creep behaviour of indium is distinctively different from other metals .
for example in aluminium , pop - in is a pre - requisite for an indentation creep .
the stress exponent before and after the pop - in was also remarkably different it changes from ~1.5 before pop - in ~6 after pop - in .
lucas and oliver have reported the value of stress exponent was 57 for indium films after pop - in . the value of ~1.5 before displacement burst is rather low compared with the usual range expected for dislocation creep , which is 38 . they have concluded that before strain burst , it was diffusional creep , and after displacement burst , it was dislocation creep .
diffusional creep can happen at the test temperature because room temperature is 0.7 tm for indium .
our results on stress exponent are also matching with the reported values . from the above analysis
, it is understood that the onset of dislocation creep phenomena occurs in indium nano / microwire kind of structures at much lower loads ( 3040 n ) compared to bulk / thin indium films ( 80120 n ) .
this could be attributed to the better crystalline quality and low dislocation density of the indium wire kind of structures .
nanomechanical properties such as indentation hardness and modulus of the indium wires and thin film calculated from the load displacement curves using oliver pharr method after pile - up correction are shown in fig .
3 . the hardness and elastic modulus of the film as well as wire as a function of normalized depth is presented in fig . 3a .
it is clear that the effect of substrates has come into picture after a critical film / wire thickness . to find out film / wire - only hardness from the composite hardness ( film + substrate ) ,
we have used korsunsky s equation to fit the experimental data . for soft films on hard substrates ,
the hardness value for the indium continuous film after the korsunsky fit was 2.55 gpa , whereas it was 1.99 gpa for the nanowires of diameter ~2000 nm .
the hardness of the 500 nm diameter of nanowire was found to be 1.4 gpa .
after the indentation projected area calibration , the hardness values of the indium thin film and wires were measured to be about 2.15 0.5 and 1.3 0.4 gpa , respectively .
the differences between these two methods could be due to the pile - up phenomenon .
oliver and pharr s proposed method is best applied when no pile - up but some sink - in occurs when the sample is indented . in the case of pile - up ,
contact depth is always more than the total depth of indentation , thus the projected area is always underestimated .
this underestimated area could overestimate both hardness and reduced elastic modulus , with a more significant effect on hardness .
the trends for indium film and nanowire with the normalized depth for a hardness and b elastic modulus [ inset : partial unloading function used to obtain depth - dependent mechanical properties of nanowires at a single spot ] summarizes the variation of mechanical properties of nanowires of different diameters and comparison with the indium thin film trend with the normalized depth for elastic modulus is shown in fig .
elastic modulus is a measure of stiffness of a material . in the present case of indium metal ,
indium nanowire and untemplated continuous thin film , both show almost same kind of behaviour but the value of elastic modulus for the film was a bit higher than the wire .
film exhibited the value of modulus around 50 gpa and it was 40 gpa for the wire of diameter 2000 nm .
even the modulus of the 500-nm - diameter wire exhibited still lower modulus than 2000-nm - diameter wire but the difference was not much .
the elastic modulus of the indium wire decreases as its diameter decreases , because of lateral deformation of the wire . increasing
the penetration depth will increase the lateral deformation and in turn significantly decreases the elastic modulus of the wire .
they found that the elastic modulus can increase , decrease or not change with decreasing wire diameter .
one is the surface modification which could arise from the change of the surface tension of the nanowires and decreasing diameter due to the large surface - to - volume ratio ; another is the change of crystallographic directions in the nanowires that also alter their elastic modulus .
chang et al . [ 39 - 45 ] have suggested that the smaller diameter nanowires could not provide sufficient elastic force to resist the pressure from the indenter in the unloading process , yielding the lower contact stiffness , resulting in the lower modulus .
besides , the nanowires subjected to nanoindentation could have the lateral deformation behaviour , which also affects the estimate of the elastic modulus .
recent report has also revealed that the elastic modulus of silicon nanowires is unaffected by their sizes , and their young s moduli of wires are close to that of bulk modulus . in our case , it should be noted that the dimension of our indium nanowires are much larger than the critical dimension at which the surface effect does play an important role on elastic properties of nanowires . in conclusion ,
it is observed that elastic modulus and hardness were found be less for indium wires than indium continuous films .
it is found from the creep studies that in the case of nanowires , onset of dislocation creep phenomena occurs at very low loads .
hardness and modulus of indium micro / nanowires decrease with the decrease in wire diameter . to our knowledge
, this is the first report on the mechanical properties of indium nanowires fabricated by trench template method and using nanoindentation technique .
thus , indentation hardness , h , can be determined from the maximum indentation load , pmax , divided by the contact area , a :
where the contact area ( a ) is a function of the penetration depth , h , and can be determined by the following equation : it may be noted that only the constant c0 is used , if it is assumed that a berkovich indenter has a perfect tip .
however , for imperfect tips , higher - order terms have to be taken into account and these are obtained from the tip - area function curve fit for a given tip . in the current case ,
the values are c0 = 24.5,c1 = 2.3425 10,c2 = 2.764 10,c3 = 2.9845 10,c4 = 3.4532 10 and c5 = 2.6574 10 .
the elastic modulus , e , of the thin film can be obtained using the following equation : where and e are poisson s ratio and elastic modulus , respectively ; and the subscripts i and s refer to the indenter and test material , respectively .
the indenter properties used in this study are ei = 1140 gpa , and poisson s ratio for the indenter is i = 0.07.er * is the reduced modulus that accounts for the fact that elastic deformation occurs in both the sample and the indenter and it can be determined from the equation where s is the stiffness of the test material , which can be obtained from the initial unloading slope by evaluating the maximum load and the maximum depth , i.e.,s = dp / dh. is a shape constant that depends on the geometry of the indenter and is 1.034 for the berkovich tip .
the contact depth can be estimated from the load displacement data using where is a constant that depends on the indenter geometry ( = 0.75 for berkovich indenter ) and hmax the displacement at the peak load .
the loading curve can be fitted with the following power - law relation , and unloading curve can be expressed as where p is the indenter load , h is the elastic displacement of the indenter , hf is the final depth, and m are fitting constants .
the maximum shear stress,max , can be expressed through the mean contact pressure ( pa ) as following from hertz contact theory , where an indentation load ( pmax ) and the penetration depth ( hc ) at known tip radius of the indenter or effective elastic modulus of films ( eeff ) .
the theoretical shear strength t of indented materials may be approximated as where s is the shear modulus .
korsunsky s model is based on energy expenditure during an indentation , and the final equation is given by where is the relative indentation depth , k is a constant related to the film thickness , hf is film hardness , hs is the substrate hardness and hc is the composite hardness .
thus , indentation hardness , h , can be determined from the maximum indentation load , pmax , divided by the contact area , a :
where the contact area ( a ) is a function of the penetration depth , h , and can be determined by the following equation : it may be noted that only the constant c0 is used , if it is assumed that a berkovich indenter has a perfect tip .
however , for imperfect tips , higher - order terms have to be taken into account and these are obtained from the tip - area function curve fit for a given tip . in the current case ,
the values are c0 = 24.5,c1 = 2.3425 10,c2 = 2.764 10,c3 = 2.9845 10,c4 = 3.4532 10 and c5 = 2.6574 10 .
the elastic modulus , e , of the thin film can be obtained using the following equation : where and e are poisson s ratio and elastic modulus , respectively ; and the subscripts i and s refer to the indenter and test material , respectively .
the indenter properties used in this study are ei = 1140 gpa , and poisson s ratio for the indenter is i = 0.07.er * is the reduced modulus that accounts for the fact that elastic deformation occurs in both the sample and the indenter and it can be determined from the equation where s is the stiffness of the test material , which can be obtained from the initial unloading slope by evaluating the maximum load and the maximum depth , i.e.,s = dp / dh. is a shape constant that depends on the geometry of the indenter and is 1.034 for the berkovich tip . the contact depth can be estimated from the load displacement data using where is a constant that depends on the indenter geometry ( = 0.75 for berkovich indenter ) and hmax the displacement at the peak load . the loading curve can be fitted with the following power - law relation , and unloading curve can be expressed as where p is the indenter load , h is the elastic displacement of the indenter , hf is the final depth, and m are fitting constants
the maximum shear stress,max , can be expressed through the mean contact pressure ( pa ) as following from hertz contact theory , where an indentation load ( pmax ) and the penetration depth ( hc ) at known tip radius of the indenter or effective elastic modulus of films ( eeff ) .
the theoretical shear strength t of indented materials may be approximated as where s is the shear modulus .
korsunsky s model is based on energy expenditure during an indentation , and the final equation is given by where is the relative indentation depth , k is a constant related to the film thickness , hf is film hardness , hs is the substrate hardness and hc is the composite hardness .
u. ramamurty , department of materials engineering , indian institute of science , for giving permission , to work with nanoindentation system , existing in his laboratory and mr .
pk acknowledges the financial support received from the ugc - cas and the upe programs and from department of science and technology ( dst ) .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
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nanomechanical properties of indium nanowires like structures fabricated on quartz substrate by trench template technique , measured using nanoindentation .
the hardness and elastic modulus of wires were measured and compared with the values of indium thin film .
displacement burst observed while indenting the nanowire .
wire - only hardness obtained using korsunsky model from composite hardness .
nanowires have exhibited almost same modulus as indium thin film but considerable changes were observed in hardness value .
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it was an honor for the editors of electronic physician , in its 8th anniversary , to organize the first edition of international conference on health sciences and medical technologies ichsmt16 .
the proposed motto of the events was:leveraging / synergizing on the strength of technology written by professor dr .
syed tajuddin syed hassan ( associate editor of electronic physician journal , universiti putra malaysia ) , which fully captured the essence of the whole conference which was focused on technology for serving the most crucial scientific domain - medicine .
it is with great pride that i can report that this conference had contributors representing fourteen different nations , namely : algeria , germany , iran , switzerland , netherlands , malaysia , canada , france , morocco , spain , kingdom of saudi arabia , pakistan , tunisia and united states of america .
author s affiliations were from several departments such as medicine , biology , physics , chemistry , computer sciences , environment , pharmacy , electrical and electronic engineering and mechanical engineering .
an exceptional inaugural session , which contained three plenary speeches , commenced on the first day before the parallel sessions .
the three speeches presented in this session were ; ( 1 ) , presented by professor dr .
wolfgang seger , and was an introduction to the importance of social health and clarification of the international classification of functioning , disability and health ( icf ) .
mehrdad jalalian , and explained how to publish scientific articles in journals , with the highest standards , and the most common mistakes made by researchers .
this presentation also covered the ethics of publishing medical research , and ( 3 ) , by professor dr .
kaouel meguenni , which highlighted cancer disease and the importance of the algerian cancer registry in several studies .
three editorials ( 1 , 4 ) and five original articles ( 510 ) were selected as the best submissions to the conferences . as an award , a free advanced version of all selected papers are planned for publication in the journal electronic physician .
the program committee redefined , after the reviewing step , ten sessions for international participants according to the contributed domains .
the platinum sponsor of the conference was the university of tlemcen with medicine faculty and sciences faculty and research laboratories .
the gold sponsors were mehr publishing group , mehrafarin scientific publishing , electronic physician journal , and acta healthmedica journal .
several models , courses , communication / group process issues , and international perspectives are reviewed within it ( 11 ) .
although there are some limits such as cultural bias , language difficulties and technical problems which can decrease the efficiency of these initiatives , international interdisciplinary education participates in the construction of the future of professionals and researchers , and adds to enrich innovation and scientific realizations .
the conferences in general are excellent places to evaluate scientific findings and to prepare for future projects and much more ( 12 ) .
ichsmt16 was an exceptional model of international interdisciplinary education and research , in which several technological innovations and medical findings were presented reviewed and commented .
an exceptional deal in international medical education between germany and algeria began its first steps at the conference .
the success of the conference was measured and inferred by the collected feedback forms , and a considerable amount of advice was collected from the contributors of these conferences . to ameliorate the quality of the next edition , we plan several additional improvements .
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the international conference on health sciences and medical technologies ( ichsmt16 ) was held in tlemcen , algeria from 2729 september 2016 .
the conference was organized by the university of tlemcen , in partnership with electronic physician journal , mehr publishing , and mehrafarin scientific publishing . there were participants from 14 nations who presented their research in poster or oral presentations .
there were also some keynote speakers who gave talks on topics such as community health , ethics of publishing medical research , and scientific writing .
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juvenile xanthogranuloma ( jxg ) is a histiocytic disorder most commonly present in the first two decades of life , especially in the neonatal period and first year of life .
prognosis , generally speaking , is good except when there is deep or multisystemic involvement .
three large clinicopathologic studies are available.13 the spectrum of histological subtypes are nicely summarized by janssen et al.2 early jxg typically presents within compact sheet - like histiocytic infiltrate , with or without scattered eosinophils , sometimes mimicking langerhans cell histiocytosis ( lch ) .
classic jxg , on the other hand , typically has foamy histiocytes with abundant cytoplasm and fine cytoplasmic vacuolation ; spectrum of fully developed touton giant cells is present , but giant cells of langerhans type or foreign body type are also possible .
the late or transitional subtype typically shows predominance of single sheets of cells reminiscent of benign fibrous histiocytoma with the addition of foamy histiocytes and giant cells .
a distinctive immunohistochemistry staining pattern includes : positive cd68 , alpha-1 antitrypsin , factor xiiia , and lysozyme ; s100 and cd1a are typically negative , in contrast to its cousin langerhans cell histiocytosis . in electron microscopy studies
forms of systemic disease are rare and they have significant clinical implications , as summarized by freyer and dehner.4 the case reported here is a newborn with multisystemic jxg involvement , illustrating the difficulties and challenges encountered diagnostically and therapeutically .
the patient is a 5-week - old , former 35-week infant who was admitted for evaluation of pancytopenia , transfusion dependence , and massive hepatosplenomegaly .
it was noted that he had a blueberry muffin rash at birth . during his first month
stay at nursery ( nicu for prematurity ) , he was doing well and was eating and thriving . physical examination was remarkable for left posterior cervical mass ( suboccipital region ) that was firm and diffuse , not discrete like a lymph node .
the abdomen was soft , nontender , distended with marked hepatosplenomegaly down to the level of the umbilicus for the liver and spleen .
diagnostic workup with abdominal ultrasound initially showed hepatomegaly with multiple hypoechoic lesions in the liver along with splenomegaly and ascites ( figure 5 ) .
evaluation of the lymph node showed a mass adjacent to the left posterior cervical musculature and there was suggestion of destruction of the adjacent occipital bone and possible intracranial extension .
mri of the brain showed destruction of the left occiput with likely lesion of the left sigmoid sinus and abutment of the left internal carotid artery with encroachment of the spinal foramen without loss of flow in the artery or extension into the spinal cord .
other tests performed for these masses , including hva and vma , were all normal .
the differential for these various lesions , including the blueberry muffin spots , which were thought to be either in situ extramedullary hematopoiesis or superficial tumor metastases , included neuroblastoma and congenital leukemia .
biopsy of the neck mass revealed a histiocytic lesion ; the lesional cells were mostly polygonal ( some with quite visible vacuolation ) with indistinct borders , plus abundant classic touton giant cells . scattered inflammatory cells , mostly lymphocytes , were observed as well .
the staining pattern of lesion cells was as follows : s100 negative , cd1a negative ; cd68 positive , cytoplasmic , alpha-1 antitrypsin positive , cytoplasmic , lysozyme positive ( strong , patchy , and cytoplasmic ) .
additional stains included : cd34 , cd35 , cd21 , and cd20 negative ; cd4 positive membranous ; factor xiiia strong positive ; fascin positive ; and cytoplasmic ( figure 4 ) .
the patient was put on protocol lch - iii for the treatment of jxg , following the arm for high risk group .
as this brief report was written , he has been off therapy for twenty months . at this point , he is doing well without any concerns and has begun walking and on regular followup . on physical examination , he was alert and well appearing ; head and neck exam was remarkable only for a small bruise on his cheek ; no mass was palpable in the left occipital region any longer .
abdomen examination demonstrated the liver was down 4 cm and spleen tip was palpable 3 to 4 cm .
his gait was considered wide based but age appropriate and the remainder of his exam was unremarkable .
pancytopenia had recovered and complete blood count ( cbc ) results were within normal ranges .
radiology followup showed decrease in size of the left occipital soft tissue mass abutting the skull base ; lung lesion and low density foci distributed throughout the liver were stable ; hepatomegaly and splenomegaly were also stable .
it is fair to state that when jxg has liver involvement , it is a relatively benign disease in its worst form . in 1996 , dehner reported eight out of 34 patient with systemic jxg had liver involvement , two neonates died with systemic jxg , among which one had liver / spleen involvement ( patient number 19).4 in dehner s 2003 survey , liver involvement claimed two out of only three deaths in 174 cases ( patients number 2 and 7).1 the kiel study reported 129 patients with five cases of systemic jxg , in which the only one death ( with disease ) was the one with liver and spleen involvement ( patient number 5).2 in 2008 , haughton reported a case of blueberry muffin baby that proved to be a systemic jxg with skin and liver involvement , and who ended up needing liver transplant.5 but successfully treated cases are available , summarized by stover in 20086 so the pattern appears to be that the systemic jxg with liver involvement carries the worst prognosis implications , even though , as a whole , the systemic jxg group generally still tends to be prognostically optimistic .
it has been observed that extracutaneous jxg tends to have less or absent multinucleated giant cells , but our case is certainly the opposite .
so the lesson still is , the microscopic appearance of this entity is varied greatly and entertain the high suspicion with any histiocytes - rich lesion is essential . from a cytogenesis point of view , jxg is related to lch but has always been considered as a distinctive entity .
the evolution of the hypotheses of its cellular origin is such an interesting topic that almost every study , major or small , paid some attention to it .
briefly , the earliest speculation in 1912 was that it has an endothelial origin;7 in 1954 , its histiocytic nature was recognized and the consensus was to place this entity under non - langerhans histiocytosis group as archetype of non - langerhans histiocytosis,810 with factor xiiia+ dermal dendrocytes as the strongest candidate originating cells;11 however , this concept has been challenged recently and plasmacytoid monocyte has been proposed as the putative origin of jxg.12 due to the rarity of the systemic jxg , treatment protocols have followed that use for lch .
histiocyte society has a clinical stratification of lch based on the extent of the disease . for patients with multiorgan involvement ,
lch - iii protocol is currently the most common treatment strategy , which was first suggested by nakatani et al.13 experience with treating systemic jxg is available through a recent review by stover , in which a lch based regimen seems to have been justified.6 clinical trials can be found on the website : h-9926-lch iii : treatment protocol of the third international study for langerhans cell histiocytosis : http://clinicaltrials.gov/ct2/show/nct00488605 .
the treatment does not always go well even though jxg generally carries a favorable prognosis even for its systemic forms .
, an infant with systemic jxg with cns involvement who first could not tolerate prednisolone and later failed vinblastine was treated successfully with cladribine.14 the similar positive responses have been reported in recurrent lch treatments.15,16 disease activity monitoring is also part of the histiocyte societys recommendation.17
because of the rarity and wide spectrum of manifestation , systemic jxg is often not considered in the initial differential diagnosis as happened in this case
. therefore , high degree of clinical suspicion and open mindedness is warranted when patients present with multisystemic symptoms suggestive of malignancy , especially with cases of similar presentation in the neonatal age group . besides the consideration of neuroblastoma ,
so called blueberry muffin rash may also hint to the possible existence of systemic jxg .
visceral multisystemic presentation with liver / spleen involvement and coagulopathy is perhaps the worst form of systemic jxg with a more aggressive behavior and worse prognosis than other similar cases in the same category ; intensified treatment , close monitoring , and followup is warranted .
a lch iii based regimen is currently recommended and worked well in our case ; if no satisfactory response is generated , an alternative treatment such as cladribine may be tried .
overall , our knowledge of systemic jxg is still very limited ; more experience with its diagnosis and treatment is necessary and expected .
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this case report originated from a case of neonatal multisystemic juvenile xanthogranuloma ( jxg ) .
the patient presented with blue muffin rush , cervical mass , bone destruction , lung nodule , hepatosplenomegaly , and coagulopathy and was successfully treated with langerhans cell histiocytosis ( lch ) based chemotherapy treatment .
similar cases in literature were reviewed and it seems that jxg , a relatively benign entity , when presented in its systemic form with liver involvement , could have an aggressive course and portend quite poor prognosis .
challenges and special consideration of the diagnosis , treatment , and future case observation are discussed .
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spinal anesthesia with a sedative drug may affect patient 's consciousness , but one study has shown that patients undergoing spinal anesthesia without the sedatives may suffer from decreased level of consciousness .
regional anesthesia techniques have several advantages , including a decreased risk of failed intubation and aspiration of gastric contents , avoidance of depressant agents , and the ability of the mother to remain awake and enjoy the birthing experience .
in addition , it has been suggested that blood loss is reduced under regional anesthesia for cesarean delivery .
spinal anesthesia has been found to be faster , more cost - effective and provide a superior block .
bispectral score ( bis ) is eeg derived parameters related to the level of sedation and loss of consciousness.[36 ] bis was recognized as a method by which the anesthesiologists were able to determine the depth of anesthesia in high - risk surgeries for the first time.[59 ] some factors affect the depth of spinal anesthesia including pregnancy , additional drugs and baricity .
when neuraxial anesthesia is administered , women require less local anesthetic than non pregnant women do reach a given dermatomal sensory level .
it was previously reported that the bis value during spinal anesthesia with isobaric bupivacaine is reduced with intrathecal fentanyl but not with intravenous or epidural fentanyl .
the bis values in patients who received intrathecal isobaric bupivacaine with fentanyl were lower than those with intrathecal hyperbaric bupivacaine and fentanyl .
there are no studies evaluating intrathecal isobaric and hyperbaric bupivacaine alone and without fentanyl with respect to bis values .
the aim of this study was to compar the effect of isobaric bupivacaine and hyperbaric bupivacaine on the bis during cesarean section with spinal anesthesia .
following ethics committee approval and obtaining informed patient consent , 84 pregnant women undergoing elective cesarean section were recruited .
this double - blind randomized controlled clinical trial was performed in beheshti medical center , isfahan , iran , during the period from march 2008 to april 2011 .
inclusion criteria were asa i or ii ( american society of anesthesiologists ) , no cardiovascular disease , no history of diabetes , no previous pregnancy complications and no history of twin pregnancy .
all patients were fasted for 8 hours preoperatively and infused intravenous preload of 10cc / kg ringer 's lactate solution before surgery .
intraoperative monitoring included pulse oximetry , automated blood pressure cuff , lead ii electrocardiogram and capnograph.[1315 ] the operating theatre nurse assistant used randomization protocol to assign participants to their respective groups .
randomization was done by a table of random numbers with minimization for age , medical and psychiatric status .
, the cerebral state monitoring ( csm ) electrodes were placed in the fronto - temporal regions as recommended by the manufacturer ( csm , new version , danmeter co , denmark ) which used for measurement of bis values , signal quality index ( sqi ) and electromyography ( emg ) . to reduce skin / electrode impedance ,
if sqi was < 50% for longer than 20% of the total study period , all data for the patient were excluded from analysis .
lumbar puncture was performed in the sitting position . a 25 gauge spinal needle ( pencil point , pajunk , germany )
was introduced into the subarachnoid space at the l3-l4 lumber level midline approach with the needle orifice cephalad .
cerebrospinal fluid was aspirated and the ready fluid 2.5 ml hyperbaric 0.5% dissolved in 8.25% glucose solution ( marcaine 5% , mylan co , france ) or 2.5 ml isobaric bupivacaine 0.5% ( mylan co , france ) was injected to subarachnoid space over the period of 15 seconds , with no barbitage .
patient was set to the left lateral position . the maximum level of sensory motor block assessed by pin prink test after spinal anesthesia .
motor block was assessed by modified bromage score ( 0 : not motorless , 1 : inability to flex the hip , 2 : inability to flex the knee , 3 : inability to flex ankle ) .
after establishment of t4 block with pin prick test and confirmation of anesthesia , cesarean section was done .
the study solutions were prepared by another researcher not involved in patient care , then injected immediately afterwards .
the spinal needle was withdrawn and patient were repositioned supine with elevation of head ( 15 - 20 ) .
bis , sqi , emg , heart rate ( hr ) , mean arterial pressure ( map ) and the core temperature ( evaluated by tympanic thermometer , braun , irt 3020 , type = 6012 , thermoscan , kronberg , germany ) was recorded at the baseline and 5 , 10 , 15 , 30 , 45 , 60 , 90 and 120 minutes after spinal injection .
if sbp was < 20% below baseline or < 100 mmhg , intravenous ( iv ) ephedrine 5 mg was given incrementally .
if heart rate was less than 50 beats / min , 0.5 mg atropine sulfate was administered iv .
the sample size was based on the previous studies , 0.90 powers and error of 0.05 .
it was calculated to be 84.the analyses were performed based on mann - whitney test and independent student 's t - test for quantitative variables . all analysis was done using spss software ( version 18 , spss inc . ,
chicago , il , usa ) . a p value of < 0.05 was considered statistically significance for all analyses .
the hyperbaric bupivacaine group included 42 subjects with mean age of 28.6 5.7 years and the isobaric bupivacaine group consisted of 42 patients with mean age of 28.2 5.7 .
two groups did not have significant difference at baseline characteristics and baseline conditions ( table 1 ) .
the heart rate , mean arterial pressure changes , temperature , emg and sqi did not have significant differences at the baseline and 5 , 10 , 15 , 30 , 45 , 60 , 90 and 120 minutes after spinal injection between two groups . from recruited subjects , 41 completed the study in each group and underwent analysis .
baseline characteristics and drug consumption of the two groups ( meansd ) flow diagram of enrolled study patients the recorded bis was not significantly different between two groups at all the time points ( table 2 ) .
difference of bis from baseline was not significant between two groups at most time points but this difference was statistically significant at the 45 minute after injection ( p = 0.047 , table 3 ) .
the changing trend was not significantly different between groups with hyperbaric and isobaric bupivacaine ( figure 2 ) .
bispecteral index during cesarean section ( meansd ) changing trend of bis values during spinal anesthesia
the effect of these drugs on bis score has been investigated but the comparison of these two kinds of drugs has not been done until now . based on our results ,
the bis score of patients undergoing intrathecal spinal anesthesia with hyperbaric or isobaric bupivacaine was not significantly different .
bis change after 45 minutes from baseline was the only significantly difference between two groups .
several theories have been proposed to explain the sedative effects of spinal anesthesia , including a direct effect of the local anesthetic , either by systemic absorption or rostral spread through the csf , attenuating stimulation of the reticular activating system in a situation where there is also decreased afferent input .
pollock et al . reported that the greatest variations from baseline bis values in non sedated patients occurred at 30 and 70 minutes after induction of spinal anesthesia , and suggested that delayed rostral spread of local anesthetics might be responsible .
a previous study in which high and low blocks ( medians t3 vs. t10 ) were produced by the injection of different doses of hyperbaric bupivacaine ( 17.5 vs. 7.5 mg ) , reported that the bis values did not change until 20 minutes after induction of spinal anesthesia , the propofol infusion having been started , after 15 min in both groups .
the authors suggested that 1520 minutes was not long enough for local anesthetics to spread rostrally in concentrations sufficient to influence the electrical activity of higher neuronal centres .
although the possibility of rostral spread of the intrathecal local anesthetics can not be excluded , there is a low probability of this occurring , particularly in the
reported that in pregnant women undergoing cesarean delivery using spinal anesthesia , 12.75 mg hyperbaric bupivacaine 0.5% with fentanyl had more sedative effect than the same dose of this drug without fentanyl which resulted in the increased anesthetic block density . in the group that had only received bupivacaine ,
the peak sedative effect was seen 35 - 45 minutes after injection , while women who had bupivacaine plus fentanyl the peak sedative effect was seen at 45 - 70 minutes .
therefore , it was concluded that spinal anesthesia in cesarean is in association with loss of consciousness especially if two drugs are used simultaneously .
intrathecal spinal anesthesia has anti anxiety effect in addition to spinal ganglion blockade and decrease afferent signals from the spinal cord .
more recent data have suggested that the reduced local anesthetic requirements predate the mechanical effects of the gravid uterus . in animal studies , chronically administered progesterone has been found to reduce anesthetic requirements .
in addition , it has been suggested that the increased concentrations of endorphins and dynorphins found in pregnant rats may be related to altered pain threshold .
this evidence suggests a multi factorial explanation for the decreased anesthetic requirement . in another study ,
0.5% and 20 micrograms fentanyl intrathecal in group i , 2.5 ml bupivacaine isobar 0.5% intrathecal and 100 micrograms fentanyl intravenous in group ii , 2.5 ml bupivacaine isobar 0.5% intrathecal and 100 micrograms of epidural fentanyl in group iii , 2.5 ml bupivacaine isobar 0.5% intrathecal in group iv and finally 3 ml bupivacaine isobar 0.5% intrathecal for group v. bis values of the group administered intrathecal fentanyl were lower than those of other groups .
in addition , the time in which the bis score was 80 or less was more than other groups . therefore , the bis value was significantly decreased only by intrathecal fentanyl for cesarean section .
in addition to combination of drugs , baricity is another known factor that affects consciousness in spinal anesthesia .
no study has compared the effect of hyperbaric and isobaric bupivacaine on the bis score . according to the present study ,
the bis scores during surgery began to reduce after intrathecal injection in both of these drugs .
moreover , the trend of bis scores was lower in hyperbaric bupivacaine group than the other . however , it was not statistically significant .
one study examined the effect of different levels of spinal anesthesia for varicose vein surgery , induced by solutions of different baricity but containing the same amount of local anesthetic agent , on the requirement for sedation with propofol . the known difference in level of spinal anesthetic block induced by solutions of different baricity but the same dose of local anesthetic
the maximum levels of block in the control hyperbaric and isobaric groups were t4 ( t3t9 ) and t10 ( t8t11 ) .
cumulative consumption of propofol to maintain the bis value was also less in the hyperbaric than in the isobaric group that was influenced by the block height , not the dose of local anesthetic .
however , this study did not demonstrate a correlation between block level and propofol requirement , because the block levels were not so diverse .
conversely , another study investigated whether the specific gravity of bupivacaine combined with intrathecal fentanyl in spinal anesthesia for cesarean section effected the bis values .
thirty one parturients scheduled for cesarean section were allocated into two groups : 0.5% isobaric bupivacaine 2 ml plus fentanyl 20 microgram ( i group , n = 14 ) or 0.5% hyperbaric bupivacaine 2 ml plus fentanyl 20 microgram ( h group ,
the lowest bis values in the i group and in the h group were 77 13 and 87 6 , respectively .
the cumulative time for bis values below 80 in the i group was longer than that in the h group .
the number of bis decreased cases , defined as the cases in which the bis values continuously fell down to or below 80 for more than 10 minutes , was higher in the i group than in the h group .
the bis values in patients who received intrathecal isobaric bupivacaine with fentanyl were lower than those with intrathecal hyperbaric bupivacaine and fentanyl . in our study
found that ephedrine but not phenylephrine , increases bispectral index values during combined general and epidural anesthesia .
second , there was no control group , where intrathecal local anesthetic was not given . in conclusion , in pregnant women undergoing elective cesarean section using spinal anesthesia , the bis during surgery began to reduce when the patients were injected by both of hyperbaric or isobaric bupivacaine and these drugs did not have different effects on the bis until 2 hours after induction of spinal anesthesia .
authors are thankful to members of research committee of department of anesthesia and intensive care of isfahan university of medical sciences for helping us in approval process of this report .
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background : hyperbaric and isobaric bupivacaine has been shown to reduce bispectral index ( bis ) during spinal anesthesia . the aim of this study was to compare the effect of isobaric bupivacaine and hyperbaric bupivacaine on the bis during cesarean section with spinal anesthesia.materials and methods : in this double - blind randomized controlled clinical trial , 84 pregnant women with asa ( american society of anesthesiologists ) class i or ii were randomly assigned to receive isobaric or hyperbaric bupivacaine .
bis was measured using electro encephalography ( eeg ) and recorded at the baseline and 5th , 10th , 15th , 30th , 45th , 60th , 90th and 120th minutes after spinal injection .
the heart rate ( hr ) , mean arterial pressure ( map ) , signal quality index ( sqi ) , electromyography ( emg ) and the temperature were also recorded during surgery.results:from recruited subjects , 41 completed the study in each group and their data were analyzed .
the bis score began to reduce until 30th minute for isobaric bupivacaine and 45th minute for hyperbaric bupivacaine after spinal injection .
the recorded bis was not significantly different between two groups at all the time points .
difference of bis from baseline was not significant between two groups at most time points except for the 45th minute after injection that it was 5.9 9 vs. 2.7 4.6 for hyperbaric and isobaric bupivacaine , respectively ( p = 0.047 ) .
the changing trend was not significantly different between groups with hyperbaric and isobaric bupivacaine.conclusion:according to the present study , the bis score during surgery began to decrease when the patients were injected spinally by both of these drugs .
the greatest decrease from baseline bis values occurred at 30 and 45 minutes after induction of spinal anesthesia .
however , hyperbaric or isobaric bupivacaine did not have different effects on the bis during spinal anesthesia .
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the effects of severe trauma , infection , and surgery result in remarkable metabolic stress on the human body .
stress associated with critical illness is characterized by the activation of inflammatory cellular mediators and the hypothalamic - pituitary - adrenal ( hpa ) axis .
the release of cortisol , catecholamines , glucagon , and growth hormone is an essential component of the general adaptation to illness and stress .
the acute response to critical illness ( energy conservation toward vital organs , modulation of the immune system , and a delay in anabolism ) is generally considered to be an appropriate and adaptive response that occurs in the first few days after insult .
mild - to - moderate stress hyperglycemia is protective because it provides a source of fuel for the immune system and brain at a time of stress .
however , many of the chronic endocrine responses result in persistent hyperglycemia and insulin resistance , which can be potentially deleterious in the long run . in combination with inadequate systemic insulin levels and insulin resistance due to the increased secretion of counter - regulatory hormones , the negative manifestations inflicted by hyperglycemia lead to life - threatening conditions in these patients
. incidences of stress - induced hyperglycemia , defined as plasma glucose levels exceeding 200 mg / ml in patients , have been documented for more than 100 years in patients experiencing severe trauma or injury .
although hyperglycemia is believed to be an adaptive stress response , long - term stress - induced hyperglycemia is linked to poor clinical outcomes and increased risk of mortality .
the underlying causes of hyperglycemia during critical illness are attributed to the increased hepatic glucose production and impaired glucose consumption by peripheral tissues as well as insufficient pancreatic insulin production .
in addition , the production and accumulation of counter regulatory hormones , such as glucagon , cortisol , catecholamines , and growth hormone , will increase lipolysis , protein breakdown , and impair glucose usage by peripheral tissues . at the cellular level , increased blood glucose levels result in mitochondrial injury and endothelial dysfunction by generating reactive oxygen species and inhibiting nitric oxide production , respectively .
recently , it has been found that the endoplasmic reticulum ( er ) stress response and its subsequent unfolded protein response are activated in various tissues under conditions related burn and severe trauma .
er stress has been identified as one of the central intracellular signaling pathways that link insulin resistance , hyperglycemia , and inflammation . in this paper
, we intend to review recent advances on the regulating effects of hyperglycemia and insulin on innate immunity , with a particular emphasis on severe burns .
in addition , we will explore the history of insulin treatment on stress - induced hyperglycemia during critical illness and update the present understanding in regard to the ongoing moderate versus intensive insulin treatment debate .
although cytokine products and reactive oxygen species produced by innate immune cells may have profound effects on glucose disposal and utilization in the periphery as well as on insulin production by the pancreas , we only focus on the effects of hyperglycemia and insulin on innate immune cells .
monocytes , macrophages , and dendritic cells are antigen - presenting cells that possess phagocytic capabilities that play a crucial role in maintaining immune homeostasis and mounting an immune response against infection .
in severely burned and septic patients , monocyte phenotype and function are disrupted , resulting in a lowered expression of hla - dr on circulating monocytes as early as 2 - 3 days after injury .
this effect of decreased hla - dr expression and cytokine production has shown to persist for 28 days in burn and septic patients .
the adverse effects of hyperglycemia on innate immunity manifest through the regulation of monocyte cytokine secretion .
this notion is well supported by previous studies using thp-1 monocyte cell line and human peripheral blood monocytes , which when cultured under high glucose conditions caused elevated expression of mcp-1 , tnf- , il-1 [ 8 , 9 ] , cox2 , ip-10 , and il-6 .
a study using primary human monocytes shows that hg - induced tnf- production is through the downregulation of cd33 .
importantly , hyperglycemia - induced abnormal cytokine production in patients with severe sepsis exacerbates the clinical outcomes of these patients experiencing stress hyperglycemia .
the proinflammatory cytokine il-6 is normally released upon infection ; however , it induces insulin resistance during conditions of hyperglycemia .
devaraj and jialal found that increased secretion of ip-10 from monocytes cultured with high concentration glucose was via tlr2 and tlr4 pathway , since blockade of tlr2 and/or tlr4 inhibited ip-10 release .
other studies further demonstrated that hg - induced tlr-2 and -4 expressions are via protein kinase c ( pkc ) activation and by the stimulation of nadph oxidase .
however , other studies showed that cytokine secretion was inhibited in the presence of higher concentration of glucose or c - peptide using in vitro culture of u937 cell line or ex vivo culture of freshly isolated leukocytes from healthy volunteers .
inhibited cytokines include il-6 , il-8 , macrophage inflammatory protein- ( mip- ) 1 , mip-1 , tnf- , and reactive oxygen species .
monocytes from healthy volunteers that were exposed for 24 hours to high concentrations of glucose ( 400 mg / dl ) presented a decreased hla - dr .
hyperglycemia has also shown to regulate other functions of monocytes , such as adhesion , migration , and transmigration .
. observed that high concentration of glucose augmented monocyte adhesion to human umbilical vein endothelial cell monolayer and increased migration .
in contrast , another study showed that adhesion of monocytes to human aortic endothelial cells was diminished in the presence of 30 mm / l glucose and c - peptide .
the phagocytic ability of innate immune system has been found to be marginally enhanced by hyperglycemia .
taken together , numerous studies have investigated the effects of hyperglycemia on monocyte functions including cytokine secretion and migration . as a result of the inconsistent findings , there is a lack of consensus on the relationship between monocytes and high glucose conditions and further investigation is required .
the discrepancies may be resulted from the different model system and exposure time among investigators .
we would like to emphasize that the effects of hyperglycemia on monocytes are not necessarily equivalent to the status of monocytes in severely injured patients , such as burned and septic patients .
macrophages are an essential component of the immune system , with three fundamental homeostatic activities : host defense , wound healing , and immune regulation .
due to the large number of macrophages in the tissues and its role as a major source of cytokines during injury , hyperactive macrophages are the leading contributors of systemic inflammatory response syndrome ( sirs ) .
interestingly , recent research has shown that macrophages are heavily involved not only in proinflammatory signaling cascades but are also pivotal in the phases of anti - inflammatory , wound healing , and sepsis in critical illness . to examine the effects of high glucose on macrophage proliferation , liu et al .
cultured monocyte / macrophage cell line wehi-3 and splenic macrophages in hyperglycemic media with various concentrations ( 5.630 mm ) of glucose .
the enhanced macrophage proliferation may result from increased csf-1 receptor ( csf-1r ) under these conditions .
in addition , hyperlipidemia has combined effect with hyperglycemia to stimulate the proliferation of macrophages since hypermetabolisms including hyperglycemia and hyperlipidemia are very common in critical illness .
in addition , hyperglycemia also enhances the immunological responses , as is shown in that hyperglycemia augmented increased cytokine production and phagocytosis in response to lps .
this effect may be associated with elevated tlr expression . extending the in vitro experiments using cell lines and isolated splenic macrophages ,
there has been an ex vivo study examining the effects of hyperglycemia on alveolar macrophage function .
in contrast to studies using cell lines , hyperglycemia significantly decreased the respiratory burst of alveolar macrophages and impaired proinflammatory cytokine secretion , such as tnf- and il-6 .
the impaired reactivity of alveolar macrophage to tlr ligands might result from hg - induced alteration of intracellular signaling and is unlikely due to the modulation of tlr expression itself .
hyperglycemia also promotes the inflammatory response by activating the nf-b pathway . using a rat model of hyperglycemia and burn injury , kulp et al .
streptozotocin - induced hyperglycemia in severely burned rats rapidly activated nf-b pathways in the liver and markedly increased liver acute - phase proteins and proinflammatory cytokines . on the contrary
f4/80(+ ) peritoneal exudate macrophages ( pems ) from mice with diabetes for 4 months displayed significantly reduced proinflammatory cytokines tnf- and il-6 production but enhanced nitric oxide ( no ) secretion when treated with ifn- and lps , while the activity of arginase in pems from diabetic mice was significantly higher than control mice when stimulating with il-4 . in summary ,
the detrimental effects of hyperglycemia on thermal injury outcome may be mediated in part by augmenting macrophage inflammation via the activation of hepatic nf-b pathway .
neutrophils are typically the first leukocytes to be recruited to the inflammatory site and are capable of eliminating pathogens by multiple mechanisms . following infection , the localization of neutrophils to the site of inflammation is crucial for the clearance of pathogens .
when considering burn shock , the inflammatory response - related hyperactivation of neutrophil contributes to oxidative cell / tissue damage and potentially initiates organ - system dysfunction and failure .
severe burn and sepsis result in an inhibition of neutrophil function including migration and neutrophil paralysis leads to increased rate of infectious complications in short - term hyperglycemic critically ill patients .
hyperglycemia induces neutrophil dysfunction by modulating one of the neutrophil biochemical pathways , myeloperoxidase ( mpo ) .
hyperglycemia also reduces neutrophil degranulation and exaggerates coagulation in healthy humans that accepted glucose infusion and injection with endotoxin .
since glucose and glutamine play a key role in neutrophil function , changes in metabolism of neutrophils under the condition of hyperglycemia may play an important role in the impaired neutrophil function observed in diabetes .
sustained decreases in neutrophil function associated with hyperglycemia are associated with the extent of hyperglycemia .
t cells , a t - cell subset expressing tcr , account for approximately 35% of all lymphoid cells found in the secondary lymphoid tissues and the blood .
they are relatively abundant in the skin epithelia , intestine , uterus , and tongue where they can account for up to 50% of the total intraepithelial lymphocyte population .
resident intraepithelial t cells are responsible for maintaining epithelial integrity , regulating homeostasis and providing a first line of defense against pathogens and injury in mice and humans .
schwacha and collaborators found that t cells play a role in neutrophil - mediated remote organ ( i.e. , lung , small intestine ) injury early after burn injury by increasing chemokine levels in both the plasma and tissues .
another study from the same group showed a 6-fold reduction in cellular infiltrate in burn wound and a marked decrease of levels of mcp-1 , il-6 , and tnf- in the wound in t cells receptor - deficient mice .
more recently , a study showed that hyperglycemia negatively impacts homeostasis and functionality of skin t cells .
hyperglycemia results in impaired skin t cell proliferation , ultimately resulting in half the normal amount residing in the epidermis .
these t cells expressed decreased levels of nr4a1 and nr4a3 , two orphan nuclear receptors that have been shown to sensitize muscle to insulin , suggesting their decreased insulin sensitivity .
the dysfunctional t cells can also result from the effects of chronic inflammatory mediators , such as tnf- , in the local environment .
overall , skin t cells recognize epithelial damage and release cytokines and growth factors that facilitate wound repair .
their activities are compromised by hyperglycemia , rendering host defense mechanisms vulnerable to further injury and infection in patients with critical illness .
insulin exerts its effects on immune cells by binding to the insulin receptor ( ir ) , that is , extensively expressed on immune cells , such as neutrophils and monocytes / macrophages . upon insulin 's binding to the ir
, insulin rapidly increases tyrosine phosphorylation of its own receptor followed by the phosphorylation of the insulin receptor substrate proteins ( irs ) .
the irs is linked to the activation of two main signaling pathways : the phosphatidylinositol 3-kinase ( pi3k)akt / protein kinase b ( pkb ) pathway and the ras / mitogen - activated protein kinase ( mapk ) pathway .
it has been shown that mice deficient in insulin have an exaggerated cytokine response to peritoneal inflammation compared to controls , indicating that insulin treatment not only decreases glucose levels but also inhibits the inflammations .
recent studies indicate that insulin levels vary in patients but that higher insulin levels may be associated with increased mortality , perhaps suggesting insulin resistance [ 37 , 38 ] . in the following section
, we will review insulin 's effects on immune function and metabolism of innate immune cells ( summarized in table 1 ) . for more detailed insulin signaling pathway , please refer to a decent review paper .
insulin is considered to be a regulator of monocyte function , which includes chemotaxis , phagocytosis , and oxidative burst capacity . in a rabbit model of burn injury
, researchers found that insulin improved the capacity for phagocytosis and oxidative burst within 3 days after burn , with no effect on chemotaxis .
a similar effect was observed in trauma patients receiving intensive insulin therapy , which showed enhanced monocyte phagocytosis .
insulin increased oxidized low - density lipoprotein phagocytosis of monocytes isolated from healthy obese participants .
the effects of insulin on the rates of glucose transport in monocytes were measured with the nbdg fluorescent d - glucose analog .
insulin caused an increase in the uptake of glucose and the expression of glucose transporter ( glut ) isoforms glut3 and glut4 in the plasma membrane .
il-8/cxcl8 is a potent chemoattractant for neutrophils and causes degranulation of neutrophil - specific granules and azurophilic granules .
these results suggest that insulin may regulate the recruitment and activity of neutrophils by inducing il-8/cxcl8 secretion from monocytes .
thp-1 monocytes incubated with insulin and palmitate together produced more il-6 and tnf- , compared to monocytes incubated with palmitate alone .
however , incubation of monocytes with insulin alone did not affect the production of il-6 or tnf- .
monocytes from healthy volunteers were treated with insulin ( concentration from 10 u to 200 u ) for 24 hours in vitro and monocytic hla - dr was significantly decreased in a dose - dependent manner .
lastly , insulin also regulates other activities of monocytes including superoxide production and the expression of tissue factor ( tf ) and mmp-9 .
it stimulates superoxide ( o2 ) production in monocytes and macrophages , which is dependent on nadph oxidases .
insulin may influence the hypercoagulability in patients by inhibiting tissue factor expression in monocyte , the principal initiator of the extrinsic coagulation pathway . generally speaking
this effect is via the release of activin a and the signaling by cytoplasmic sh2-containing inositol 5-phosphatase ( ship )
. it also modulates tissue inflammation by reducing macrophage accumulation in visceral adipose tissue in mice .
another study also showed that insulin inhibits cytokine secretion ( tnf- and il-1 ) by macrophage and improves its survival .
pretreatment of cells with specific covalent inhibitor of phosphoinositide 3-kinases significantly inhibited insulin - mediated cell survival and bclxl expression . in addition , the enhancing effect of insulin on bclxl expression is dose - dependent .
furthermore , macrophages from mice with streptozotocin- ( stz- ) induced diabetes display a dysfunctional phenotype , reduced cd86 expression , and proinflammatory cytokines such as tnf- and il-6 production but enhanced nitric oxide ( no ) secretion .
these functional changes of macrophages could be efficiently reversed by insulin treatment and this effect is dependent on the activities of akt and erk .
recent study shows that insulin promotes human macrophage foam cell formation by increasing type ii scavenger receptor cd36 and decreasing the expression of the atp - binding cassette transporter abca1 . as a result , it leads to 2 - 3-folds more cholesterol accumulation within a short period by increasing oxidized ldl uptake and decreasing cholesterol efflux to apolipoprotein a1 ( apoa1 ) .
insulin also promotes foam cell formation by accelerating endocytic uptake of advanced glycation end products ( age ) proteins .
in addition , insulin specifically promotes the protein degradation of lrp1 and therefore decreases lrp1 expression on macrophages .
the decreased expression of lrp1 impairs the cellular internalization of alpha-2-macroglobulin , which may modulate cytokine secretion by macrophage .
in vitro study indicated that insulin regulated isolated neutrophil cytokine secretion .
activin a , a transforming growth factor- family cytokine , plays a crucial role in regulating the onset and severity of many inflammatory conditions .
bone marrow - derived neutrophil precursors contained 7-fold higher concentrations of activin a than bone marrow mononuclear cells .
these isolated neutrophils could release activin a in response to tnf-. however , production of activin a would be blocked upon pretreatment with insulin .
the in vivo effects of insulin on neutrophils were conducted in healthy subjects under strict euglycemia and physiological insulinemia .
they found that insulin increased the total number of neutrophils and the number of these expressing cd11b , cd15 , cd62l , and cd89 , whereas the density of these molecules was downregulated .
in addition , insulin increased pmn function including chemotaxis , phagocytosis , and bactericidal capacities .
interestingly , although insulin stimulated phagocytosis and bactericidal activity in young - adult subjects , these effects were compromised in the elderly subjects .
studies of patients who underwent major surgery showed that insulin treatment not only significantly decreased the level of blood glucose , but it also increased the number of neutrophils in the circulation as well as their ability to ingest foreign particles .
stress hyperglycemia leads to an increased incidence of infection and higher morbidity and mortality in severely traumatic patients . to manage hyperglycemia in patients with severe trauma and illness , van den berghe and colleagues conducted the first clinical study of iit over 10 years ago
the study showed that maintaining blood glucose at or below 110 mg / dl by iit reduces morbidity and mortality among critically ill patients in the surgical intensive care unit . with a total of 1548 patients enrolled , iit reduced mortality rate from 8% with conventional treatment to 4.6% .
intensive insulin protocols in thermally injured patients have shown improved wound healing by 6.9% in the early stages in comparison to burn controls . severely burned pediatric patients had reduced urinary tract infections and sepsis in the iit group with a positive association with survival . early control of hyperglycemia is essential since a lack of early glycemic control ( mean daily blood glucose < 150 mg / dl in at least two consecutive days by postburn day 3 ) was associated with higher mortality . despite the vast benefits of tight glycemic control with iit , it is accompanied by a mandate for critically monitoring and awareness of sudden fluctuations in blood glucose .
there was no overall impact on hospital or icu length of stay in severely burned paediatric patients who received iit . in addition , there was a significant concern regarding the use of iit in managing elevated blood glucose in traumatic brain injury patients .
iit did not result in reduced cerebral metabolic rate , but it did increase markers of neural metabolic distress and showed no improvement in mortality .
more recently , iit in both critically ill neurological and stroke patients showed more episodes of hypoglycemia and little to worsening effect of patient outcomes compared to nonaggressive approaches .
there has been an ongoing debate regarding the effectiveness of iit versus modest insulin treatment .
in fact , hyperglycemia can be safely avoided using a moderate glycemic control protocol without inducing hypoglycemia . using a retrospective approach ,
kutcher and colleagues concluded that the two treatments had no significant impact on multiorgan failure and mortality .
however , the moderate regime had scarce hyperglycemia episodes , low glucose variability , and intermediate blood glucose ranges of hypoglycemia .
along with the numerous investigations of insulin therapy to manage stress hyperglycemia , there are numerous other treatment options that have been studied .
the use of metformin in treating hyperglycemia decreases endogenous glucose production and increases glucose clearance and oxidation .
the use of other agents in combination with insulin , such as glucagon - like peptide-1 ( glp-1 ) analogue exenatide , has shown to reduce the amount of insulin required to achieve euglycemia .
lastly , the lipolysis agonist fenofibrate treatment can reduce insulin resistance and , when used in combination with insulin , reduces hypoglycemic episodes with clear improvements in skeletal muscle insulin signaling , glucose oxidation , and mitochondrial function .
there is compelling evidence for the multifaceted effect of hyperglycemia treatment and the respective outcomes in critically injured and burn patients .
patients will benefit most from the use of moderate insulin treatment regimens with rigorous attention being given in the first few days of injury to obtain blood glucose levels within the well - established target ranges ( summarized in table 2 ) .
future prospective randomized trials need to place emphasis on the frequency of hypoglycemic and hyperglycemic episodes and the extreme changes in glucose variability to determine the detrimental impact on survival .
stresses - induced insulin resistance and hyperglycemia represent adverse sequelae resulting from trauma and critical care injuries . long - term exposure to stress - induced hyperglycemia
is linked to an increased incidence of infections and sepsis , multiorgan failure , and mortality .
insulin has not only anabolic , but also anti - inflammatory and immune regulatory properties as shown in figure 1 . the interaction or integration of hyperglycemia and insulin on the innate immune cells should be investigated desirably in the future .
a recent study indicates that genomic responses in mouse models poorly mimic human inflammatory diseases including burn and sepsis . in this regard , it is the priority for translational research to take advantage of the clinical samples rather than relying on mouse models to study human inflammatory diseases . furthermore , to increase the study depth we suggest that future studies should examine the effects of the combined effects of hyperglycemia and hyperlipidemia on innate immune cells since hyperlipidemia is also the hallmark of critical illness .
glycemic control established early ( 2 - 3 days after trauma or burn ) in critically ill hyperglycemia patients leads to reduce mortality .
the combination of insulin treatment with other agents will reduce the insulin dose and hypoglycemic episodes . a better understanding of the roles played by hyperglycemia and insulin in the regulation of innate immunity will guarantee a more rational and an effective insulin treatment for these patients .
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hyperglycemia ( hg ) and insulin resistance are the hallmarks of a profoundly altered metabolism in critical illness resulting from the release of cortisol , catecholamines , and cytokines , as well as glucagon and growth hormone .
recent studies have proposed a fundamental role of the immune system towards the development of insulin resistance in traumatic patients .
a comprehensive review of published literatures on the effects of hyperglycemia and insulin on innate immunity in critical illness was conducted .
this review explored the interaction between the innate immune system and trauma - induced hypermetabolism , while providing greater insight into unraveling the relationship between innate immune cells and hyperglycemia .
critical illness substantially disturbs glucose metabolism resulting in a state of hyperglycemia .
alterations in glucose and insulin regulation affect the immune function of cellular components comprising the innate immunity system .
innate immune system dysfunction via hyperglycemia is associated with a higher morbidity and mortality in critical illness . along with others
, we hypothesize that reduction in morbidity and mortality observed in patients receiving insulin treatment is partially due to its effect on the attenuation of the immune response .
however , there still remains substantial controversy regarding moderate versus intensive insulin treatment .
future studies need to determine the integrated effects of hg and insulin on the regulation of innate immunity in order to provide more effective insulin treatment regimen for these patients .
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a seven - year - old boy presented to the emergency department at the winnipeg children s hospital ( winnipeg , manitoba ) on august 7 , 2013 , with a five - day history of fever ( up to 39.5c ) and a headache .
he experienced no other meningeal or respiratory tract symptoms and there was no nausea , vomiting or diarrhea .
no rash , jaundice or icterus had been noted by his parents . his medical history consisted of multiple congenital anomalies related to a midline defect syndrome that had not been formally diagnosed .
these consisted of hydrocephalus treated with a ventriculoperitoneal shunt ; panhypopituitarism ; partially corrected tetrology of fallot and dextrocardia ; and asplenia secondary to mid - gut malrotation , which was surgically corrected at two weeks of age .
the patient had travelled with his relatives to the southeast corner of manitoba to stay at a cabin four weeks before the onset of symptoms .
he did not recall specific tick bites but had numerous mosquito bites during the 48 h he was there .
the patient had received blood transfusions for his surgeries during his first month of life , but not after . screening blood tests , including electrolyte , urea and creatinine levels ,
were all within normal limits . his white blood cell count , hemoglobin and platelet levels were also normal .
a manual slide review was performed due to abnormalities consistent with his asplenia , and a parasite believed to represent plasmodium falciparum was noted .
numerous ring - form trophozoite parasites were observed within erythrocytes , and a lack of pigment and occasional tetrads / maltese cross formations were noted ( figure 1 ) . based on these findings , and a lack of a significant travel history , identification was deemed to be consistent with babesia species .
twenty - four hours after initial evaluation , the patient was notified to return to the emergency department and the pediatric infectious diseases service was consulted . at this point ,
he was diagnosed with mild babesiosis and prescribed a six - week course of atovaquone and azithromycin .
follow - up bloodwork was performed one week into his treatment course . at that time
, the patient continued to have headache and intermittent , nonspecific abdominal pain . a mild anemia and slightly increased transaminase levels and bilirubin were noted . a blood specimen was collected and sent to the national microbiology laboratory for confirmation of babesia microti infection , and to rule out infection with b burgdorferi and/or anaplasma phagocytophilum .
while polymerase chain reaction ( pcr ) was negative for the latter two organisms , real - time pcr was performed using primers that target the chaperonin - containing t - complex eta ( cct ) ( 1 ) and subsequently confirmed using a second real - time pcr assay targeting the 18s ribosomal rna gene ( in - house / applied biosystems , usa ) . to generate sequence data ,
nested pcr was performed using primers for the 18s ribosomal rna gene ( 2 ) and the sequence of this product was compared with those in genbank ( national center for biotechnology information , www.ncbi.nlm.nih.gov/genbank/ ) , confirming the diagnosis .
the patient was medically reassessed every two weeks for the six weeks of his therapy .
the medication was well tolerated , with only mild gastrointestinal intolerance . aside from the previously mentioned mild laboratory abnormalities , the patient responded well to treatment . on final
follow - up visit at six weeks , the patient had fully recovered to baseline activity and health .
babesiosis is a rare intraerthrocytic parasitic disease in canada , caused primarily by b microti in north america and babesia divergens in europe , though cases of babesiosis have also been attributed to babesia duncani on the pacific coast of the united states .
it is primarily transmitted to humans through the bites of nymphal or adult female black - legged and western blacklegged ticks ( 3 ) , ixodes scapularis and pacificus , respectively .
these tick species also serve as the vector for b burgdorferi and a phagocytophilum ( 4 ) .
a diverse variety of mammal species can be infected and serve as reservoirs for b microti ( 5 ) , although in most localities white - footed mice , peromyscus leucopus , appear to be the most important .
states in close proximity to manitoba have reported numerous cases of babesiosis ( 68 ) .
for example , minnesota reported 41 cases of confirmed or probable babesiosis in 2012 ( 6 ) while wisconsin had 46 in 2011 ( 7 ) .
a population of blacklegged ticks , i scapularis , infected with the agents of lyme disease , b burgdorferi , and anaplasmosis , a phagocytophilum , has been known to be established at the location where our patient had stayed , since 2006 ( 4 ) . since 2010 , blacklegged ticks infected with b microti have been collected from six different localities in manitoba where populations of these ticks have recently become established .
the overall prevalence of b microti detected in blacklegged ticks collected from the established populations in manitoba was 1.8% ( six of 326 tested ) ; however , at sites where b microti was detected , the prevalence was as high as 10% .
b microti has also been detected in the tissues from field - collected rodents at two of these localities .
comparison of sequence data from the b microti dna from the ticks and rodents , as well as from the reported case , revealed at least 98% homology with those in genbank , indicating that the strains of b microti in manitoba are similar to those reported in the united states .
the detection of b microti in blacklegged ticks in multiple years and from the tissues of resident rodents suggests that this pathogen has become endemic in some localities in the province .
in addition , all blacklegged ticks submitted through the ongoing passive tick surveillance program in canada ( 9 ) have been screened for b microti infection since late 2012 and infected bird - borne ticks have been observed in ontario ( n=1 ) , although the prevalence of infection is very low .
these surveillance data support the interpretation that the risk of human exposure to b microti - infected blacklegged ticks is currently low across much of central and eastern canada ; however , this pathogen is likely becoming endemic in some of many recently established blacklegged tick populations in canada ( 10 ) including those in manitoba .
excellent descriptions of key clinical features and laboratory investigations for babesiosis have been summarized elsewhere ( 3,11,12 ) .
this represents the first confirmed case of tick - borne b microti infection acquired within canada
. imported ( 13 ) and transfusion - acquired ( 14 ) b microti infections have been previously described .
locally acquired human babesiosis in canada has important implications for clinical , laboratory and transfusion medicine as well as public health .
clinically , it should be considered in individuals at highest risk , including those at the extremes of age ( < 1 year of age and > 50 years of age ) , the immunocompromised ( congenital , hiv - infected , organ / blood and marrow transplant or immunosuppressive / chemotherapeutic medications ) , and in asplenic individuals in particular .
persistent fever without diagnosis , flu - like illness during spring and summer months ( outside of influenza season ) , and an appropriate travel history , history of exposure to blacklegged ticks and history of blood transfusion in the preceding six months are key features that should prompt clinicians to consider babesiosis .
laboratory practitioners should suspect b microti when pleomorphic and predominantly intraerythrocytic parasites with hyperparasitized erythrocytes are noted . it is further distinguished from p falciparum by the lack of pigment or gametocytes
. intraerthrocytic or maltese cross tetrads are highly suggestive of babesia species , particular when other clinical information is inconsistent with p falciparum . microscopic diagnosis of b microti should be supplemented with pcr - based confirmation by reference laboratories capable of full characterization .
clinicians are encouraged to provide appropriate travel and blacklegged tick exposure history on laboratory requisitions to assist the laboratory in prioritization of appropriate testing .
b microti seroprevalence studies and screening of i scapularis populations in canada are critical tools to identify emerging areas of endemicity and keep practitioners informed of the risks patients may face . currently , babesiosis is not a nationally notifiable disease but it is reportable in manitoba . since january 2011
, babesiosis has been a nationally reportable disease in the united states . in our opinion ,
given its emerging nature and potential to infect the canadian blood supply , babesiosis is a good candidate to be made nationally reportable in canada .
the present report is the first published case of a patient with tick - borne babesiosis acquired within canada .
fortunately , infection with b microti is readily detected by routine microscopy supplemented by appropriate clinical history , and can be confirmed by pcr . although our patient fared well with standard treatment of atovaquone and azithromycin , it is important to consider this infection , particularly in individuals at higher risk for severe disease , because babesiosis can be fatal .
human infection remains a rare event but is likely to increase as the range of blacklegged ticks expands in central and eastern canada .
it is prudent to add babesiosis to the nationally notifiable disease list in canada to further track this emerging infection .
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infection with babesia microti can cause severe illness , particularly among asplenic individuals . blacklegged and western blacklegged ticks ( ixodes scapularis and ixodes pacificus , respectively ) are the vector through which b microti is transmitted .
the distribution of these ticks in canada has increased over the past several years . in this article , the authors present the first case of babesiosis in canada that was not diagnosed following travel to an area in which the disease is endemic .
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the introduction of highly active antiretroviral therapy ( haart ) has not only improved longevity in hiv - infected individuals but in addition has had a significant impact on the rate of mother - to - child transmission of hiv infection ( mtct ) .
vaginal delivery contributes 6070% of mtct , breastfeeding contributes 2030% , while in - utero infection could occur in less than 10% of mtct . without preventive intervention , about 2540% of infants born to hiv - positive mothers will contract the virus .
following introduction of haart , the rates of mother - to - child transmission of hiv infection has practically crashed to less than 2% [ 3 , 4 ] .
however , the success of haart , like any medication , is dependent on both the intrinsic properties of the drugs and the individual 's ability to take the medication as prescribed .
this is particularly true in the prevention of mother - to - child transmission , where the consequence of failing to achieve viral suppression is the transmission of the virus to the baby .
adequate adherence to the prescribed antiretroviral medications is essential to achieving maximal viral suppression necessary to prevent mtct .
adherence rates exceeding 95% are necessary , in order to maximize the benefits of antiretroviral therapy .
higher levels of drug adherence are associated with improved virological , immunological , and clinical outcome .
poor adherence to antiretroviral drugs during pregnancy can lead to suboptimal viral suppression , development of viral resistance , higher risk of mother - to - child transmission , and mother - to - child transmission of resistant hiv strains . interrupting medication permits the virus to resume rapid replication and as many as 10 viral particles
this allows resistant mutant strains to be generated which are no longer responsive to available antiretroviral drugs , posing a public health danger .
adherence to antiretroviral drugs poses unique challenges to hiv infected persons particularly in pregnant women .
several methods have been used to measure adherence , but no gold standard has been established .
available methods include pill counts , self - report , prescription refills , medication event monitoring system ( mems ) , biological markers , and assays [ 4 , 9 ] .
though several studies in nigeria have evaluated the factors associated with nonadherence to antiretroviral therapy among hiv - positive adults [ 1012 ] , only one from literature search studied antiretroviral adherence issues in hiv - positive pregnant women .
they deduced from their study that the determinants of nonadherence to antiretroviral drugs in hiv - positive pregnant women were low level of education , nondisclosure of hiv status , and longer duration of therapy .
this study , therefore , seeks to find out if there is good adherence to antiretroviral drugs among hiv - positive pregnant women .
the main objective of this study is to determine the level and factors that influence adherence to antiretroviral drugs among hiv - positive pregnant women accessing pmtct services in lagos nigeria .
research questions
what is the level of adherence to antiretroviral drugs among pregnant women?what are the barriers to adherence in pregnancy?what factors facilitate adherence among pregnant women ?
what is the level of adherence to antiretroviral drugs among pregnant women?what are the barriers to adherence in pregnancy?what factors facilitate adherence among pregnant women ?
what are the barriers to adherence in pregnancy ? what factors facilitate adherence among pregnant women ?
the study was conducted at the hiv treatment centre of the nigerian institute of medical research ( nimr ) , lagos , a federal government of nigeria comprehensive hiv care and treatment centre .
nimr is supported by aids prevention initiative in nigeria ( apin ) through the united states president 's emergency fund for aids relief ( pepfar ) , funding since 2004 .
the centre provides an outpatient prevention of mother - to - child - transmission of hiv infection ( pmtct ) services , in addition to adult and pediatric hiv services .
pregnant women who tested positive to hiv are referred from the nimr hiv counseling and testing centre or other public and private health institutions to the pmtct clinic which takes place once a week , on wednesdays .
intrapartum care is provided for these women in collaboration with some hospitals within lagos metropolis which include lagos university teaching hospital idi araba , lagos state university teaching hospital ikeja , general hospital surulere , general hospital apapa , general hospital ikorodu , havana specialist hospital surulere , and rao specialist hospital surulere .
health workers from the collaborating centres have been trained on intrapartum care of hiv - positive mothers both by nimr and the national hiv programme . each woman on the pmtct programme
is referred to any of the collaborating centres , close to their place of residence at 36 weeks or as soon as possible for late booking .
the referral note usually contains detailed information about their chosen mode of delivery , infant feeding choice , client 's viral load , and cd4 count .
infant postexposure prophylaxis drugs and mother 's antiretroviral drugs are given to the women on referral .
the women are sent back to the centre at 2 weeks postdelivery with a completed case record form ( crf ) designed specifically to capture all delivery - related information .
the home - based care team contacts any of the women who did not report back 2 weeks after the expected date of delivery .
a semistructured questionnaire was administered to the women who met the inclusion criteria and gave consent to participate in the study .
adherence was measured by expressing the number of doses taken as a percentage of the number of doses prescribed .
for example if 20 doses are prescribed and 19 doses are taken , adherence is 95% .
all pregnant women with known gestational age either by date or early ultrasound seen during the study period and who gave informed consent were enrolled . at the time of the conduct of the study ,
the study aimed for a precision of 5% for a proportion of 50% using 95% confidence interval , therefore , a sample size of 70 subjects was required . in order to increase the power of the study
, we decided to enroll all pregnant hiv - positive women who attended pmtct clinic between 1st september and 31st november 2009 provided the minimum sample size of 70 is met .
the enrolled women were interviewed using a semistructured questionnaire containing closed and open - ended questions .
the open - ended questions were included to give the women opportunity to freely express themselves without boxing them into closed answers .
information on demographics , socioeconomic characteristics , knowledge of hiv , and antiretroviral drug medication , adherence pattern , reasons for missing drugs , and factors that encourage adherence were also contained in the questionnaire .
the p value was based on 95% confidence intervals ( ci ) ; a p value > 0.05 was not significant ( ns ) .
descriptive analyses were first performed followed by bivariate analyses of the determinant factors associated with good adherence .
the variables that were found significant at this level were added to a multivariate logistic regression model and those with a p - value < 0.05 were considered significant in the final multivariate model , calculating odds ratios ( or ) and 95% confidence interval ( ci ) .
ethical approval for the study was obtained from the institutional review board , nigerian institute of medical research , lagos , nigeria .
written informed consent was obtained from all women for the use of their data for study however , women who declined consent to participate in study were provided care but excluded from research .
the clinic patients are organized into an independent support group of people living with hiv ( positive life organization of nigeria ) that ensures that patients are not stigmatized and discriminated against .
this group ensures that no patient is denied requisite care because of failure to participate in any of our studies including this study .
during the three months study period , one hundred and seventy eligible hiv - positive pregnant women consented and were interviewed .
one hundred and thirty seven ( 80.6% ) of the interviewed women reported achieving adherence level of greater or equal to 95% using 3 day recall method , with a nonadherent rate of 19.4% .
majority of the women were aged 3034 years ( 52.4% ) , married ( 77.1% ) , have at least one living child ( 70.0% ) , employed / working ( 76.5% ) , had at least secondary education ( 81.1% ) , and from the three major ethnic tribes of yoruba , igbo , and hausa ( 84.1% ) .
one hundred and forty seven ( 86.5% ) women had disclosed their hiv status and their disclosure was to their partners in most of the cases ( 97.3% ) .
the majority of the women were on first line haart regimen ( 58.2% ) , while the rest of the women were either on non - haart prophylactic regimen ( 24.7% ) or 2nd line haart regimen ( 17.1% ) .
only 20.0% and 22.9% of the women have had previous pmtct experience and were within the first trimester , respectively .
their knowledge of hiv and antiretroviral therapy were very good as over 85% of the respondent had very good knowledge .
the use of treatment support was relatively common as greater than half of the women had a treatment supporter ( 55.9% ) who is the husband in 89.5% of the cases .
the outcome of subanalysis of reasons given for adhering to the antiretroviral drugs as prescribed , among the women that had adherence level greater 95% , is shown in table 3 .
the desire to ensure that the unborn child is protected from hiv infection was the greatest motivation ( 51.8% ) .
some of the written expression of the women , expressing the desire to protect the child as the motivation for adherence includes
i do not want to live with the guilt of infecting my baby , and i learnt my baby will not be infected if i take my drugs religiously .
the desire to remain healthy and alive was the other motivator for adhering to the arv drugs ( 21.2% ) .
i want to live long train my children and fulfill my life dreams , to keep me healthy , as i was always on admission in the hospital before i started treatment .
the reasons given by the nonadherent respondents for missing or skipping their drugs are shown in table 4 .
forgetfulness ( 57.6% ) , tight work schedule ( 39.4% ) , and fear of being identified as hiv positive ( 63.6% ) were the common reasons for skipping or missing drugs among the nonadherent women .
i get stuck in traffic sometimes on my way home from work and am not comfortable carrying my drugs around or taking them in the public .
table 5 shows the bivariate analysis of some possible factors associated with good drug adherence .
of the nine variables , only four variables which are marital status ( p = 0.023 ) , disclosure of hiv status ( p = 0.000 ) , good knowledge of hiv , and art ( p = 0.001 and having a treatment supporter ( 0.002 ) were found to be significantly associated with good adherence .
however , after subjecting the variables found to be significantly associated with good adherence to multiple logistic regressions while controlling for other potential confounders , only hiv status disclosure ( odds ratios : 6.1 ; ci : 2.811.6 ) and having a treatment partner ( odds ratios : 2.5 ; ci : 1.36.7 ) retained their association with good adherence .
effective strategies to reduce mother - to - child transmission of hiv infection are well known and well established [ 1 , 3 ] .
these include the use of arv drugs , avoiding unplanned and unwanted pregnancy in hiv - positive women , safe delivery and infant feeding options , reduction of unwarranted and unnecessary surgical intervention during pregnancy and labor , prevention of prolonged rupture of membrane , and so forth [ 1 , 3 ] .
research has also shown that at undetectable viral load , it is possible to achieve zero mother to child transmission of hiv infection .
antiretroviral drugs can only achieve the required effect at adherence level of at least 95% .
poor adherence to antiretroviral drugs has been reported to be the major challenge to achieving the goal of antiretroviral therapy .
many factors have been cited as reasons for nonadherence in hiv - positive adults [ 1012 , 1518 ] , but relatively few adherence studies have been done in hiv positive pregnant women [ 4 , 19 ] . apart from factors preventing adherence in nonpregnant adults ,
the nausea and vomiting of pregnancy and possible effect of the drugs on the fetus are additional factors that makes adherence in pregnancy a challenge .
we conducted this study not only to provide this scarce information on adherence in pregnancy but also to generate information that will assist during adherence counseling for pregnant hiv positive women accessing pmtct services .
the nonadherence rate of 19.4% among our cohort , though comparable to 21.7% reported by igwegbe et al . in south eastern nigeria is much lower than 37.1% and 37.4% reported by olowookere et al . and shaahu et al .
, respectively , from southwestern nigeria where present study was conducted . considering that our study and that of igwegbe et al .
was among pregnant women unlike the two studies by olowookere et al . and shaahu et al .
it is not surprising that women are willing and ready to do anything , to ensure the wellbeing of their offspring .
the above statement was confirmed in this study in which 51.8% of the adherent women gave the reason of protecting their unborn child from hiv infection as the major motivator for taking their drug as prescribed .
the effect of vomiting and nausea of pregnancy was not noticed in this study , as majority of the women are referred patients after hiv diagnosis . at the time , these women present to nimr hiv treatment centre , the nausea and vomiting of pregnancy would have subsided .
the free services offered at nimr , compared to other centres , may have played a great role , as cost of drugs and laboratory services have been shown to be a major barrier to antiretroviral drug adherence [ 18 , 20 ] .
the adherence rate of 99% reported by jones and barthlomew among 194 pregnant women where services were completely free gives credence to this assertion .
the adherence level of 99% reported above also showed that we need to do a lot of work on issues of adherence .
presently , all patients are counseled prior to the initiation of antiretroviral therapy , but there is need to reevaluate counseling methods and techniques where necessary , aiming to achieve the 99% adherence level reported by jones and barthlomew .
encouraging the women to come along with a treatment supporter for the counseling sessions prior to initiation of antiretroviral therapy preferably the partners would help in educating the partners appropriately and improving adherence in the long run .
apart from reviewing the counseling techniques , the knowledge of the adherence counselors also needs to be evaluated as their beliefs and attitudes are central to effective counseling . among the nonadherent women , similar reasons reported in other studies for missing drugs were found [ 4 , 10 , 11 , 18 ] .
it , therefore , shows that pregnancy related factors are not the reasons for missing antiretroviral drugs during pregnancy , but as a result of other personal and sociocultural factors .
stigma and discrimination remain an important factor militating against quality hiv care , as 63.6% of women in this study expressed the reason for missing their drugs as afraid of being identified as hiv positive .
there is , therefore , the need for continued campaign against stigma and discrimination if we must improve adherence to antiretroviral drugs and uptake of other hiv - related services .
the findings of hiv status disclosure and having a treatment support as factors associated with good adherence after controlling for potential confounders is in agreement with previous studies . with the disclosure of hiv status to partners , who in most cases is the husband
, he will not only provide support but will act as treatment partner for the spouse .
we , therefore , need to encourage these women , to disclose their status to get the maximal benefit of disclosure .
it is important , however , to note that women should not be forced to disclose their status , as hiv status disclosure has been reported to be accompanied by partner violence . instead women who decline to disclose should be counseled and encouraged until they feel safe to disclose .
it is important to state that self - report used to measure adherence is not the gold standard for adherence measurement , but it has been reported to be sufficient enough to assess adherence when pill count is not possible and electronic devices and blood arv blood measurement are not feasible .
in conclusion , this study has shown that good adherence is achievable during pregnancy , however , more has to be done to achieve adherence rate of 99% reported elsewhere . the identified reasons for nonadherence and the effects of status disclosure and having a treatment partner will be used to improve adherence counseling process .
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background .
the need for a high level of adherence to antiretroviral drugs has remained a major hurdle to achieving maximal benefit from its use in pregnancy .
this study was designed to determine the level of adherence and identify factors that influence adherence during pregnancy
. method .
this is a cross - sectional study utilizing a semistructured questionnaire .
bivariate and multiple logistic regression models were used to determine factors independently associated with good drug adherence during pregnancy .
result . 137 ( 80.6% ) of the interviewed 170 women achieved adherence level of 95% using 3 day recall .
the desire to protect the unborn child was the greatest motivation ( 51.8% ) for good adherence . fear of being identified as hiv positive ( 63.6% ) was the most common reason for nonadherence .
marital status , disclosure of hiv status , good knowledge of art , and having a treatment supporter were found to be significantly associated with good adherence at bivariate analysis .
however , after controlling for confounders , only hiv status disclosure and having a treatment partner retained their association with good adherence .
conclusion .
disclosure of hiv status and having treatment support are associated with good adherence . maternal desire to protect the child was the greatest motivator for adherence .
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the incidence of obstetric brachial plexus injury ( obpi ) is about 1.51 per 1000 live births in the united states and reports vary from 0.38 to 5.8 per 1000 live births .
many of these injuries are transient ; however , most of the obpi patients never recover full function and develop permanent injuries [ 2 , 4 , 5 ] . in reports conducted by pediatricians and specialists , with follow - up times greater than 3 years , the reported proportion of injuries that remain permanent varies from 50 to 90% [ 68 ] . risk factors for injury include shoulder dystocia , macrosomia ( defined as birth weight greater than 4500 g [ 912 ] ) instrument - assisted delivery , and downward traction of the fetal head [ 1 , 7 , 8 ] . yet in a database search of over 11 million births , it was found that most children with neonatal brachial plexus palsy did not have known risk factors . in obstetrics , presentation of shoulder dystocia is often emergent because the reported risk factors for its occurrence are not good predictors of it [ 13 , 14 ] .
therefore we seek to examine the most prevalent risk factors found in a population of patients with permanent obpi that necessitated surgical treatment to attempt to identify better predictors of injury and to elucidate the pathophysiology of obpi .
recently , studies have determined risk factors specifically for permanent brachial plexus injury including birth weight , delivery methods [ 16 , 17 ] , and maternal factors [ 16 , 17 ]
. risk factors for permanent injury may also contribute to the secondary glenohumeral and scapular deformities that result from neonatal brachial plexus injury . it has already been shown that clavicular fractures at birth are associated with bony deformities of the glenohumeral joint . instrument - assisted delivery , birth weight , shoulder dystocia , and lack of finger movement at birth have not been evaluated for their association with shoulder deformity .
patients with permanent obstetric brachial plexus injuries frequently develop bony deformities which are caused by muscle imbalance on the developing bony elements of the infant shoulder developing from the asymmetrical brachial plexus injury , with the upper plexus ( c5 - 6 ) being injured more commonly than the lower ( c8-t1 ) . these secondary deformities , including internal rotator and adductor contractures , glenohumeral dysplasia , humeral head posterior subluxation or dislocation , and/or scapular elevation and rotation , cause major long - term morbidity requiring surgical correction to improve limb function . in studies that quantify obstetric brachial plexus deformities , the most common measurements are posterior subluxation of the humeral head , glenoid retroversion , scapular deformity ( shear ) , and glenoid shape from ct or mri images [ 2023 ] .
this study will explore potential risk factors of permanent brachial plexus injury and determine if these risk factors can predict the development of osseous deformities .
during a 19-month period between august 2006 and april 2008 , 249 patients from various locations in the united states , asia , and europe were surgically treated for the sequelae of permanent obstetric brachial plexus injury at our institute .
241 patients were selected for this study ; of the 8 patients excluded , 4 patients were excluded due to insufficient data , and 4 patients sustained their injury during caesarian section or breech delivery .
all the patients in this study were injured severely enough to develop shoulder deformities that required surgical reconstruction .
all of the patients in the study have had at least one reconstructive surgery related to the initial nerve injury .
all surgeries were performed by the same surgeon ( rkn ) , whose practice has focused on reconstructive surgery in this population for the past 12 years . there were 122 ( 51% ) girls and 119 ( 49% ) boys with an average age of 5.6 years at the time of visit , ranging from 5 months to 27 years .
information regarding instrument use during delivery , birth weight , and shoulder dystocia was obtained from the patient 's parent or guardian during the initial evaluation .
presence of deformity was confirmed with analysis of ct and/or mri scans obtained prior to surgical treatment .
posterior subluxation ( measured as phha , percent humeral head anterior to the scapular line , see figure 1 ) , glenoid version ( angle difference between the glenoid and a line 90 to scapular line ) , and shear ( scapular hypoplasia , elevation and rotation measured as percent scapula superior to the clavicle , see figure 2 ) were measured from either ct or mri scans as previously described [ 20 , 21 ] .
measurements of phha and version were not available for 49 patients and shear was not available for 89 patients . increasingly severe deformity is associated with phha values decreasing from 50% , version values decreasing from 0 , and shear values increasing from 0% .
glenoids were divided into 2 groups based on the presence ( more deformed ) or absence ( less deformed ) of a pseudoglenoid in ct or mri images .
a pseudoglenoid is present when the glenoid is biconcave or shows a posterior concavity concentric with the humeral head in axial images .
therefore , data including the transiently injured population was gathered from published studies for comparison .
national birth statistics were obtained from the centers for disease control and prevention website for the year 2003 .
macrosomia was defined as > 4,500 g [ 17 , 25 , 26 ] and appropriate for gestational age as < 3,750 g .
averages are reported as the mean one standard deviation and proportions are provided with 95% confidence intervals determined by the modified wald method .
the proportion of injuries that remain permanent was pooled from multiple studies into two groups , studies conducted by obstetricians ( n = 5 ) and studies conducted by pediatricians and specialists ( n = 5 ) .
our data showed that the average birth weight was 4085 476 g ( min 3005 g , max 5812 g ) and birth weight was normally distributed ( p = 0.73 ) .
macrosomia was present in 20% ( 1626% , 49/241 ) of the patients ; 55% ( 4861% , 132/241 ) weighed between 3750 and 4499 g at birth ; 25% ( 2031% , 60/241 ) weighed less than 3750 g at birth ( see table 1 ) . in macrosomic patients ,
glenoid version was significantly worse ( p = 0.001 ) and posterior subluxation was not significantly different ( p = 0.06 ) , but lower when compared to patients below 4500 g ( table 2 ) .
the severity of the shear deformity as determined by scapular elevation did not differ between these two groups ( table 2 ) .
the rate of documented shoulder dystocia among children with permanent brachial plexus injury was 97% ( 9398% , table 1 ) .
birth weight was significantly higher in patients with documented shoulder dystocia ( 4100 474 g ) than those without documented shoulder dystocia ( 3634 307 g ; p = 0.005 ) .
posterior subluxation and glenoid version were both worse in children with documented shoulder dystocia , but the difference was not significant because of the small number of patients in the group without documented shoulder dystocia ( n = 8 , table 2 ) .
instruments ( forceps , vacuum , or both ) were used in 41% ( 3547% ) of the deliveries studied ( table 1 ) .
the average birth weight was indistinguishable for all instrument delivery categories ( vacuum : 4013 443 g , forceps : 3997 424 g , both instruments : 4124 661 g , no instruments : 4125 467 g ) .
the mean birth weight in instrument - assisted deliveries ( 4032 489 g ) was not significantly different from spontaneous deliveries ( 4125 467 g , p = 0.11 ) .
the rate of instrument use decreased in higher birth weight categories ( table 1 ) .
the shear deformity was significantly worse ( p = 0.04 ) in all instrument - assisted deliveries as compared to spontaneous deliveries .
the significance was lost when forceps or vacuum extraction used alone was compared separately to spontaneous delivery ( p = 0.17 , 0.30 , resp . ) but was maintained when both instruments were used for the same delivery ( p = 0.03 ) .
phha and version did not differ between instrument - assisted and spontaneous deliveries ( p = 0.45 , 0.79 , resp .
no finger movement at birth was observed in 68% ( 6173% ) of the patients ( table 1 ) .
mean birth weight among patients with no finger movement was 4089 477 g ; mean birth weight among those with finger movement was 4075 478 g ( p = 0.88 ) . lack of finger movement at birth was not associated with macrosomia , documented shoulder dystocia , or instrument use ( p = 0.82 , 0.47 , and 0.91 , resp .
phha and version measurements in children with no finger movement at birth indicated a less severe shoulder deformity in comparison with those with finger movement at birth ( p = 0.046 , 0.001 , resp .
the glenoid fossa was also less deformed ; a higher proportion of less severely deformed glenoids ( non - pseudoglenoid ) was observed in patients without finger movement ( 81% , 7387% ) than in those with finger movement ( 67% , 5577% ) , although this difference was not significant ( p = 0.055 ) .
risk factors for sustaining an obstetric brachial plexus injury have been previously identified ; however , the most pertinent risk factors , those associated specifically with permanent injury , have only recently been studied [ 3 , 1517 , 27 ] .
the proportion of injuries that remain permanent was thought to be less than 10% ; however , a review by pondaag , et al . concluded that the most commonly reported recovery rates were based upon inadequate study methodologies . in studies focusing on temporary versus permanent obstetric brachial plexus injury ,
the criteria for permanent injury are often not fully described , the follow - up periods are inadequate , or the end - stage evaluation is not performed by a brachial plexus specialists .
indeed , the proportion of injuries that remain permanent is significantly lower among studies conducted by obstetricians ( 13% , 1017% , 55/419 ) [ 3 , 16 , 17 , 27 , 29 ] than pediatricians and orthopedic surgeons ( 51% , 4358% , 86/170 ) [ 68 , 30 , 31 ] ( p < 0.0001 ) . for these reasons , data gathered from permanent injury in this study were contrasted with findings common to all brachial plexus palsies rather than data specific for transient palsies .
birth weight , shoulder dystocia , instrument use , and finger movement at birth were evaluated as potential risk factors for permanent injury and predictors of future osseous shoulder deformity .
the average permanent injury birth weight in this study was 4085 476 g , which is lower than the average birth weight reported in the literature for brachial plexus injuries at 4265 480 g , 4227 g , 4205 608 g , and 4500 625 .
additionally , the rate of macrosomia in this study was 20% ( 1626% ) which is less than a previously reported rate for brachial plexus injuries at 29% ( 2038% ) . among patients with documented shoulder dystocia in this study ,
the rate of macrosomia was 21% ( 1627% , 49/233 ) ; lower than previous findings of gherman et al .
, at 26% and 28% , for shoulder dystocia - associated brachial plexus injury [ 15 , 34 ] .
thus , high birth weight is a risk factor for shoulder dystocia [ 35 , 36 ] and brachial plexus injury in general [ 1 , 37 ] , but it is an unreliable predictor of permanent injury .
this was further demonstrated by comparing the birth weight distributions of permanent brachial plexus injuries with all brachial plexus injuries ( figure 1 ) .
it is not implied that birth weights in permanent injury are similar to the normal population ; birth weight among injured patients is higher than average , as demonstrated in figure 3 .
glenoid retroversion was significantly more severe in macrosomic patients , which suggests that although macrosomia can not reliably predict permanent injury , macrosomia is associated with the development of a more severe glenohumeral deformity .
posterior subluxation was also more severe in macrosomic patients , although not significantly ( table 2 ) .
permanent injury is not exclusive to large infants ; 80% ( 7484% ) of the patients in this study were not macrosomic and 43% ( 104/241 , 3749% ) weighed less than 4000 g at birth ( table 1 ) .
52% ( 126/241 , 4658% ) of the patients were below the estimated 90th percentile for birth weight at 40-week gestation and 11% ( 26/241 , 715% ) were below the 50th percentile .
our findings indicate that nonmacrosomic fetuses frequently experience shoulder dystocia and develop permanent obpi , despite the fact that macrosomia is said to be one of the primary indicators of permanent obpi .
the average birth weight in this study was indistinguishable from the average birth weight reported for brachial plexus injuries .
higher birth weight does not , therefore , affect the prognosis of brachial plexus injury .
infants with birth weights normal for gestational age are still susceptible to severe permanent brachial plexus injuries .
it is important that delivering caregivers consider that nonmacrosomic babies may sustain injury to the brachial plexus . during the process of delivery
, maneuvers causing the twisting and extension of the fetal head , which result in the stretching of the fetal neck , may be responsible for obpi .
report evidence , based on computational modeling of intrauterine forces , of increased brachial plexus stretch caused by lithotomy positioning during delivery , while acute flexion of the hips in the supine position ( mcrobert 's maneuver ) resulted in 53% less brachial plexus stretch .
since the brachial plexus is the most complex peripheral neural unit , every effort needs to be taken to ensure that the brachial plexus is not injured during delivery . in our study
the rate of instrument use was 41% ( 3548% ) , which is 4-fold higher than the 10% predicted for all vaginal deliveries in the united states [ 25 , 43 ] ( table 1 ) .
instrument use in our permanently injured patient group is in agreement with rates previously reported for all brachial plexus injuries in chauhan et al .
instrument use is therefore equally represented in both temporary and permanent brachial plexus injuries . as shown in table 1 ,
the rate of instrument use consistently decreased with increasing birth weight from 47% ( < 3750 g ; 35%60% ) to 33% ( > 4500 g ; 21%41% ) .
, is contrary to a report published in the american college of obstetricians and gynecologists technical bulletin that instrument use is higher in the delivery of macrosomic fetuses [ 25 , 44 ] . therefore , we found forceps / vacuum delivery to be an independent risk factor for obpi , regardless of birth weight .
this finding points to the fact that higher birth weight by itself does not cause obpi , while it may be associated with certain conditions that cause obpi .
this finding also supports the high correlation with forceps delivery and obpi reported by foad et al . .
our data shows that the shear deformity was significantly more severe in instrument - assisted deliveries , especially when both instruments were used .
however , posterior subluxation and glenoid retroversion were not significantly different between spontaneous and instrument - assisted deliveries .
this result suggests a mechanism of injury or presence of a risk factor that uniquely affects the development of a shear deformity and is associated with the sequential use of instruments .
it has been proposed that the sequential use of instruments is associated with severe shoulder dystocia and brachial plexus injury .
it is , however , unlikely that an increase in shoulder dystocia with sequential instrument use can explain these findings because the shear deformity is not more severe in patients with documented shoulder dystocia .
doumouchtsis and arulkumaran found a strong association between downward traction of the fetal head and obpi ; even without shoulder dystocia they stated that substantial forces were found to be used in many obpi cases .
as traction forces increase , exogenously applied lateral flexion during delivery places force on the brachial plexus .
these reports are consistent with our findings , which show that a significant number of our patients suffering from obpi underwent an instrumented delivery .
nerves in the brachial plexus that are associated with scapular elevation may be considerably more susceptible to injury during instrument assisted deliveries .
it is well established that shoulder dystocia is a significant risk factor for brachial plexus injuries .
the shoulder dystocia rate among all brachial plexus injuries in the united states , though found to be the most significant risk factor , was reported at a significantly lower rate ( 18% ) than in this study ( 97% ) .
our results are similar to a smaller series of permanent obpp patients which showed a shoulder dystocia rate of 94% .
it should be noted that there is no universal definition of shoulder dystocia , hence the variable incidence noted in literature .
true shoulder dystocia was coined by gross et al . as deliveries requiring , in addition to downward traction and episiotomy , maneuvers to deliver the shoulders .
shoulder dystocia was the most prevalent risk factor in our patients ; almost all the children in this study had documented shoulder dystocia and the rate in macrosomic patients was 100% .
shoulder dystocia is , therefore , closely associated with the most severe cases of permanent obstetric brachial plexus injuries .
lack of finger movement indicates a severe initial injury that extends to c8/t1 and affects the entire brachial plexus .
all the muscles in the shoulder are weakened and muscle imbalances are greatly reduced in these injuries compared to c5/c6 injuries that retain finger movement .
severe bony deformities caused by muscle imbalances during a time of rapid growth should thus develop more often in children with finger movement at birth .
posterior subluxation and glenoid retroversion were both significantly less severe in patients with no finger movement at birth .
moreover , the proportion of patients who had no finger movement at birth was higher in non - pseudoglenoid shoulders ( 68% ) than in the pseudoglenoid shoulders ( 51% ) . taken together , the results show that lack of finger movement at birth is actually protective against bony deformities of the shoulder , although the patient suffers extensive functional impairment .
permanently injured patients with finger movement at birth develop more severe bony deformities of the shoulder than patients without finger movement at birth due , in part , to asymmetrical muscle action on developing bony structures .
the strengths of this study are a large sample size ( n = 241 ) and a rigorous definition of permanent brachial plexus injury .
risk factors associated with permanent brachial plexus injury should be magnified among patients requiring surgical reconstruction .
this study is unique in that it examines the relationship between risk factors at birth and future deformities , as measured by radiographic characteristics .
it also determined the value of finger movement at birth , a simple and rapid clinical test , as a predictor of outcome .
the birth data in this study is , however , based on retrospective information which was obtained from interviews with patient families .
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purpose . to examine the most prevalent risk factors found in patients with permanent obstetric brachial plexus injury ( obpi ) to identify better predictors of injury
. methods .
a population - based study was performed on 241 obpi patients who underwent surgical treatment at the texas nerve and paralysis institute .
results .
shoulder dystocia ( 97% ) was the most prevalent risk factor .
we found that 80% of the patients in this study were not macrosomic , and 43% weighed less than 4000 g at birth .
the rate of instrument use was 41% , which is 4-fold higher than the 10% predicted for all vaginal deliveries in the united states .
posterior subluxation and glenoid version measurements in children with no finger movement at birth indicated a less severe shoulder deformity in comparison with those with finger movement .
conclusions .
the average birth weight in this study was indistinguishable from the average birth weight reported for all brachial plexus injuries .
higher birth weight does not , therefore , affect the prognosis of brachial plexus injury .
we found forceps / vacuum delivery to be an independent risk factor for obpi , regardless of birth weight .
permanently injured patients with finger movement at birth develop more severe bony deformities of the shoulder than patients without finger movement .
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prostate cancer is the most common solid organ malignancy in men in the united states and the second leading cause of cancer death . over the past 80 years
, radical retropubic prostatectomy ( rrp ) has been the most common form of surgical treatment for prostate cancer .
however , in the last 8 years , there has been a paradigm shift in surgically treated prostate cancer with the adaptation of robot - assisted laparoscopic prostatectomy ( ralp ) .
because of the presence of three - dimensional magnification and tools with 7 degrees of freedom that can duplicate hand movements with high accuracy , ralp has gained notable popularity in both the united states and europe ; furthermore , it is estimated that more than 75% of radical prostatectomies are performed by use of the da vinci robotic platform ( intuitive surgical inc . , sunnyvale , ca , usa ) .
the feasibility , safety , and early functional efficacy of ralp have been well documented . a recent population - based analysis comparing ralp and rrp procedures
undertaken between 2003 and 2005 concluded that men undergoing ralp experienced significantly fewer 30-day complications , blood transfusions , and anastomotic strictures and shorter lengths of stay . in another study ,
other studies have also consistently shown that blood loss and length of hospital stay are less in patients who undergo ralp than in those who undergo rrp
. however , there is lack of uniformity in documenting and reporting complications that may result in incomplete capture of data and may make comparisons among different surgical approaches or institutional series problematic .
menon et al . compared the complications at one institution between rrp performed by one group of surgeons and ralp performed by a different group of surgeons .
although the study was prospective and covered the same period , inter - surgeon differences in technique may have weakened the ability to objectively compare differences in complications . moreover , most studies did not use a formal reporting system for complications or a uniform grading system . in this study
, we compared the complications of rrp with ralp performed by a single surgeon using the modified clavien system .
three hundred forty - one rrps and 524 ralps were performed by a single surgeon ( c.s.k . ) between july 2007 and august 2012 .
the conducting surgeon had substantial experience with rrp but no experience with ralp at the start of the study period ; in fact , he did not have any experience with laparoscopic radical prostatectomy .
in korea , the cost to the hospital for ralp is much greater than that for rrp because rrp is covered by national insurance , whereas ralp is not .
therefore , the choice of surgical method was made on the basis of counseling with patients , considering the severity and extent of their disease and their characteristics .
the preoperative workup for both groups consisted of serum prostate - specific antigen measurement , 10- to 12-core biopsies for cancer detection , magnetic resonance imaging , and a bone scan .
prostate volume and clinical stage were evaluated by transrectal ultrasonography and digital rectal examination , respectively .
comorbidities ( e.g. , diabetes mellitus , hypertension , chronic obstructive pulmonary disease , and cardiovascular disease ) and social history were assessed .
tumors were graded histologically according to the gleason grading system , and the pathological stage was defined according to the tnm staging classification . on the fifth to sixth postoperative day , cystography with anteroposterior and lateral views was performed to evaluate the anastomotic tightness .
if there was no leak as evidenced by cystography , the urethral catheter was removed .
briefly , a lower midline abdominal incision was made , entering the space of retzius .
thereafter , for the cases in which a nerve - sparing procedure was attempted , the surgeon dissected the lateral aspects from the prostate allowing the neurovascular bundles to retract laterally .
the attachments between the bladder and the prostate were then divided and the surgical specimen was removed .
the vesicourethral anastomosis with six separate sutures was typically performed over a 20-fr urethral catheter . in ralp
, pneumoperitoneum was established by using a veress needle and five trocars were then inserted .
the prostate was dissected with an antegrade approach , beginning with a bladder neck - sparing procedure . for all potent patients ,
a continuous suture was performed during the creation of the vesicourethral anastomosis with two 3 - 0 monocryl sutures tied together , as described by van velthoven .
medical and surgical complications were retrospectively collected for all patients through a combination of institutional electronic medical records , including operative and nursing notes , discharge summaries , outpatient and emergency room visits , and written correspondence with patients regarding complications .
any complications that occurred within 90 days of surgery were analyzed and categorized as either medical or surgical .
this system determines the severity of a complication by using a scale with five grades .
grade i complications are designated as any deviation from the normal postoperative course that do not require extra therapy ( with the exception of antiemetic , antipyretic , analgesic , and antidiarrheal drugs ) .
grade ii complications necessitate pharmacologic treatment with drugs other than the drugs mentioned for the grade i complications .
grade iii complications are defined as complications necessitating surgical , endoscopic , or radiologic intervention .
this grade is subdivided into grades iiia and iiib on the basis of the need for general anesthesia .
grade iv complications are life - threatening and necessitate intensive care , often leaving the patient with residual disability . a grade v complication represents the death of a patient as the result of the complication .
the two - tailed pearson chi - square test was used to assess differences in the variables between the rrp and ralp groups .
three hundred forty - one rrps and 524 ralps were performed by a single surgeon ( c.s.k . ) between july 2007 and august 2012 .
the conducting surgeon had substantial experience with rrp but no experience with ralp at the start of the study period ; in fact , he did not have any experience with laparoscopic radical prostatectomy .
in korea , the cost to the hospital for ralp is much greater than that for rrp because rrp is covered by national insurance , whereas ralp is not .
therefore , the choice of surgical method was made on the basis of counseling with patients , considering the severity and extent of their disease and their characteristics .
the preoperative workup for both groups consisted of serum prostate - specific antigen measurement , 10- to 12-core biopsies for cancer detection , magnetic resonance imaging , and a bone scan .
prostate volume and clinical stage were evaluated by transrectal ultrasonography and digital rectal examination , respectively .
comorbidities ( e.g. , diabetes mellitus , hypertension , chronic obstructive pulmonary disease , and cardiovascular disease ) and social history were assessed .
tumors were graded histologically according to the gleason grading system , and the pathological stage was defined according to the tnm staging classification . on the fifth to sixth postoperative day , cystography with anteroposterior and lateral views was performed to evaluate the anastomotic tightness .
if there was no leak as evidenced by cystography , the urethral catheter was removed .
briefly , a lower midline abdominal incision was made , entering the space of retzius .
thereafter , for the cases in which a nerve - sparing procedure was attempted , the surgeon dissected the lateral aspects from the prostate allowing the neurovascular bundles to retract laterally .
the attachments between the bladder and the prostate were then divided and the surgical specimen was removed .
the vesicourethral anastomosis with six separate sutures was typically performed over a 20-fr urethral catheter . in ralp
, pneumoperitoneum was established by using a veress needle and five trocars were then inserted .
the prostate was dissected with an antegrade approach , beginning with a bladder neck - sparing procedure . for all potent patients ,
a continuous suture was performed during the creation of the vesicourethral anastomosis with two 3 - 0 monocryl sutures tied together , as described by van velthoven .
medical and surgical complications were retrospectively collected for all patients through a combination of institutional electronic medical records , including operative and nursing notes , discharge summaries , outpatient and emergency room visits , and written correspondence with patients regarding complications .
any complications that occurred within 90 days of surgery were analyzed and categorized as either medical or surgical .
this system determines the severity of a complication by using a scale with five grades .
grade i complications are designated as any deviation from the normal postoperative course that do not require extra therapy ( with the exception of antiemetic , antipyretic , analgesic , and antidiarrheal drugs ) .
grade ii complications necessitate pharmacologic treatment with drugs other than the drugs mentioned for the grade i complications .
grade iii complications are defined as complications necessitating surgical , endoscopic , or radiologic intervention .
this grade is subdivided into grades iiia and iiib on the basis of the need for general anesthesia .
grade iv complications are life - threatening and necessitate intensive care , often leaving the patient with residual disability . a grade v complication represents the death of a patient as the result of the complication .
the two - tailed pearson chi - square test was used to assess differences in the variables between the rrp and ralp groups .
all statistical analyses were performed by using ibm spss ver . 21.0 ( ibm co. , armonk , ny , usa ) .
the clinical stage , d'amico classification , pathological stage , pathological gleason grade , and treatment characteristics of all patients are also shown in table 1 .
the mean hospital stay ( 7.9 days vs. 10.1 days , p<0.001 ) and mean urethral catheter duration ( 6.2 days vs. 7.5 days , p<0.001 ) were significantly shorter in the ralp group than in the rrp group , respectively .
the postoperative complications for the two groups by use of the clavien classifications are shown in table 2 .
the complication rate of the ralp group was lower than that of the rrp group ( 27.3% vs. 68.0% , respectively ) . among them , there were more major complications defined as clavien grade iii or more in the rrp group ( 3.4% vs. 7.6% , respectively ) . in the ralp group ,
clavien grade iii complications included anastomotic leakage , infected lymphocele , wound dehiscence , surgical reintervention , and anal abscess .
clavien grade iii complications in the rrp group also included anastomotic leakage , wound dehiscence , and urethral dilation .
concerning clavien grade iv complications , one patient in the rrp group was taken to the intensive care unit ( icu ) after surgery because of the occurrence of stroke .
three cases of stroke and icu care owing to small bowel injury and postoperative bleeding were included in the clavien grade iv complications in the ralp group .
the incidence of medical complications ( table 3 ) was not significantly different between the two groups .
the rates of cardiac problems , pneumonia , urinary tract infections , respiratory distress , respiratory failure , acute renal failure , and cerebrovascular accidents were similar in both groups .
urinary retention was more common in the rrp group ( 7.0% vs. 2.7% , respectively ; p=0.002 ) .
the need for perioperative blood transfusion was also significantly higher in the rrp group ( 42.2% vs. 6.3% , respectively ; p<0.001 ) .
conversely , wound repairs ( 4.1% vs. 0.2% , p<0.001 ) and extravasation of contrast medium at cystography ( 10.0% vs. 2.1% , p<0.001 ) were more common in the rrp group than in the ralp group , respectively .
there was no significant difference in femoral neuropathy ( rrp , 2.1% ; ralp , 2.5% ; p=0.682 ) , postoperative ileus ( rrp , 1.8% ; ralp , 2.3% ; p=0.593 ) , reoperation ( rrp , 0.6% ; ralp , 0.4% ; p=0.664 ) , or postoperative icu care ( rrp , 0.3% ; ralp , 0.6% ; p=0.554 ) rates between the groups .
reports from several centers have demonstrated the superiority of ralp over open and laparoscopic prostatectomy with respect to surgical outcomes [ 15 - 17 ] .
compared rrp and a robotic approach ( 30 patients in each group ) and reported transfusion rates of 17% and 7% and complication rates of 6% and 6% , respectively . in a similar study , ahlering et al . , comparing the newly introduced ralp with rrp , observed satisfying outcomes in terms of complication rates ( 6.7% vs. 10% , respectively ) .
. also showed the superiority of ralp ( 105 patients ) over rrp ( 103 patients ) with respect to postoperative transfusion rates ( 1.9% vs. 14% , respectively ) .
however , in the aforementioned studies , multiple surgeons performed the rrp and ralp procedures , and the perioperative complications were not classified by use of the clavien system .
compared complications arising from rrp and ralp procedures performed by the same surgeon ; however , the study was limited by a small sample size .
we found that the incidence of clavien grade iii - iv complications was significantly lower in the ralp group ( 3.4% ) than in the rrp group ( 7.6% ) .
this finding is notable because of the large sample size , the inclusion of a single surgeon series , and the use of the clavien system for classification of the complications .
the previously noted advantages of ralp were confirmed in the present study , in which two comparative groups of patients were managed by use of an identical postoperative regimen .
the conducting surgeon had abundant experience ( 10 years ) in rrp but had no experience in ralp , or in laparoscopic radical prostatectomy , at the start of the study period .
thus , effects of the initial learning curve would be expected to be present in the ralp data .
the fact that the complication rates were lower in the ralp group despite the inclusion of a learning curve adds weight to the argument supporting this surgical method in the treatment of prostate cancer .
the rate of wound repair after rrp in this study was 4.1% , which was approximately 20 times higher than the rate in the ralp group ( 0.2% ) .
the incidences of wound repairs in the united states and europe have been reported to be 0.3% to 3.2% after rrp , whereas two multicenter studies from japan have reported a higher incidence of between 6.0% and 7.5% .
the skin incision in rrp is much longer than the short umbilical incision used in ralp for specimen retrieval and this difference might be important when trying to explain the higher incidence of wound repair in the rrp group .
although these were not culture - confirmed complications and therefore seroma could not be separated from true wound infections , the more frequent rate of wound infection in the rrp group than in the ralp group could be another possible cause of the higher wound repair rate with rrp .
we found that the incidence of extravasation of contrast medium during cystography was higher in the rrp group ( 10.0% ) than in the ralp group ( 2.1% ) .
one possible explanation for the lower extravasation rate in the ralp group is that a watertight anastomosis was more likely with the robotic technique owing to better visualization and the use of a running anastomosis .
similarly , the urinary retention rate of the rrp group ( 7.0% ) was significantly higher than that of the ralp group ( 2.7% ) .
reported that patient- or surgery - specific risk factors for urinary retention were not identified , but they suggested that acute urinary retention develops as the result of edema at the anastomosis .
we believe that the running anastomotic suture of ralp might facilitate initial voiding because earlier healing to a watertight state could lead to an earlier decrease in edema .
our suggestion is supported by a previous study showing that running anastomotic suture of laparoscopic prostatectomy allowed early catheter removal .
considering the duration of hospitalization , our values were far longer in both study arms than those reported in series from the united states , which probably reflected the different pathways of management . specifically , owing to the differences in economic health systems and cultural backgrounds , korean patients often stay in the hospital until the urinary catheter is removed , whereas patients in the united states are usually discharged quickly in the case of an uneventful early postoperative course .
comparing transfusion rates from different hospitals can be misleading , because the indication for blood transfusion may vary with clinical praxis , but the transfusion rate of the rrp group in our study was much higher than that reported in other studies .
blood transfusion rates of between 3% and 34% have been reported in rrp patient series [ 27 - 29 ] .
arai et al . reported that only 6.9% of patients who had predonated an autologous blood subsequently received an allogenic transfusion in an rrp series .
by contrast , among the patients who donated no autologous blood before rrp , 45% subsequently received an allogenic transfusion .
it is likely that the transfusion rate of our study was high because the patients did not receive autologous transfusion , granulocyte - stimulating factor , or cell saver .
moreover , in the early phase of this study , the patients tended to receive transfusion for rapid recovery if their hemoglobin was lower than 10 g / dl after surgery , even though postoperative bleeding was not suspected .
however , more recently , patients underwent blood transfusion only when their postoperative hemoglobin was lower than 8 g / dl or had a tendency to decrease .
thus , the transfusion rates of the rrp and ralp group were decreased to 18.8% and 0% in the last 50 cases .
irrespective of the change in the transfusion rate , the difference in transfusion rate between the rrp and ralp groups was significant in our study .
one explanation for the seven times lower transfusion rate in the ralp group ( 6.3% ) than in the rrp group ( 42.2% ) could be a tamponading effect resulting from the pneumoperitoneum , which is beneficial in preventing venous bleeding , the most common source of blood loss during rrp .
loss of refinement in the technique of vascular control during rrp may also have contributed to the result .
there are challenges in implementing a randomized trial because patients are usually unwilling to be randomly assigned to different treatments and may only accept robotic surgery .
however , the clinical characteristics of the patients were not significantly different between the two groups in the present study .
this could , of course , have influenced the results , but this must be considered unlikely as an explanation of the difference in complications between these two groups . as previously discussed , the lack of surgical experience with ralp compared with the significant experience with rrp must also be considered when reviewing the outcomes from this study .
furthermore , only complications that occurred within 90 days of the surgery were included in this report .
late complications such as bladder neck contractures and the need for further surgery for urinary incontinence should be considered in further studies .
the introduction of ralp at our institution resulted in a decreased number of patients with urinary retention , extravasation of contrast medium during cystography , and wound repair after surgery .
our results show that ralp is associated with a lower complication rate than is rrp , especially the rate of major complications .
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purposeto compare the complications of radical retropubic prostatectomy ( rrp ) with those of robot - assisted laparoscopic prostatectomy ( ralp ) performed by a single surgeon for the treatment of prostate cancer.materials and methodsthe postoperative complications of 341 patients who underwent rrp and 524 patients who underwent ralp for prostate cancer at the asan medical center between july 2007 and august 2012 were retrospectively reviewed and compared .
complications were classified according to the modified clavien classification system.resultsralp was associated with a shorter length of hospital stay ( mean , 7.9 days vs. 10.1 days , p<0.001 ) and duration of urethral catheterization ( 6.2 days vs. 7.5 days , p<0.001 ) than rrp .
major complications ( clavien grade iii - iv ) were less common in the ralp group than in the rrp group ( 3.4% vs. 7.6% , p=0.006 ) .
there were no significant differences in medical complications between procedures .
considering surgical complications , urinary retention ( 7.0% vs. 2.7% , p=0.002 ) and wound repair ( 4.1% vs. 0.2% , p<0.001 ) were more common after rrp than after ralp .
extravasation of contrast medium during cystography was more common in the rrp group than in the ralp group ( 10.0% vs. 2.1% , p<0.001).conclusionsralp is associated with a lower complication rate than rrp .
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the pilot protests that though the incapacity of the king had been discussed in the parliament , and a committee ( the lords sidmouth , liverpool , eldon , and others ) appointed to oversee matters of governance , and worried that when the independent character and condition of a british sovereign ( could ) thus be made subservient with impunity to any faction of the state , or to the views of any ministers or men , be they who they may be , the british constitution ( was ) not merely shaken , it ( was ) dissolved , and the reign ( was ) given to every revolutionary projector , who may seek to raise himself hereafter upon the ruins of his country , and that the situation makes the sovereign a slave of his servants .
this implies , in a sense , a belief that the king , whether sane or not , could not be deprived of his absolute power .
the threat to the state posed by this questioning was larger than the threat raised by the debate between rationality and irrationality .
the newspaper was also concerned about the restrictions placed upon the king and the machinations of the ministers and other pretenders under a weak king .
notwithstanding all the outrages upon the rights , the character , and the feelings of the regent , it ( was ) still suggested that he ought to keep those ministers in office .
they warn the prince regent that his concurrence was tantamount to high treason against himself , and compare it to the fate of ferdinand who was deposed by napoleon . despite these views , the parliamentary processes went on , and the king was divested of his executive role by the regency act ( as mentioned above ) on february 5 , 1811 .
he was , however , not deprived , of either his liberty or position , and remained the king until his death in 1820 .
the madras courier was obviously unaware of all this even by july 1811 , and clearly news did not travel as fast as it now does ! why did henry cole retain this newspaper cutting ?
just the previous year , in a letter to his sister , florence , he describes certain events recounted by mr .
his physician , concerning a rajah ( king veera rajendra of coorg ) near mysore , who had probably developed a paranoid disorder . in 1809 , about the middle of november last , the rajah having found out a formidable conspiracy that was against his life , ordered 2000 men to be killed , without any trial , and all male children to have their heads bashed on rocks . on the 16 november
, the rajah ordered a certain number to be slain ( for what reason i can not ascertain ; only that his faculties must have been deranged at the time he issued the above inhuman order ) .
the said rajah has also become very suspicious and ordered many of his concubines to be executed ( by bamboo tube being driven into the private parts of his concubine and molten lead poured in ) ; while the eunuch who was found fondling her was ordered to be buggered to death
the two accounts , preserved in the same set of documents by arthur cole , regarding events in coorg in 1809 and london in 1810 , highlight the tension between madness and a sense of political order .
the account in the madras courier emphasizes that the paramount power of the regent can not , and should not , be restricted by any other process , parliamentary or medical , as it was absolute , even though the king was insane .
the process in britain disregarded this opinion , appropriate medical and social interventions were undertaken , and neither the state nor its people , had to suffer , unlike the thousands killed in coorg . in the case of the rajah ,
the same unquestioned obedience to absolute power had terrifying consequences because there were no processes in place to question the deranged mind of the ruler .
these issues of freedom and constraints as well as civic rights and responsibilities , as they apply to those with mental illness , continue to be debated even now .
a clause of sanity , being a prerequisite for rulers , has been inserted into most political and civil processes , so that those wielding political authority could only be the sane .
the impact of this formalization of prejudice against the mentally ill is quite obvious , as the trickle - down effect gradually barred the mentally ill from any employment , civic role or even elementary processes like voting .
interestingly , a parallel narrative has been constructed around the widening of the ambit of mental illness to include a variety of conditions and behaviors , so that what we have now is a projection that one in four people may have had some sort of mental illness at some point of their lives .
the distinction between the madness of yesteryear and some common mental disorders of today has progressively become more blurred . at another level , arthur cole 's letters reveal a somewhat unhappy and lonely , even miserable , existence after moving to india .
his letters to his sister , and to his mother , talked of the cultural issues linked to migration .
like many middle - class emigrants , his primary motive was to secure financial stability , and india was the preferred place to get rich quickly , especially for the younger sons of middle - level nobility .
his initial exposure to indian life in calcutta in 1801 was more depressing than encouraging , and he described it as living in a society ( where there was ) scarcely a person in which ( he felt ) the least relish for . he wrote that a kind of apathy had a hold of him , and that he could not shake it off .
he did , however , mention that he was getting on as well as possible , and though ( he ) neither liked the country or the people he spent his time pleasantly .
he proceeded to pass an examination in hindustanee in 1804 ( a precursor to the - international english language testing system , now essential for the journey in the other direction ) , at fort william in calcutta ( the certificate is signed by buchanan , the famous doctor who wrote on mysore and malabar ) and moved to southern india , serving in madras and mysore .
it was only when he moved to mysore that he optimistically notes that his prospects ( were ) much improved since ( his ) last ( letter ) , and that now he had
however , the hopes kept receding , and by 1810 , he was still concerned that his return to england would be protracted for a century .
he wrote that if in 10 years he could save 20,000 pounds ( current value 62 million pounds ! ) it ( would ) do , and that he was not ambitious but want ( ed ) a modest fortune . despite all this , sleepy mysore , got the better of him , and by 1811 he was forced to say that everything in this country that you could feel interested in ( went ) on humdrumically ,
photograph copyright manuscripts and archives research library ( m and arl ) , trinity college , dublin it would be tempting to diagnose arthur cole as suffering from the common mental disorder , diagnosable as depression , based on the ennui and pessimism that marks his letters home . in the 19 century , migrating for economic betterment to a richer nation ( the possibilities of acquiring a
modest saving of 20,000 pounds was more likely in india than in ireland ) did not prevent cole from feeling listless , bored , and unsure of how he fitted in .
he also suffered from frequent bouts of headaches , weight loss , and colicky pains .
readers of this journal who have migrated from india to the west may have some empathy for cole 's predicament !
the movement of people during the phase of colonial expansion brought societies with very different traditions of dealing with madness , and psychological distress , face to face .
the experience of madness , under feudal governance and attempts to restrict its effects by democratic processes , is still debated .
migration itself , whether from the seat of colonial power , or from one erstwhile colony ( ireland ) to another ( india ) , also created an interface wherein cultural dissonance could be seen to produce symptoms , even of what we might today call depression .
the experiences of , and attitudes toward , severe mental illness and the common mental disorders are often said to have changed , but have they ?
the two accounts , preserved in the same set of documents by arthur cole , regarding events in coorg in 1809 and london in 1810 , highlight the tension between madness and a sense of political order .
the account in the madras courier emphasizes that the paramount power of the regent can not , and should not , be restricted by any other process , parliamentary or medical , as it was absolute , even though the king was insane .
the process in britain disregarded this opinion , appropriate medical and social interventions were undertaken , and neither the state nor its people , had to suffer , unlike the thousands killed in coorg . in the case of the rajah , the same unquestioned obedience to absolute power had terrifying consequences because there were no processes in place to question the deranged mind of the ruler .
these issues of freedom and constraints as well as civic rights and responsibilities , as they apply to those with mental illness , continue to be debated even now .
a clause of sanity , being a prerequisite for rulers , has been inserted into most political and civil processes , so that those wielding political authority could only be the sane .
the impact of this formalization of prejudice against the mentally ill is quite obvious , as the trickle - down effect gradually barred the mentally ill from any employment , civic role or even elementary processes like voting .
interestingly , a parallel narrative has been constructed around the widening of the ambit of mental illness to include a variety of conditions and behaviors , so that what we have now is a projection that one in four people may have had some sort of mental illness at some point of their lives .
the distinction between the madness of yesteryear and some common mental disorders of today has progressively become more blurred .
at another level , arthur cole 's letters reveal a somewhat unhappy and lonely , even miserable , existence after moving to india .
his letters to his sister , and to his mother , talked of the cultural issues linked to migration .
like many middle - class emigrants , his primary motive was to secure financial stability , and india was the preferred place to get rich quickly , especially for the younger sons of middle - level nobility .
his initial exposure to indian life in calcutta in 1801 was more depressing than encouraging , and he described it as living in a society ( where there was ) scarcely a person in which ( he felt ) the least relish for .
he wrote that a kind of apathy had a hold of him , and that he could not shake it off .
he did , however , mention that he was getting on as well as possible , and though ( he ) neither liked the country or the people he spent his time pleasantly .
he proceeded to pass an examination in hindustanee in 1804 ( a precursor to the - international english language testing system , now essential for the journey in the other direction ) , at fort william in calcutta ( the certificate is signed by buchanan , the famous doctor who wrote on mysore and malabar ) and moved to southern india , serving in madras and mysore . it was only when he moved to mysore that he optimistically notes that his prospects ( were ) much improved since ( his ) last ( letter ) , and that now he had hopes .
however , the hopes kept receding , and by 1810 , he was still concerned that his return to england would be protracted for a century .
he wrote that if in 10 years he could save 20,000 pounds ( current value 62 million pounds ! ) it ( would ) do , and that he was not ambitious but want ( ed ) a modest fortune . despite all this , sleepy mysore , got the better of him , and by 1811 he was forced to say that everything in this country that you could feel interested in ( went ) on humdrumically ,
photograph copyright manuscripts and archives research library ( m and arl ) , trinity college , dublin it would be tempting to diagnose arthur cole as suffering from the common mental disorder , diagnosable as depression , based on the ennui and pessimism that marks his letters home . in the 19 century , migrating for economic betterment to a richer nation ( the possibilities of acquiring a
modest saving of 20,000 pounds was more likely in india than in ireland ) did not prevent cole from feeling listless , bored , and unsure of how he fitted in .
he also suffered from frequent bouts of headaches , weight loss , and colicky pains .
readers of this journal who have migrated from india to the west may have some empathy for cole 's predicament ! the movement of people during the phase of colonial expansion brought societies with very different traditions of dealing with madness , and psychological distress , face to face .
the experience of madness , under feudal governance and attempts to restrict its effects by democratic processes , is still debated .
migration itself , whether from the seat of colonial power , or from one erstwhile colony ( ireland ) to another ( india ) , also created an interface wherein cultural dissonance could be seen to produce symptoms , even of what we might today call depression .
the experiences of , and attitudes toward , severe mental illness and the common mental disorders are often said to have changed , but have they ?
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what happens if a king becomes mentally ill ?
excerpts from the personal papers of arthur henry cole , resident to the kingdom of mysore in 1809 , open up fascinating insights into the madness of rulers , in neighboring coorg and faraway london , and ways in which different societies responded to this . musings on legal capacity and restrictions imposed on account of insanity , as well as migration and ennui in imperial colonies inevitably follow .
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noncommunicable chronic disease ( ncd ) is a global health crisis , with physical inactivity a leading behavioral risk factor common to obesity , type 2 diabetes mellitus , cardiovascular disease , and metabolic syndrome.14 obesity has reached epidemic levels in australia , with approximately 60% of the adult population overweight or obese.5 management of ncd is a growing public health challenge , with australian primary health care systems designed to deliver acute , symptom - driven care , while chronic disease management is arguably more resource intensive .
australian primary health care involves four main types of services and providers : 1 ) general practice , 2 ) community health services , 3 ) private allied health providers , and 4 ) indigenous community - controlled health services .
general practice is the predominant provider of primary health care , with general practitioners essentially the gatekeepers to a range of other health services .
however , an effective framework for lifestyle modification and collaborative interdisciplinary care in primary health settings for chronic disease management is lacking.6,7 for example , a significant practice gap exists in the delivery , maintenance , and follow - up of patients for whom lifestyle modification has been prescribed.8 general practitioners may provide excellent advice on physical activity and dietary changes , but are less likely to provide tailored exercise or ongoing support given a lack of time , understanding of the physiological and metabolic responses to exercise , or understanding of patients readiness to change behaviors.9 lifestyle intervention is underemphasized in the undergraduate training of general practitioners and other health professionals , as are interprofessional practice and collaboration.9,10 increasingly , the demands placed on physicians time and a lack of training in the area may limit their ability to effectively counsel patients on the benefits of exercise and comprehensive lifestyle risk - factor modification.3,6 the burgeoning evidence base for both the benefits of physical activity for health and physical inactivity as a risk factor for ncds1113 underpins support for the revision of ncd models of care .
clinical programs should have a greater emphasis on lifestyle modification incorporating exercise is medicine
principles,14 as physical activity and exercise have traditionally been coupled with other public health agendas and often not fully recognized as a stand - alone , public health priority.2 the introduction of accredited exercise physiologists ( aeps ) specialists in exercise interventions and physical activity education into primary health care settings has the potential to greatly enhance existing health care delivery .
this can be achieved through interprofessional practice , referring to a clinician whose role is exclusively in physical activity and exercise prescription to ensure tailored and appropriate advice , and creating a focus on behavior change as the core component of all clinical programs for both the prevention and management of ncd.15
numerous benefits of interdisciplinary teams in primary health care settings have been cited , including increased learning and development of health care professions , better utilization of resources and improved cost - effectiveness , and improved self - management and health outcomes for patients.10,16 interdisciplinary team care , which advocates lifestyle modification for chronic disease and supports high levels of self - efficacy and self - management in patient cohorts , has been associated with higher levels of treatment compliance and improved health outcomes.7,17,18 to a certain extent , interdisciplinary teams are a result of increasing levels of specialization , although while individuals of the team may be specialists , the overall knowledge is broader and interprofessional collaboration is stronger.16 care provided by interdisciplinary teams is becoming increasingly complex and often challenging , necessarily requiring the application of a specialized body of knowledge.8 unfortunately , physical activity and exercise advice provided in standard care practice is often the least comprehensive component of care . currently , no single clinician is dedicated to providing exercise , physical activity , and health education,8 and recommendations made to patients are based on the national physical activity guidelines for australians,19 which outline the minimum levels of physical activity required to gain a health benefit .
provision of these guidelines needs to be expanded to provide more comprehensive , tailored recommendations to individuals who may be suffering from complex conditions and/or injuries to support behavior change.15 although evidence for effective interventions is lacking in the literature , lifestyle modification programs , with individualized counseling and exercise prescription , have been successful in south africa , canada , and the uk.9,18,20 small - scale exercise referral schemes have also been successful in both the uk for sedentary individuals with chronic conditions20 and in australia with the healthy eating activity and lifestyle ( heal ) program.21 participants in the heal program have successfully achieved weight reduction , improvements in blood pressure and physical fitness parameters , and importantly , have maintained behavior change as measured by a 5-month post - program follow - up.21
aeps are allied health professionals who specialize in the prescription of clinical exercise interventions for patients at high risk of developing , or with existing , chronic and complex medical conditions and/or injuries.22 this emerging exercise physiology discipline has adopted the global initiative
exercise is medicine , founded by the american college of sports medicine , and endorsed by professional bodies and practicing clinicians across many countries including australia , south africa , canada , the usa , germany , new zealand , and the uk.22,23 the aims of aep interventions are to prevent or manage chronic disease or injury , and assist in restoring optimal physical function , health , and wellness.22 the scope of responsibilities for the aep includes diagnostic and functional testing , exercise prescription , exercise supervision , patient counseling , education , and outcomes analysis . in addition , aeps may address barriers to maintaining exercise compliance , including psychosocial factors , family or job responsibilities , orthopedic or musculoskeletal limitations , or other deterrents to regular exercise.3 exercise physiologists are currently widely employed in cardiac rehabilitation programs , but increasing opportunities exist in chronic disease management , including weight management , osteoporosis , pre - diabetes and type 2 diabetes mellitus management , and cancer rehabilitation.8 cardiac rehabilitation programs designed and facilitated by exercise physiologists have been highly successful in changing exercise and physical activity behaviors ; preventing or delaying subsequent cardiac arrest ; improving exercise tolerance , muscle atrophy , and circulation ; improving quality of life ; and significantly reducing risk factors for comorbidities in this patient population.3,12 this established model of care is transferrable to other areas of rehabilitative health and is hypothesized to produce the same health benefits .
the exercise is medicine initiative aims to raise the profile of exercise physiology and incorporate physical activity and exercise as standard components of a disease prevention and treatment paradigm in australia , improving community health and reducing long - term health care costs.23 more information about aeps and exercise is medicine can be found at http://exerciseismedicine.org.au/ and http://www.essa.org.au ( accessed november 27 , 2013 ) .
extensive evidence exists with respect to growing levels of obesity and associated ncds such as heart disease and diabetes and the positive effect of physical activity on the prevention and management of these conditions.15,24,25 equally , significant evidence is emerging with respect to the value of an interprofessional approach in the prevention and management of ncds.10 a small but growing body of evidence also exists indicating the positive impact of the addition of aeps to interprofessional practice , which supports exercise physiology as an invaluable but currently underutilized discipline and essential resource in interventions designed to combat all chronic and noncommunicable diseases , not restricted to coronary heart disease.3,8,26 opportunity also exists for further research in this area of ncd prevention and management and exercise is medicine .
the potential for exercise physiology to add measurable value in interventions designed to combat chronic and noncommunicable diseases is significant , and opportunities exists for aeps to contribute to current chronic disease management in australian primary health care settings .
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noncommunicable diseases ( ncds ) , such as obesity and type 2 diabetes mellitus , are a growing public health challenge in australia , accounting for a significant and increasing cost to the health care system .
management of these chronic conditions is aided by interprofessional practice , but models of care require updating to incorporate the latest evidence - based practice . increasing research evidence reports the benefits of physical activity and exercise on health status and the risk of inactivity to chronic disease development , yet
physical activity advice is often the least comprehensive component of care . an essential but as yet underutilized player in ncd prevention and management
is the accredited exercise physiologist , a specialist in the delivery of clinical exercise prescriptions for the prevention or management of chronic and complex conditions . in this article , the existing role of accredited exercise physiologists in interprofessional practice
is examined , and an extension of their role proposed in primary health care settings .
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telemedicine can be defined as the use of telecommunication technologies to provide medical information and service .
historically , it started more than 4 decades ago to allow communication and discussion between physicians of small hospitals and those of large tertiary care hospitals for non - acute cases where advanced treatment must be planned and agreed on . therefore , telemedicine started in an asynchronous module where data can be stored and forwarded and then reviewed offline .
however , acute care has developed significantly in the last few decades as well as telecommunication technologies , which made synchronous telemedicine possible and in a different format from telephone - based to audio - video teleconference , and this may go further with the attachment of peripheral devices for robotic surgery or procedures under tele - guidance .
we can define telestroke as the emergency use of telecommunication technologies to provide appropriate acute stroke therapy at a distance .
a telestroke program using telephone consultation is cheap and takes less time to initiate and to complete , however , it is associated with significant limitations and the risk of making a wrong diagnosis or at least inaccurate acute stroke related deficit evaluation , and then the wrong thrombolysis decision .
most of the current telestroke programs use synchronous two - way audio - video teleconferences , and this is our area of interest in this article .
the patient is clinically evaluated using the national institute of health stroke scale ( nihss ) and the brain image is reviewed by a stroke neurologist from a distance , and then a thrombolysis decision is taken . in one randomized
controlled study , the audio - video teleconference was compared to the telephone - based telestroke and was found to be more sensitive , more specific , and more likely to lead to the right thrombolysis decision.3,4 after confirming the diagnosis of acute stroke , there are 2 important measures to increase the recanalization rate with significant clinical benefit : thrombolysis rate ( percentage of acute stroke patients who received iv t - pa within the therapeutic window ) and iv t - pa needle time ( time from stroke onset to iv t - pa bolus ) .
currently , 2 options exist for acute stroke patients care in medically under served areas : either urgent patient transfer to a tertiary care hospital where acute stroke treatment can be administered , or managing the patient at the local hospital .
patient transfer can be via ambulance or air evacuation , which is associated with high transportation costs , smaller increase in thrombolysis rate , and smaller chance for significant recovery if they were treated , because most likely the iv t - pa needle time will be within the last third of the therapeutic time - window , which is associated with higher number needed to treat.5 the second option necessitates telestroke technology so stroke experts share with local physicians the duty of applying evidence - based acute stroke therapy .
we need to look here for 2 measures , the validity of patient assessment using the nihss through teleconference , and the effectiveness , including success , and complication rate , of telestroke based intravenous thrombolysis .
few studies have looked into the accuracy of patient assessment using the nihss . in one study,6
the nihss scores of 20 patients were compared to their nihss scores carried out remotely through a broadband - connected workstation .
both assessments were carried out by qualified neurologists . paired t tests and pearson correlation coefficients were very strong ( r=0.9552 , p=0.0001 ) indicating that high scores in bedside correlate with high scores remotely .
for the second measure , we will review published data from major telestroke programs in europe and north america .
the reach telestroke network started in 2003 in georgia , usa.7 by 2005 , the network had 7 hospitals in rural areas connected to the medical college of georgia via one - way video and two - way audio teleconference system . between march 2003 and may 2005
, 194 acute stroke consultations were made within the 3-hour therapeutic window and 30 patients with a median nihss of 12.5 were treated with iv t - pa .
rapid improvement defined as a drop of 4 or more nihss points within 24 hours occurred in 18 out of 30 treated patients , and no symptomatic intracerebral hemorrhage occurred .
the study interestingly showed a significant reduction in onset to treatment after one year of operation from 143 to 111 minutes .
the most common reasons for not treating were rapid improvement , nihss was below 4 , beyond therapeutic window , or presence of seizure or hemorrhage prior to treatment .
some other data recently published represents the experience of the university of pittsburg medical center telestroke network with its 12 spoke facilities through audio - video conferencing equipment.8 between july 2008 and december 2009 , 351 telestroke consultations were performed .
eighty - three stroke patients received iv t - pa at the spoke hospitals , and 59 patients at the hub center .
the 2 groups were similar in their baseline characteristics including median nihss scores and mean onset to treatment time .
the mean arrival to treatment time was longer in the telestroke patients ( 67.8 versus 98.9 minutes ) .
the study showed no statistical significant difference between the 2 groups in favorable outcomes , symptomatic intracerebral hemorrhage , and mortality rates .
the northern alberta telestroke service started in 2003.9 by 2007 , 7 spoke centers were connected to the university of alberta hospital ( 4 via audio - video teleconference , and 3 via telephone only ) . in their first 2 years of experience , the telestroke was activated 210 times and only 44 patients were candidates for thrombolysis using iv
ten ( 23% ) of them were assessed by the er physician at the spoke centers without video assessment by a stroke neurologist .
sixteen ( 40% ) patients had a modified rankin scale less than 2 by 90 days from iv t - pa , and 9 deaths occurred within 90 days from iv thrombolysis . only 2 ( 4.5% ) of the treated patients had t - pa complications with intracerebral hemorrhage , and the rest died due to other mechanisms .
the telemedical project for integrative stroke care ( tempis ) was established in 2003 serving a large area in south east bavaria , germany.10 it comprised 12 spoke hospitals connected to 2 academic centers in munich via video teleconference and ct / mri image transfer .
currently , tempis is considered to be the largest telemedical stroke service in the world .
tempis was associated with significant improvement of acute in - hospital stroke treatment in terms of quality indicators such as rapid brain imaging , thrombolysis rate , assessment of swallowing disorders , and early rehabilitation therapy.11 when patients served with tempis were compared to similar acute stroke patients within the same region , but not served with telemedicine , tempis showed significant reduction in death and dependency outcome at 3 , 12 , and 30 months,10,12 without a significant difference in mortality rate alone .
the thrombolysis rate in most of the published data of different telestroke networks ranges from 18 - 51%.7 - 10 reasons for no iv t - pa include stroke onset beyond 270 minutes , presence of contraindication that was not recognized by the er physician at the spoke hospital , or patient having a stroke mimic like seizure with todd s paralysis , cns neoplasm with sudden deterioration , cns infection , and others .
considering the threat of these acute stroke mimics , on - call stroke neurologists are still of great benefit to the patients and the spoke hospitals .
the small waste of telestroke resources on acute stroke mimics is well compensated by the gain of managing these urgent neurological conditions despite the fact that some patients were required to be transferred to the university hospital anyway . with time and experience
there are 3 nonexclusive methods of clinical operation of telestroke : drip and keep , which means after telestroke consultation and approval for iv t - pa , the iv t - pa is infused at the spoke center followed by admission there for observation and stroke work up .
this requires a local stroke unit with at least minimal acceptable quality of care with multidisciplinary teams to have an integrative telestroke network .
a follow up assessment by a stroke neurologist can also be carried out by telestroke , or through outpatient visit to the hub center a few weeks later . for further complications during the acute phase of treatment , urgent telestroke consultation can be initiated again by the treating physician at the hub hospital . the second method is the ship method , which is recommended for patients with an acute stroke with contraindication for iv t - pa , but that can still have intraarterial t - pa , or an endovascular procedure , and mechanical removal / disruption of the clot ( for example : patients on anti - coagulation with inr > 1.6 ) . here , the patient gets transferred directly to the hub center for urgent treatment and intervention .
the third way is drip and ship and indicated for those acute stroke patients who are candidates for iv t - pa .
however , the consulting spoke hospital can not provide appropriate post - tpa care or stroke workup .
the patient can have the iv t - pa infused locally , and then shipped to the hub center .
developing telestroke networks may better encourage more spoke centers to participate if they choose drip and ship since it put less load on the spoke centers , and with time and experience , spoke centers can start admitting patients after thrombolysis .
it is also important to mention that the type of clinical practice can be influenced by financial incentives and reimbursements for physicians and hospitals , and by clear job descriptions and duties at each end of the telestroke network , in case of complications or medico - legal claims .
the quantity and type of manpower required for a telestroke network varies , and depends on the proposed size of the network , type of clinical operation , and complexity of care .
based on experience , each hub center needs 4 or more stroke neurologists , one of them assigned as the medical director of the network , one telestroke manager , one data manager , 2 telestroke nurses , and 2 information technologists , plus the clinical and administrative back up support from the tertiary hospital . for each spoke center
, you need to involve at least the emergency physicians , emergency nurses directed by the head of er there , plus a program manager . for drip and keep practice , the multidisciplinary teams including the internists should be added .
effective and interactive relationships between different network sites , frequent sites visits between hub and spoke personnel , continuing medical educational activities , and sharing data , and successful stories are mandatory for sustainability and further development .
the recommended quality and performance measures for acute stroke units should be used to assess quality and performance of the telestroke networks plus other measures that are specific to telestroke , such as those concerned with communication ( table 1 ) .
the director of the telestroke network must obtain the data regularly from each spoke , and hub center , and discuss them at least once annually with all network staff in a transparent fashion with the intention to improve quality and reduce failures .
professionals of each telestroke network should also compare their network performance with other networks nationally and internationally .
today , most tertiary care hospitals are either practicing some form of telemedicine or considering it soon .
the practice can be limited to certain health care networks of similar financial and legal jurisdiction , or may go beyond different states borders .
therefore , the first legal issue is getting the license from an appropriate body based on the defined map of telemedicine practice , and making sure the practice does not exceed that map .
the second challenge is staff accreditation as different hospitals may have different regulations and requirements .
for example in saudi arabia , although the saudi commission for health specialties has a set of regulations concerning staff accreditation to designated positions , tertiary care hospitals like king khalid university hospital have added further requirements . therefore ,
mutual agreement on staff accreditation between the spoke and hub centers is needed with defined rules and areas of practice , and then hospitals do not need to change their accreditation requirements in order to practice telemedicine .
the third challenge is agreement on sharing electronic medical records and patient s data without breaking rules of confidentiality and privacy .
this agreement can be initiated by the hospitals boards , but needs to be approved by the appropriate licensing / legal body .
as the telestroke network expands and more cases are treated , the hub stroke neurologists may become overwhelmed between their regular hospital duties and telestroke cases , but they will still be legally responsible for delivering the telestroke service on time ; otherwise , they may be blamed for negligence .
the director of the telestroke network needs to adequately estimate the required number of stroke neurologists based on the size of the population served , and ensure a fair and stable reimbursement system ; otherwise , staff - recruitment failure may occur . a medical error or failure to serve the acute stroke patient on time
can be related to the staff at the spoke center , staff at the hub center , technology related , patient related , or mixed . therefore , a 24/7 documentation and auditing system is needed .
the job description of every staff member within the network must be clearly written and defined .
regular telestroke morbidity and mortality educational activities must run and be shared between all network sites to enhance the learning curve at different levels as well as discussing successful stories .
for example in the usa alone , the direct and indirect costs associated with stroke for 2008 are approximately us$65.5 billion.13 fagan and colleagues14 showed that acute stroke treatment with iv t - pa is associated with an incremental cost savings of us$8,000 per qaly gained , and telestroke was proved to increase thrombolysis rates mainly in patients living in suburban and rural areas.15 there are 2 cost categories associated with telestroke : equipment costs and telestroke running costs , including physician s compensations and salaries for network employees .
the annual telestroke hub equipment cost is approximately us$16,204/site , and the annual telestroke spoke equipment costs are approximately us$5,309/site,16 which are cheap when we consider today s medical costs .
when looking into the cost - effectiveness of telestroke , we consider the telestroke running costs from one side , which are more or less predictable per network s size , versus the thrombolysis - related savings , such as less or no disability , shorter length of stay at hospital , and savings from less patients transportation .
the cost - effectiveness ratio can be non - constant and affected by the network s performance and number of cases treated .
too reduce its costs further , it can be part of a synchronous telemedicine network so some resources are shared with other acute medical consultation services . in conclusion , telestroke is the only solution in many countries to serve suburban areas and to increase the thrombolysis rate .
new regulations are needed to ensure its legality and stability , and initiatives should come from stroke neurologists , er physicians , and medical directors of small hospitals .
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despite developments in acute stroke therapies , stroke continues to be a leading cause of death and disability worldwide .
one major limitation from intravenous thrombolysis with tissue plasminogen activator ( t - pa ) is patient s arrival to the emergency room at a tertiary care hospital after the therapeutic time - window , which is generally 270 minutes .
the problem is worse for people living in suburban areas where stroke expertise can be missing .
therefore , telestroke networks were developed at several sites where a stroke neurologist at a tertiary hospital participates through synchronous audio - video teleconference in confirming diagnosis of stroke , assessing risks and benefits of giving iv t - pa , and making the decision with the patient and emergency physician at the local hospital . in this article , we will review the experience of major telestroke networks in north america and europe , and the evidence of its safety and cost - effectiveness .
telestroke complexity , with regard to practice , manpower , quality assurance , and legal issues will be discussed briefly .
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since their introduction in the 1960s , the anthracyclines have gained a central role in the treatment of non - hodgkin 's lymphoma ( nhl ) . in particular , the use of doxorubicin in the standard rituximab , cyclophosphamide , doxorubicin , vincristine , and prednisone ( r - chop ) regimen means that a large proportion of patients with aggressive lymphomas receive anthracycline therapy . in this paper , a brief history of the development of anthracyclines , their use in nhl , and the management of anthracycline - related cardiotoxicity are presented .
the potential of anthracyclines was first recognized in 1960 , when federico arcamone and co - workers filed a patent application for the fermentative production and recovery of an antibiotic obtained from streptomyces krestomyceticus . in 1963 ,
a novel cytotoxic compound derived from s. peuceticus was isolated simultaneously by researchers in italy and france .
the italian group gave the compound the name daunomycin , the french group named it rubidomycine .
a hydroxylated derivative of daunorubicin was subsequently isolated by the italian group and called adriamycin .
it was later renamed doxorubicin to conform to the established naming convention . in preclinical studies ,
this new antitumor antibiotic was shown to have greater activity against solid murine tumors and a higher therapeutic index than daunorubicin .
early clinical evaluation of doxorubicin demonstrated its efficacy in both adults and children with acute and chronic leukemias .
the first key demonstration of the efficacy of chop in lymphoma was published in 1984 .
armitage and co - workers reported the results of a clinical trial in 75 patients treated with chop for diffuse histiocytic lymphoma ; 51% achieved complete remission and 23 of the complete responders were alive and in complete remission at a median of 38 months after stopping treatment .
ten years later , the southwest oncology group and the eastern cooperative oncology group published the results of a prospective phase iii trial comparing chemotherapy regimens commonly used at that time to treat advanced nhl .
chop was therefore compared with macop - b ( methotrexate with leucovorin rescue , doxorubicin , cyclophosphamide , vincristine , prednisone , and bleomycin ) , promace - cytabom ( prednisone , doxorubicin , cyclophosphamide , etoposide , followed by cytarabine , bleomycin , vincristine , methotrexate with leucovorin rescue ) , and m - bacod ( methotrexate with leucovorin rescue , bleomycin , doxorubicin , cyclophosphamide , vincristine , and dexamethasone ) in 1138 patients ( 899 eligible for efficacy analysis ) .
there were no significant differences in the efficacy of these regimens , but chop was associated with improved safety and tolerability compared with the other regimens ( figure 1 ) .
the authors therefore concluded that chop remains the best available treatment for patients with advanced - stage intermediate - grade or high - grade nhl .
the efficacy of chop in the standard 21-day treatment cycle could be further improved by the use of a 14-day treatment cycle with granulocyte - colony stimulating factor ( g - csf ) support in both young and elderly patients .
subsequently , it was found that the addition of rituximab to the chop regimen further increased its efficacy , and that six cycles of r - chop plus two additional cycles of rituximab were as effective as eight cycles of r - chop .
figure 1overall survival of patients with non - hodgkin 's lymphoma receiving three different treatment regimens ( 3-year estimates of survival ) .
cyclophosphamide , doxorubicin , vincristine , prednisone ; m - bacod methotre - xate with leucovorin rescue , bleomycin , doxorubicin , cyclo - phosphamide , vincristine , and dexamethasone ; promacecytabom
prednisone , doxorubicin , cyclophosphamide , etoposide , followed by cytarabine , bleomycin , vincristine , methotrexate with leucovorin rescue ; macop - b methotrexate with leucovorin rescue , doxorubicin , cyclophosphamide , vincristine , prednisone , and bleomycin . reprinted with permission from fisher ri et al .
comparison of a standard regimen ( chop ) with three intensive chemotherapy regimens for advanced non - hodgkin 's lymphoma .
overall survival of patients with non - hodgkin 's lymphoma receiving three different treatment regimens ( 3-year estimates of survival ) . chop
cyclophosphamide , doxorubicin , vincristine , prednisone ; m - bacod methotre - xate with leucovorin rescue , bleomycin , doxorubicin , cyclo - phosphamide , vincristine , and dexamethasone ; promacecytabom
prednisone , doxorubicin , cyclophosphamide , etoposide , followed by cytarabine , bleomycin , vincristine , methotrexate with leucovorin rescue ; macop - b methotrexate with leucovorin rescue , doxorubicin , cyclophosphamide , vincristine , prednisone , and bleomycin . reprinted with permission from fisher ri et al .
comparison of a standard regimen ( chop ) with three intensive chemotherapy regimens for advanced non - hodgkin 's lymphoma .
although the phase i and ii studies did demonstrate electrocardiographic changes in more than half of 97 patients who were prospectively studied , these abnormalities were subclinical , no symptoms were mentioned , and additional research did not suggest any cumulative cardiac toxicity of doxorubicin .
however , 10 years after these first studies , cardiotoxicity started to be identified as a late side - effect of cumulative doses of anthracyclines .
an early analysis of this problem demonstrated a correlation between the cumulative dose of doxorubicin and the probability of developing congestive heart failure ( chf ) ( figure 2 ) .
subsequent studies confirmed that the incidence of chf increased from 3% at a cumulative doxorubicin dose of 400 mg / m to 18% at a cumulative dose of 700 mg / m .
moreover , chf was often irreversible . risk factors for cardiotoxicity included : previous exposure to anthracyclines , patient age , and a history of cardiovascular disease .
it is the main factor limiting anthracycline use , and can result in discontinuation or non - use of treatment for patients who might otherwise benefit from this therapy .
figure 2risk of developing congestive heart failure ( chf ) in relation to the cumulative dose of doxorubicin . reprinted with permission from von hoff dd et al .
risk of developing congestive heart failure ( chf ) in relation to the cumulative dose of doxorubicin . reprinted with permission from von hoff dd et al
treatment strategies designed to reduce anthracycline cardiotoxicity have included modification of the dosing regimen , treatment with cardioprotective agents , and the use of liposome - encapsulated forms of doxorubicin . as higher cumulative doses of doxorubicin
are associated with a greater risk of chf , then maintaining doses below a maximum cumulative dose may reduce patients ' risks .
nevertheless , such maximum doses can not be used alone as safe limits for drug treatment , as cardiac dysfunction has been seen in patients receiving lower than maximal doses , and some patients are more susceptible to cardiotoxicity than others .
thus , long drug infusions instead of a single injection have been shown to be less cardiotoxic . however , this approach is more laborious than conventional treatment , particularly when doxorubicin is used in combination with other agents .
several studies have investigated the use of dexrazoxane , an iron - chelating agent that scavenges free radicals , as a means of limiting anthracycline - related cardiotoxicity . initially , there were concerns that this agent might adversely affect the antitumor activity of doxorubicin .
however , a subsequent systematic review of nine randomized trials found that dexrazoxane significantly reduced patients ' risk of developing chf , without affecting the efficacy of doxorubicin , in terms of response rate or survival .
the authors therefore concluded that dexrazoxane treatment should be considered in patients at high risk of cardiotoxicity .
two liposome - encapsulated forms of doxorubicin are currently available : pegylated liposomal doxorubicin and non - pegylated liposomal doxorubicin ( npld ) .
liposomes are microscopic structures consisting of one or more concentric lipid bilayers , which encapsulate the active substance .
these lipid structures can not escape the vascular space in areas that have tight capillary junctions , such as the heart muscle , but can reach other tissues .
in particular , npld is preferentially delivered to the liver , spleen , bone marrow , and lymph nodes , compared with relatively little entry to cardiac muscle and the gastrointestinal mucosa .
this treatment may therefore reduce the cardiac toxicity of doxorubicin while maintaining its antitumor efficacy . in patients with newly diagnosed aggressive nhl ,
substitution of npld into the chop regimen , to give comp , produced a complete response rate of 67.4% .
importantly , the use of comp in patients at risk of cardiotoxicity has also produced promising results .
a retrospective analysis of rituximab - comp ( r - comp ) treatment for patients who either had concurrent cardiac diseases or had been pretreated with anthracyclines demonstrated a complete response rate of 76% .
a prospective international study of r - comp in elderly patients with aggressive lymphoma gave a complete remission rate of 57% , and patients showed no signs of cardiotoxicity . left ventricular ejection fraction did not change during treatment , and the r - comp regimen was not associated with any increase in acute cardiac toxicity .
a meta - analysis of two studies comparing free and liposomal doxorubicin ( in patients with breast cancer , not nhl ) showed a significantly lower rate of both clinical heart failure , and clinical and subclinical heart failure combined , in patients treated with liposome - encapsulated doxorubicin .
the potential of anthracyclines was first recognized in 1960 , when federico arcamone and co - workers filed a patent application for the fermentative production and recovery of an antibiotic obtained from streptomyces krestomyceticus . in 1963 ,
a novel cytotoxic compound derived from s. peuceticus was isolated simultaneously by researchers in italy and france .
the italian group gave the compound the name daunomycin , the french group named it rubidomycine .
a hydroxylated derivative of daunorubicin was subsequently isolated by the italian group and called adriamycin .
it was later renamed doxorubicin to conform to the established naming convention . in preclinical studies ,
this new antitumor antibiotic was shown to have greater activity against solid murine tumors and a higher therapeutic index than daunorubicin .
early clinical evaluation of doxorubicin demonstrated its efficacy in both adults and children with acute and chronic leukemias .
the first key demonstration of the efficacy of chop in lymphoma was published in 1984 .
armitage and co - workers reported the results of a clinical trial in 75 patients treated with chop for diffuse histiocytic lymphoma ; 51% achieved complete remission and 23 of the complete responders were alive and in complete remission at a median of 38 months after stopping treatment .
ten years later , the southwest oncology group and the eastern cooperative oncology group published the results of a prospective phase iii trial comparing chemotherapy regimens commonly used at that time to treat advanced nhl .
chop was therefore compared with macop - b ( methotrexate with leucovorin rescue , doxorubicin , cyclophosphamide , vincristine , prednisone , and bleomycin ) , promace - cytabom ( prednisone , doxorubicin , cyclophosphamide , etoposide , followed by cytarabine , bleomycin , vincristine , methotrexate with leucovorin rescue ) , and m - bacod ( methotrexate with leucovorin rescue , bleomycin , doxorubicin , cyclophosphamide , vincristine , and dexamethasone ) in 1138 patients ( 899 eligible for efficacy analysis ) .
there were no significant differences in the efficacy of these regimens , but chop was associated with improved safety and tolerability compared with the other regimens ( figure 1 ) .
the authors therefore concluded that chop remains the best available treatment for patients with advanced - stage intermediate - grade or high - grade nhl .
the efficacy of chop in the standard 21-day treatment cycle could be further improved by the use of a 14-day treatment cycle with granulocyte - colony stimulating factor ( g - csf ) support in both young and elderly patients .
subsequently , it was found that the addition of rituximab to the chop regimen further increased its efficacy , and that six cycles of r - chop plus two additional cycles of rituximab were as effective as eight cycles of r - chop .
figure 1overall survival of patients with non - hodgkin 's lymphoma receiving three different treatment regimens ( 3-year estimates of survival ) . chop
cyclophosphamide , doxorubicin , vincristine , prednisone ; m - bacod methotre - xate with leucovorin rescue , bleomycin , doxorubicin , cyclo - phosphamide , vincristine , and dexamethasone ; promacecytabom
prednisone , doxorubicin , cyclophosphamide , etoposide , followed by cytarabine , bleomycin , vincristine , methotrexate with leucovorin rescue ; macop - b methotrexate with leucovorin rescue , doxorubicin , cyclophosphamide , vincristine , prednisone , and bleomycin . reprinted with permission from fisher ri et al .
comparison of a standard regimen ( chop ) with three intensive chemotherapy regimens for advanced non - hodgkin 's lymphoma .
overall survival of patients with non - hodgkin 's lymphoma receiving three different treatment regimens ( 3-year estimates of survival ) .
cyclophosphamide , doxorubicin , vincristine , prednisone ; m - bacod methotre - xate with leucovorin rescue , bleomycin , doxorubicin , cyclo - phosphamide , vincristine , and dexamethasone ; promacecytabom
prednisone , doxorubicin , cyclophosphamide , etoposide , followed by cytarabine , bleomycin , vincristine , methotrexate with leucovorin rescue ; macop - b methotrexate with leucovorin rescue , doxorubicin , cyclophosphamide , vincristine , prednisone , and bleomycin . reprinted with permission from fisher ri et al
. comparison of a standard regimen ( chop ) with three intensive chemotherapy regimens for advanced non - hodgkin 's lymphoma .
although the phase i and ii studies did demonstrate electrocardiographic changes in more than half of 97 patients who were prospectively studied , these abnormalities were subclinical , no symptoms were mentioned , and additional research did not suggest any cumulative cardiac toxicity of doxorubicin .
however , 10 years after these first studies , cardiotoxicity started to be identified as a late side - effect of cumulative doses of anthracyclines .
an early analysis of this problem demonstrated a correlation between the cumulative dose of doxorubicin and the probability of developing congestive heart failure ( chf ) ( figure 2 ) .
subsequent studies confirmed that the incidence of chf increased from 3% at a cumulative doxorubicin dose of 400 mg / m to 18% at a cumulative dose of 700 mg / m .
moreover , chf was often irreversible . risk factors for cardiotoxicity included : previous exposure to anthracyclines , patient age , and a history of cardiovascular disease .
it is the main factor limiting anthracycline use , and can result in discontinuation or non - use of treatment for patients who might otherwise benefit from this therapy .
figure 2risk of developing congestive heart failure ( chf ) in relation to the cumulative dose of doxorubicin . reprinted with permission from von hoff dd et al .
risk of developing congestive heart failure ( chf ) in relation to the cumulative dose of doxorubicin . reprinted with permission from von hoff dd et al .
treatment strategies designed to reduce anthracycline cardiotoxicity have included modification of the dosing regimen , treatment with cardioprotective agents , and the use of liposome - encapsulated forms of doxorubicin . as higher cumulative doses of doxorubicin
are associated with a greater risk of chf , then maintaining doses below a maximum cumulative dose may reduce patients ' risks .
nevertheless , such maximum doses can not be used alone as safe limits for drug treatment , as cardiac dysfunction has been seen in patients receiving lower than maximal doses , and some patients are more susceptible to cardiotoxicity than others .
thus , long drug infusions instead of a single injection have been shown to be less cardiotoxic . however , this approach is more laborious than conventional treatment , particularly when doxorubicin is used in combination with other agents .
several studies have investigated the use of dexrazoxane , an iron - chelating agent that scavenges free radicals , as a means of limiting anthracycline - related cardiotoxicity . initially , there were concerns that this agent might adversely affect the antitumor activity of doxorubicin .
however , a subsequent systematic review of nine randomized trials found that dexrazoxane significantly reduced patients ' risk of developing chf , without affecting the efficacy of doxorubicin , in terms of response rate or survival .
the authors therefore concluded that dexrazoxane treatment should be considered in patients at high risk of cardiotoxicity .
two liposome - encapsulated forms of doxorubicin are currently available : pegylated liposomal doxorubicin and non - pegylated liposomal doxorubicin ( npld ) .
liposomes are microscopic structures consisting of one or more concentric lipid bilayers , which encapsulate the active substance .
these lipid structures can not escape the vascular space in areas that have tight capillary junctions , such as the heart muscle , but can reach other tissues .
in particular , npld is preferentially delivered to the liver , spleen , bone marrow , and lymph nodes , compared with relatively little entry to cardiac muscle and the gastrointestinal mucosa .
this treatment may therefore reduce the cardiac toxicity of doxorubicin while maintaining its antitumor efficacy . in patients with newly diagnosed aggressive nhl ,
substitution of npld into the chop regimen , to give comp , produced a complete response rate of 67.4% .
importantly , the use of comp in patients at risk of cardiotoxicity has also produced promising results .
a retrospective analysis of rituximab - comp ( r - comp ) treatment for patients who either had concurrent cardiac diseases or had been pretreated with anthracyclines demonstrated a complete response rate of 76% .
a prospective international study of r - comp in elderly patients with aggressive lymphoma gave a complete remission rate of 57% , and patients showed no signs of cardiotoxicity . left ventricular ejection fraction did not change during treatment , and the r - comp regimen was not associated with any increase in acute cardiac toxicity .
a meta - analysis of two studies comparing free and liposomal doxorubicin ( in patients with breast cancer , not nhl ) showed a significantly lower rate of both clinical heart failure , and clinical and subclinical heart failure combined , in patients treated with liposome - encapsulated doxorubicin .
as higher cumulative doses of doxorubicin are associated with a greater risk of chf , then maintaining doses below a maximum cumulative dose may reduce patients ' risks .
nevertheless , such maximum doses can not be used alone as safe limits for drug treatment , as cardiac dysfunction has been seen in patients receiving lower than maximal doses , and some patients are more susceptible to cardiotoxicity than others .
thus , long drug infusions instead of a single injection have been shown to be less cardiotoxic . however ,
this approach is more laborious than conventional treatment , particularly when doxorubicin is used in combination with other agents .
several studies have investigated the use of dexrazoxane , an iron - chelating agent that scavenges free radicals , as a means of limiting anthracycline - related cardiotoxicity . initially , there were concerns that this agent might adversely affect the antitumor activity of doxorubicin .
however , a subsequent systematic review of nine randomized trials found that dexrazoxane significantly reduced patients ' risk of developing chf , without affecting the efficacy of doxorubicin , in terms of response rate or survival .
the authors therefore concluded that dexrazoxane treatment should be considered in patients at high risk of cardiotoxicity .
two liposome - encapsulated forms of doxorubicin are currently available : pegylated liposomal doxorubicin and non - pegylated liposomal doxorubicin ( npld ) .
liposomes are microscopic structures consisting of one or more concentric lipid bilayers , which encapsulate the active substance .
these lipid structures can not escape the vascular space in areas that have tight capillary junctions , such as the heart muscle , but can reach other tissues .
in particular , npld is preferentially delivered to the liver , spleen , bone marrow , and lymph nodes , compared with relatively little entry to cardiac muscle and the gastrointestinal mucosa .
this treatment may therefore reduce the cardiac toxicity of doxorubicin while maintaining its antitumor efficacy . in patients with newly diagnosed aggressive nhl ,
substitution of npld into the chop regimen , to give comp , produced a complete response rate of 67.4% .
importantly , the use of comp in patients at risk of cardiotoxicity has also produced promising results .
a retrospective analysis of rituximab - comp ( r - comp ) treatment for patients who either had concurrent cardiac diseases or had been pretreated with anthracyclines demonstrated a complete response rate of 76% .
moreover , only one patient included in this analysis developed acute grade iii cardiotoxicity . a prospective international study of r - comp in elderly patients with aggressive lymphoma gave a complete remission rate of 57% , and patients showed no signs of cardiotoxicity . left ventricular ejection fraction did not change during treatment , and the r - comp regimen was not associated with any increase in acute cardiac toxicity .
a meta - analysis of two studies comparing free and liposomal doxorubicin ( in patients with breast cancer , not nhl ) showed a significantly lower rate of both clinical heart failure , and clinical and subclinical heart failure combined , in patients treated with liposome - encapsulated doxorubicin .
moreover , the introduction of liposome - encapsulated doxorubicin may allow their use to be extended to patients at risk of cardiotoxicity who would benefit from this treatment .
a key challenge now in nhl is to maximize treatment outcomes , through new biological approaches such as gene profiling , and through optimal use of therapies across the diverse spectrum of lymphomas .
this challenge to clinicians was the theme of an international workshop , moving forward with new data and approaches : a fresh
look at anthracyclines in breast cancer and nhl , which took place in meldola , italy , in september 2010 .
this meeting reviewed recent data on the use of anthracyclines in the treatment of breast cancer and nhl , with the aim of providing guidance on how the management of these diseases can be improved .
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anthracyclines have a central role in the management of non - hodgkin 's lymphoma ( nhl ) . the cyclophosphamide , doxorubicin , vincristine , and prednisone ( chop ) treatment regimen has been the standard of care for more than 20 years .
further improvements have been made to the efficacy of this chemotherapy by reducing the dosing interval and adding rituximab to the regimen .
a major limitation to the use of anthracyclines is the development of cardiotoxicity as a late adverse event .
strategies to reduce cardiac events include changes to the dosing schedule for doxorubicin , use of the chelating agent dexrazoxane and the use of liposome - encapsulated doxorubicin .
this latter strategy has demonstrated good efficacy and reduced cardiotoxicity in patients with nhl , including those at risk of developing cardiac effects .
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older adults with type 2 diabetes ( t2 dm ) are at increased risk for developing micro- and macrovascular complications ( e.g. , retinopathy , neuropathy , nephropathy , and cardiovascular disease ) .
recent studies suggested that t2 dm is associated with higher risk of cognitive dysfunction , dementia , and depression in the elderly [ 2 , 3 ] .
the cause of cognitive impairment and depression in type 2 diabetes is unknown , but it is most likely multifactorial .
chronic hyperglycemia , cerebral microvascular disease , severe hypoglycemia , and an increased prevalence of macrovascular disease could play significant role in pathophysiology of brain disturbances .
diabetes is associated with an increased release of inflammatory cytokines , and the excess inflammation may be neurotoxic .
depression , mild cognitive impairment ( mci ) , and dementia are highly prevalent chronic conditions associated with social , medical , and economic burdens .
although there are several epidemiological studies that have reported the prevalence of mild cognitive impairment ( mci ) or depressive syndrome in elderly diabetic population little is known about the comorbidity of these conditions [ 5 , 6 ] .
data about identification potentially modifiable risk factors for cognitive impairment and depressive syndrome in elderly people with type 2 diabetes are also poor and could be important for future diabetes health care initiatives . therefore ,
the aim of the current study was to ( 1 ) estimate the prevalence of mci , depressive mood , and its comorbidity in the elderly subjects with t2 dm and ( 2 ) find potential demographic , clinical , and biochemical risk factors associated with cognitive impairment and depressive syndrome .
a survey was conducted among unselected 276 elders who attended to the outpatient clinic belonging to the department of internal medicine and diabetology , university hospital no . 1 in lodz , poland .
we included patients aged 65 and over with diabetes type 2 diagnosed minimum 1 year earlier , subjects who had been able to understand and cooperate with study procedures .
the exclusion criteria were diagnosed depression or dementia , use of possible or known cognition - impairing drugs in the previous 3 months , presence of neoplasm , constant alcohol or substance abuse , severe visual , mobility , or motor coordination impairment , history of head trauma , inflammatory or infectious brain disease , and severe neurological or psychiatric illness . written consent was obtained from the participants at the beginning of the study .
the first part of visit included a morning blood draw after a 1012-hour overnight fast , blood pressure measurements , height and weight assessment , and complete physical examination .
then patients had eaten a breakfast followed by capillary glucose level measuring to ensure that participants were not hypoglycemic at the time of cognitive testing .
weight and height were measured to calculate body mass index ( bmi = weight / height ( kg / m ) ) .
the systolic and diastolic blood pressures ( mmhg ) were measured with the patient in sitting position after 5 minutes of rest .
the detailed medical history of diabetes type 2 was taken and includes diabetes duration , currant treatment for diabetes and complications if present , family history of diabetes , comorbid diseases of the patient ( hyperlipidemia , hypertension , cardiovascular disease , lung disease , cancer , and gastrointestinal tract diseases ) , and their treatment .
diabetic vascular complications were assessed based on the existence of nephropathy , retinopathy , neuropathy , cardiovascular disease ( cvd ) , and stroke .
hypertension was defined as either a history of hypertension or use of any antihypertensive agents , and hyperlipidemia was defined as use of any lipid lowering agent or an untreated serum ldl cholesterol level 2.6 mmol / l or / and triglycerides 1.7 mmol / l .
after overnight fasting , blood samples were taken by venipuncture to assess serum levels of glycosylated hemoglobin ( hba1c ) , total cholesterol , triglycerides , low - density lipoprotein cholesterol ( ldl - c ) , and high - density lipoprotein cholesterol ( hdl - c ) .
all participants underwent the following tests : the montreal cognitive assessment ( moca ) to evaluate the cognitive impairment , long version of the geriatric depression scale ( gds-30 ) to assess the depressive mood , katz basic activities of daily living ( badl ) , and lawton instrumental activities of daily living ( iadl ) questionnaires to collect information on daily activities [ 9 , 10 ] .
the moca tests 8 cognitive domains , visual - spatial ability , attention , executive function , immediate memory , delayed memory , language , abstraction , calculation , and orientation , for a maximum total score of 30 .
the normal moca score is 26 , with one point added if the subject has fewer than 12 years of formal education .
the moca is better than other tools to detect mci in the elderly patients with type 2 diabetes .
mci was diagnosed based on criteria established in the 2006 european alzheimer 's disease consortium which are currently available standard test [ 12 , 13 ] .
the cut - off points for moca scores ( 19/30 ) are recommended for the diagnosis of dementia in epidemiological studies .
patients with score 19 and below were excluded from the study as dementia and sent to psychiatrist for further care .
the criteria mentioned above included also absence of major repercussions on daily life ( in our study , measured by katz badl and lawton iadl ) .
scores range from 0 to 9 was considered as normal and from 10 to 19 was considered to have depressive symptoms .
patients with score 20 and above were excluded from the study as severe depressive symptomatology and sent to psychiatrist for further diagnosis . according to criteria mentioned above 276 older subjects with diabetes type
2 were selected into groups : patients with mci , patients without mci , patients with depressive mood , patients without depressive mood , patients with mci and depressive mood , and patients without mci and depressive mood .
the study was operated in accordance with the world medical association 's declaration of helsinki .
each participant was assigned a number by which he / she was identified to keep his or her privacy .
the approval was obtained from the independent local ethics committee of medical university of lodz .
the descriptive statistics for the categorical variables were tested using the chi - square and the continuous variables using the student 's t - test or the mann - whitney u tests whenever applicable .
three multivariate logistic regression models were generated to identify the influential factors associated with the presence of mci , depressive syndrome , and both .
the dependent factors were set as having mci or having depressive mood or having both mci and depressive mood .
the univariate analysis included the variables that were found to be significant in the models .
the independent variables entered in the model at step one were gender , age , education , marital status , smoking status , physical activity , duration of diabetes , the body mass index , hba1c and lipids levels , treatment of dm 2 , micro- and macrovascular complications , previous ha or use of ha drugs , hyperlipidemia , number of comorbid conditions , and presence of hypoglycemia .
odds ratios ( or ) were computed and presented with the 95% interval of confidence ( ci ) . the nonsignificant predictors ( p > 0.05 ) were subsequently removed on a backward stepwise basis .
the demographic and clinical characteristics of the study group have been presented in table 1 .
the prevalence of mci in elderly patients with type 2 diabetes was 31.5% ( n-87 ) , and the prevalence of subjects with depressive syndrome was 29.7% ( n-82 ) .
we also evaluated 25 participants ( 9.1% ) with both mci and depressive mood . compared with the group without mci ,
patients with mci were significantly older and less educated and had a longer duration of diabetes ; more were diagnosed with cardiovascular disease , hypertension , hyperlipidaemia , retinopathy , nephropathy , and other comorbidities and more had a history of hypoglycemia .
the level of hba1c and triglycerides was higher and hdl was lower in mci group .
patients with depressive syndrome were more likely to be female , single , and current and past smokers and had less physical activity , higher bmi , and a longer duration of diabetes ; more were diagnosed with neuropathy and other comorbidities , had a history of hypoglycemia , and were treated with insulin .
the level of total cholesterol and ldl cholesterol was higher in group with depressive mood .
the characterization of 25 subjects with both mci and depressive syndrome is presented in table 1 .
the univariate logistic regression models revealed many factors which are associated with the diagnosis of mci , depressive syndrome , and both ( table 2 ) .
finally we constructed three multivariate logistic regression models to determine the predictors of mci , depressive status , and both ( table 3 ) .
the first model showed that variables which increased the likelihood of having been diagnosed with mci were higher level of hba1c , presence of previous cvd , hypertension , retinopathy , increased number of comorbidities , and less years of formal education .
significant predictors of having a depressive mood included female gender , single marital status , current and past smoking status , lack of physical activity , higher bmi and total cholesterol level , increased number of comorbidities , presence of history of hypoglycemia , and insulin treatment .
factors associated both with mci and depressive syndrome were as follows : female gender , single marital status , past smoking status , retinopathy , presence of previous cvd or stroke , increased number of comorbidities , and insulin treatment .
type 2 diabetes is a complex metabolic disease , caused by reduced insulin sensitivity and relative insulin deficiency .
coexisting disorders , including hypertension , dyslipidaemia , and obesity , contribute to the severity of type 2 diabetes .
the cause of diabetes - related brain dysfunction is difficult to establish because of the prevalence of several comorbidities , each of which might affect it .
there are many studies which had investigating prevalence of cognitive function and depression disorder in type 2 diabetes .
however none of them had described the coexistence of mci and depressive symptoms in diabetic subjects and factors associated with the presence of both these conditions .
the prevalence of mci in our study was 31.5% and it was comparable to other data in the worldwide literature .
one population - based study reported that the incidence of mci in diabetic subjects was around 28% .
a meta - analysis of longitudinal studies showed that diabetes increased the risk of mild cognitive impairment by 21% .
data from large population studies suggest that chronic hyperglycaemia is a risk for cognitive impairment in diabetes type 1 and type 2 [ 15 , 16 ] . in agreement with many other results
our study reported that higher level of hba1c was a risk factor for diagnosis of mci .
the pathomechanism underlying this process is complex and can include increased formation advanced glycation end products ( ages ) , oxidative stress and inflammation , accumulation of sorbitol , and development of a hyperosmotic state in nerve cells that leads to edema and impaired brain function , impaired insulin homeostasis in the brain , and insulin resistance syndrome .
chronic hyperglycaemia and long duration of diabetes are both associated with increased development of cognitive dysfunction , as is the presence of vascular risk factors ( e.g. , hypertension , hypercholesterolaemia , and obesity ) and microvascular and macrovascular complications .
the edinburgh type 2 diabetes study showed that general cognitive ability was significantly lower in people with moderate - to - severe diabetic retinopathy than in those without retinopathy . in our study presence of retinopathy increased a likelihood of having mci or both mci and depressive mood .
the underlying mechanism of the association between hypertension and cognitive impairment in diabetics might include cerebrovascular diseases and vascular dysfunction .
patients with diabetes have an increased risk in thrombotic stroke , and vascular disease has long been hypothesized to contribute to abnormalities in cognition in such patients [ 4 , 19 ] .
the hypertension can cause vascular occlusion and compromise cerebral infarction , alter the structure of cerebral blood vessels , and change blood supply to the brain [ 20 , 21 ] .
we had also noticed that patients with mci had higher level of triglyceride and lower level of hdl cholesterol .
the hyperlipidemia was significant predictor of diagnosis with mci . in agreement with our results ,
other studies had showed similar association and explained that hyperlipidemia itself causes denaturation of neurons responsible for cognitive function or accelerates the progression of atherosclerosis , which leads to mci [ 22 , 23 ] .
previous studies have shown that diabetic patients with lower levels of education have more long - term complications , more cardiovascular diseases , and a greater risk of mortality . in our study
we noticed also a high prevalence of depressive symptoms ( 29.7% ) among elderly patients with type 2 diabetes . since depression can cause cognitive deficits , patients with diagnosed depression
other studies performed in elderly general population ( but not only diabetics ) had showed also high prevalence [ 6 , 25 ] . in the cardiovascular health study cognition study approximately 41% of participants had depressive mood . in the large population study performed among japanese elders
the authors had found high coexistence rate for depression and mci and concluded that individuals with mci were more likely to develop depressive symptoms and vice versa . in agreement with many other results our study revealed significant predictors of having a depressive mood : female gender , single marital status , current and past smoking status , lack of physical activity , higher bmi , and total cholesterol level [ 26 , 27 ] . some researchers also indicated that depression is highly prevalent among diabetics and the risk of depression might be increased in the presence of other comorbid conditions .
we found that the strongest predictors of being diagnosed with mci and depressive symptoms were micro- and macrocomplications of diabetes like presence of retinopathy , previous cardiovascular disease , and stroke .
other data had also showed that diabetic complications and mortality were greater among depressed patients .
the microvascular and macrovascular complications of diabetes are augmented by the presence of depression in diabetes thus contributing to the increased mortality rate in this population .
it have been hypothesized that small vessel diseases in the brain ( white matter lesions and lacunae ) affect cognitive function in older diabetics without overt dementia or symptomatic stroke . the contribution of small vessel disease to depression has been also described .
pathogenesis other researchers had found that depressive symptoms were associated with increased risk of mci , and this association was independent of underlying vascular disease .
the authors had explained that depressive symptoms in the absence of overt cognitive impairment may reflect the early signs of a neurodegenerative disease or that depression leads to damage in the hippocampus through a glucocorticoid cascade .
although there have been many significant demographic , clinical , and biochemical risk factors associated with cognitive impairment and depressive syndrome the causative mechanisms of diabetes on brain 's complication are still unclear .
systematic screening and early intervention strategies could result in the identification of high - risk patients and the improvement of the effectiveness of the treatment .
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the aim of the study was to estimate the prevalence of mild cognitive impairment ( mci ) , depressive syndrome cases , and its comorbidity , and to identify predictors of these conditions .
methods .
276 diabetics elders were screened for mci and depressive symptoms .
detailed information of history of diabetes , and data of bmi , hba1c , and blood lipids were collected .
results .
the prevalence of mci was 31.5% , depressive syndrome was 29.7% , and mci with coexisting depressive mood was 9.1% .
the logistic regression analysis revealed that variables which increased the likelihood of having been diagnosed with mci were : higher hba1c level , previous cvd , hypertension , retinopathy , increased number of comorbidities , and less years of formal education .
significant predictors of having a depressive mood included female gender , single marital status , current and past smoking status , lack of physical activity , higher bmi and total cholesterol level , increased number of comorbidities , history of hypoglycemia , and insulin treatment .
factors associated with both mci and depressive syndrome were female gender , single marital status , past smoking status , retinopathy , previous cvd or stroke , increased number of comorbidities , and insulin treatment .
conclusions .
depressive symptoms , mci , and its comorbidity are common in elderly subjects with type 2 diabetes .
systematic screening could result in the identification of high - risk patients .
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the membrane is modeled as a lipid slab between two water regions using two implicit solvent models . the first one , imm1 , uses an empirical energy function , parameterized with experimental data .
imm1 primarily accounts for solvent exclusion effects and assumes that total solvation energy is the sum of group contributions .
it partially accounts for electrostatic interaction by using a distance dependent dielectric function allowing for screening of electrical charge .
the second model , gbsw , decomposes the solvation free energy into nonpolar and electrostatic components .
the nonpolar component is approximated as being proportional to the accessible surface area of the protein .
boltzmann method by a generalized born equation for the calculation of the solvation energy for an isolated charge in some dielectric medium . in our simulations ,
initial coordinates were obtained from the x - ray structure of tm segment of vpu ( pdb i d : 1pje ) . for both imm1 and gbsw ,
four independent simulations were carried out with a membrane thickness d equal to 15 , 19 , 23 , and 27 , respectively .
the initial structure was subjected to 100 steps of steepest - descent minimization followed by 500 steps of adopted - basis newton
the system was centered at the midpoint of the membrane as the origin and the helical axis of vpu aligned to the z - axis ( the normal to the membrane plane ) .
the system was heated from 0 to 298.15 k. subsequently , a 60-ns trajectory was generated using langevin dynamics .
algorithm was used to constrain bonds involving hydrogen , allowing for a 0.002-ps integration time step .
free - energy profiles for the tilt angle relative to the z - axis were determined by means of umbrella sampling and the weighted histogram analysis method ( wham ) .
this method has been extensively used to determine free - energy profiles , and as such , the details related to theoretical background and implementation are not included here ( see kumar et al . 1992 ; roux 1995 ) .
briefly , umbrella sampling involves applying a bias to restrict sampling along some reaction coordinate . to obtain potential of mean force ( pmf ) profiles , the reaction coordinate is divided into a number of sampling windows , and in each window , simulations are biased to sample conformations around some value of the reaction coordinate .
statistics are collected in each window , and then wham is used to unbias and combine statistics from all windows to generate the desired pmf profiles along the entire range of the reaction coordinate . in this study ,
the biasing potential was of the following form:\documentclass[12pt]{minimal }
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\begin{document}$$ v\left ( \theta \right)\ , = \,\frac{k}{2}\left ( { \theta \ , - \,\theta_{0 } } \right)^{2 } , $ $ \end{document}where k is the force constant , is the tilt angle ( defined as the angle subtended by the vector along the helical and the vector normal to the membrane ; fig . 1 ) ,
statistics were collected in 36 sampling windows in the range 0 = [ 0 , 90 ] , with a bin size of 2.5. in a typical window , a force constant of 250 kcal / mol / deg was used .
the first 1 ns was discarded , and the other 3 ns was used to collect statistics.fig .
schematic representation of the tilting of a tm protein under conditions of hydrophobic mismatch when membrane thickness ( d ) is smaller than the length of the helix ( l ) . in response to mismatch ,
the helix tilts relative to the normal of the membrane ( here the z direction ) such that hydrophobic side chains are buried inside the membrane protein reorientation in response to hydrophobic mismatch .
schematic representation of the tilting of a tm protein under conditions of hydrophobic mismatch when membrane thickness ( d ) is smaller than the length of the helix ( l ) . in response to mismatch ,
the helix tilts relative to the normal of the membrane ( here the z direction ) such that hydrophobic side chains are buried inside the membrane
the variation of the helix tilt as a function of time and membrane thickness , d , is summarized in fig . 2 .
2b ) , the helix quickly tilts in response to positive hydrophobic mismatch , and it thereafter fluctuates about their mean . in the case of imm1 , as the membrane thickness is decreased from 27 to 15 , the tilt angle , measured as the time average of the length of the trajectory , increased from 9 to 45 ( fig .
similarly , in the case of gbsw , as the membrane thickness is decreased from 27 to 15 , the tilt angle increased from 18 to 56 ( fig .
these values show very good agreement with mean tilt angles for both imm1 and gbsw . when cos is plotted against d , a roughly linear relationship is revealed , in agreement with park and opella ( 2005 ) ( fig .
these results corroborate those of other molecular dynamics simulation studies on -helical tm proteins . specifically , using an explicit lipid representation for the membrane , goodyear et al .
( 2005 ) observed that for a synthetic peptide initially oriented along the normal of membrane the helix tilts in response to positive hydrophobic mismatch , with an average tilt angle comparable to the experimentally determined value.fig .
tilt angles are shown as a function of time for vpu simulated in the imm1 ( a ) and gbsw ( b ) implicit membrane models .
results are shown for simulations where d ( membrane thickness ) is 15 ( black ) , 19 ( blue ) , 23 ( green ) , and 27 ( red ) . shown on each plot
is a plot of cos( ) vs. d determined using results from imm1 simulation ( red ) , gbsw ( black ) simulation , and experiment ( blue ) ( color figure online ) tilt angle time series .
tilt angles are shown as a function of time for vpu simulated in the imm1 ( a ) and gbsw ( b ) implicit membrane models .
results are shown for simulations where d ( membrane thickness ) is 15 ( black ) , 19 ( blue ) , 23 ( green ) , and 27 ( red ) . shown on each plot
is a plot of cos( ) vs. d determined using results from imm1 simulation ( red ) , gbsw ( black ) simulation , and experiment ( blue ) ( color figure online ) the next phase of the study involved the determination of free - energy profiles as a function of the tilt of the -helical peptide . by means of umbrella sampling ,
the free energy as a function of tilt angle was determined for angles ranging from 0 to 90 for membrane thicknesses of 15 , 19 , 23 , and 27 , respectively .
as mentioned earlier , if we assume that the optimal tilt angles a helix adopts in a membrane represent its energetically favorable orientations according to the free energy computed with the implicit solvent model , then by using these free - energy profiles , we can predict tilt angles . in the case of imm1
, for d = 15 , 19 , 23 , and 27 , the absolute minimum was located at 41 , 31 , 17 , and 11 , respectively ( fig .
gbsw , for d = 15 , 19 , 23 , and 27 , the absolute minimum was located at 51 , 41 , 29 , and 14 , respectively ( fig .
free - energy profiles are shown as a function of tilt angle ( ) calculated using the imm1 ( a ) and gbsw ( b ) implicit membrane models . shown are profiles calculated where d ( membrane thickness ) is 15 ( black ) , 19 ( blue ) , 23 ( green ) , and 27 ( red ) ( color figure online ) potential of mean force profiles .
free - energy profiles are shown as a function of tilt angle ( ) calculated using the imm1 ( a ) and gbsw ( b ) implicit membrane models . shown
are profiles calculated where d ( membrane thickness ) is 15 ( black ) , 19 ( blue ) , 23 ( green ) , and 27 ( red ) ( color figure online ) the fact that the results obtained from the imm1 and gbsw models so closely resemble the experiment is significant because it implies that the implicit models used for this system contained all the necessary physics as it relates to electrostatic and hydrophobic interaction within the membrane region .
although more detailed interactions such as anchoring interactions between peptides and lipids ( e.g. , tryptophan residues interacting with the polar head group region ; de planque et al .
2003 ) can compete with the fundamental free - energy driving force captured in imm1 and gbsw , we believe that our study shows that imm1 and gbsw , at least for systems as simple as the one used in this project , certainly embody the necessary functional form and parameters to describe the tilt process as a function of positive hydrophobic mismatch . imm1 and gbsw thus prove promising for membrane proteins , given the savings in computer time , while at the same time accurately recreating the solvent effects of the cell membrane and the surrounding aqueous regions .
they were both able to recreate the tilting of the -helical tm peptide vpu as function of hydrophobic mismatch that was observed experimentally , and they both exhibit a linear relationship between cos and d , in agreement with park and opella ( 2005 ) .
quantitatively , however , imm1 and gbsw give slightly different tilt angles for a given membrane thickness . in general , imm1 gave values lower than the experimental observed values , while gbsw gave higher values ( fig 2 ) .
this is also reflected in the free - energy profile , where qualitatively the models mirrored each other , but quantitatively there were slight difference ( gbsw predicted the minima to exist at higher for any given d ; fig .
the pmfs for imm1 and gbsw also differ as the tilt angle approaches 80. for imm1 , the barrier to tilting beyond the minima is larger as the membrane thickness decreases .
the trend is reversed slightly in the case of gbsw , but in this region , the difference amounts to a few kcal / mol . in terms of the performance of the algorithms ,
this can be attributed to the fact that gbsw involves the time - consuming calculation of the accessible surface area , which is used to approximate the nonpolar contribution to the solvation energy .
( 2005 ) was derived and recently applied to the vpu helix ( ulmschneider et al .
the basic hypothesis of that study was that each residue on its own would prefer a certain depth inside the membrane bilayer .
potentials of mean force along the membrane normal were derived for each amino acid by fitting gaussian functions to residue position distributions from known tm structures .
the resulting implicit membrane potential was tested on various membrane proteins as well as single trans - membrane helices .
all membrane proteins were found to be at an energy minimum when correctly inserted into the membrane .
in our computational test , the use of two widely used implicit membrane models , imm1 and gbsw , was sufficient to reproduce the experimentally measured tilting ( park and opella 2005 ) of an -helical peptide that occurs in response to positive hydrophobic mismatch . umbrella sampling - based free - energy profile calculations to obtain the distribution of the peptide tilt angles relative to the membrane proved to be a reliable means of gaining information concerning the free energy of the system .
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free - energy profiles describing the relative orientation of membrane proteins along predefined coordinates can be efficiently calculated by means of umbrella simulations .
such simulations generate reliable orientational distributions but are difficult to converge because of the very long equilibration times of the solvent and the lipid bilayer in explicit representation .
two implicit lipid membrane models are here applied in combination with the umbrella sampling strategy to the simulation of the transmembrane ( tm ) helical segment from virus protein u ( vpu ) .
the models are used to study both orientation and energetics of this -helical peptide as a function of hydrophobic mismatch .
we observe that increasing the degree of positive hydrophobic mismatch increased the tilt angle of vpu .
these findings agree well with experimental data and as such validate the solvation models used in this study .
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wilson and jungner have defined 10 criteria for screening of chronic diseases such as dm and dr .
the reported prevalence of dr in india ranges from 17.6% to 28.2% . with this prevalence
, the number of people with dm is likely to increase to 79.4 million and people with dr is likely to increase to 22.4 million by the year 2030 . both dm and dr
are relatively less , between 10% and 12% , in rural india compared to urban india but not small enough to ignore . considering that prevalence of dr at a moderate level of 20% in urban india and at a lower level of 10% in rural india and assuming that 70% of indians live in rural areas , the incidence of dr in rural india could rise to 10.97 million by the year 2030 .
in addition , there is an acute shortage of skilled manpower to screen dr in rural india .
traditionally , the two popular methods of dr screening are either ophthalmologist led or optometrist led . both methods are effective and are used both in india and globally . however , with the large emerging burden , it may no longer be the cost effective method of dr screening .
hence , a cost - effective dr screening is required in rural india , and this is possible with the existing and emerging technology of telemedicine .
the american telemedicine association has described four categories of dr screening from identification of mild npdr ( category 1 ) to the one that exceeds the ability of early treatment diabetic retinopathy study photographs ( category 4 ) [ table 2 ] .
american telemedicine association diabetic retinopathy telescreening categories dr screening is done by direct and indirect ophthalmoscopy and fundus photography .
it is also convenient because it does not need the ophthalmologist to be always present in the screening site ; the stored photographs could be examined later , and when in doubt , could be confirmed with another expert .
the technological improvements in fundus photography from analog to digital , from 30 to 45 to 60 field in most digital cameras , and from mydriatic to nonmydriatic retinal cameras have made the fundus photography as the most accepted form and standard of care in dr telescreening . in past , satellite connection has been used for transmission of the fundus photographs for teleconsultation . with improvement in internet and wire - free connection , the current generation of cameras is capable of transmitting the fundus images at fraction of cost incurred in the past .
we have established such a system of receiving the fundus photographs obtained in a hand - held nonmydriatic fundus camera placed at centers managing diabetes , and the images are both qualified and quantified for dr in the image reading center , l. v. prasad eye institute imaging laboratory and analysis center ( lilac ) .
certified technicians grade the images and the specialist 's opinion is sought in difficult ones .
this has further reduced the ophthalmologist 's time required to screen these patients . in a pilot study ,
the lilac received fundus photographs of 229 diabetic subjects over 6 months ; images of 32 subjects were ungradable ( small pupil , lens opacities , etc .
, ) and 94 were diagnosed to having treatable dr ( unpublished data ) . with increasing and powerful internet services planned in india , this method could be the most cost - effective method of dr telescreening in rural areas .
a few indian studies have confirmed the superiority of fundus photography in teleophthalmology of dr care and good patient satisfaction .
some indian investigators have used the services of the indian space research organization designed teleophthalmology vehicles to transmit images from outreach locations to the dr reading and grading center at the base eye care facilities . while this facility may not be available to everyone and everywhere , the internet or cloud - based facility will be cost effective that delivers equally reliable information .
the current lacunae in telescreening of dr in india are lack of enough broadband capacity in india .
as india is progressing in digital technology , we hope that this deficiency will be effectively addressed .
we foresee two important future developments ; one , further miniaturization of fundus camera , superior picture quality , and reduced cost of the cameras ; two , autodetection of fundus lesions in dr . this will empower and enable the patients for informed decision - making .
there are many attempts currently made for autodetection of retinal lesions . while we are on the threshold of witnessing a revolution in dr screening , the knowledge , attitude , and practice ( kap ) toward diabetes and retinopathy needs considerable improvement ( authors unpublished data ) .
we are less likely to benefit from these disruptive technological unless the kap also improves .
prevalence of dr in rural india ( or the world ) is not too different than the urban people .
however , the rural population is disadvantaged for not having a similar access to skilled personnel , particularly a specialist ophthalmologist similar to their urban counterpart . the modern technology of digital nonmydriatic photography , particularly the smartphones , fast expanding broadband transmission of these fundus pictures , and smarter autodetection of the retinal lesions will make the rural diabetic patients no more remain disadvantaged .
the success of these new developments partly rests on similar improvement in the kap of the rural folk .
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diabetic retinopathy ( dr ) is a growing concern in india .
the first step in management of dr is timely screening . with 10% prevalence in rural india ,
11 million people are likely to have dr by the year 2030 . with limited resources and skilled manpower
, it will not be possible to have routine eye examination to identify and treat these patients on a regular basis .
telemedicine is a possible answer in these situations where patients could be remotely screened and appropriately advised . with the advent of several technological advances such as low cost hand - held nonmydriatic camera , increased capabilities of the smartphones to take external eye and retinal photographs coupled with improving broadband connectivity ; teleophthalmology in the management of dr could be a reality in the not too distant future .
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natural proteins with over 35% sequence similarity tend to fold into similar conformations , yet several evolutionarily related natural protein pairs with up to 40% similarity have been observed to produce substantially different topologies .
two sequences bearing the same length and only three nonidentical residues were posted as sequential targets in the recent 8th community - wide experiment on the critical assessment of techniques for protein structure prediction ( casp8 ) .
targets t0498 and t0499 therefore posed a riddle for the international protein fold prediction community to determine whether the conformations of these 95% identical sequences maintain the same topological folds or adopt different ones .
the proteins were artificially produced in the group of john orban and philip bryan as a study of the tolerance of sequence identity to maintain the 3- and / folds of streptococcal protein g domain a ( ga ) and domain b ( gb ) , respectively .
the two 16% identity domains of protein g were brought together in sequence space first by adding terminal tails to ga to make it equal in length to gb and then by progressively mutating sites of nonidentity .
the key in this approach was linking each fold to its natural function : human serum albumin binding for the 3- ga fold and igg binding for the / gb fold .
this linkage of fold to function allowed the application of powerful biologic selection methods to determine clusters of sites in each protein , which could be substituted with the corresponding amino acid in the other protein .
more recently , two 95% identical sequences possessing the same fold , ga95 and gb95 , were designed and provided as the two casp8 targets discussed here .
the designed protein pairs maintain the fold and specific binding function of the proteins from which they were derived , with immeasurable structural or functional character of the domain represented in the alternate protein .
structural genomics consortia contribute thousands of new protein structures each year , yet previously unobserved folds are seldom found .
this pattern seems to indicate that the majority of folds that can be detected by current laboratory techniques have already been observed .
the completeness of the structural fold space has been addressed using a subset of 1,489 proteins covering the protein data bank at the level of 35% sequence identity ; all but two folds can be resolved using templates found within the same set .
thus , template - based modeling appears to be feasible given the best template(s ) within the set .
the best performing freely available fold recognition web server methods are maintained by yang zhang within the local meta - threading server ( lomets ) fold recognition pipeline of i - tasser ( iterative threading assembly refinement algorithm ) , the best performing protein structure prediction server in the past two casp experiments . as an isolated meta - threading server , lomets uses local implementation to avoid the destructive aspects of internet dynamic regulation corrupting so many meta - servers .
the nine methods of lomets are representative of the fold recognition field ( normally targeted toward naturally occurring proteins ) and can be summarized as various combinations of the following : comparing target to known structure sequence profiles , secondary structure preferences , environmental fitness , pairwise contact probabilities , structure profiles , simulated mutations , single - body or residue - specific knowledge - based potentials , and profile hidden markov models ( hmms ) .
most web server groups predicted both t0498 and t0499 to adopt the / fold of protein gb ( figure 1a ) .
for example , our own predictions for t0498 did not significantly resemble the target structure [ 37.2 global distance test total score ( gdt - ts ) ; figure 1b , left ] , yet all five of our predictions for t0499 were within the top 10 total predictions ( 88.4 gdt - ts ; figure 1b , right ) .
the models for t0499 exemplify progress in another major challenge in protein structure prediction : refinement of model quality from the best template .
( a ) only four out of over 150 contributing team groups recognized the difference in fold caused by three nonidentical residues in the 56 residue proteins : hhpred ( cyan ) , feig ( black ) , foldpro ( blue ) , and coma ( others are in orange ) .
the results are shown in global distance test ( gdt ) plot format , in which the alpha carbon atoms of the predicted model and experimental structure are spatially aligned within distance cutoffs of 0.5 , 1 , and 1.5 up to 10 , such that lower lines denote higher accuracy .
a common trend of these four groups was to predict the alternate fold as a lower confidence model .
most groups correctly identified the gb95 t0499 fold , yet most models were no better than random for ga95 t0498 .
the ability for four automated servers to disentangle this riddle provides a positive outlook for the fold recognition field .
( b ) predictions made by our group ( purple ) for t0498 and t0499 compared with the experimental structures for ga95 and gb95 ( cyan ) , respectively . while our predictions were among the very best for t0499/gb95 ( the only group with all five submissions in the top 10 ) , the incorrect fold assignment led to highly inaccurate predictions for t0498/ga95 . as with so many other protein structure prediction groups
ca , alpha carbon ; ga95 , the artificial protein with ga fold and 95% sequence identity to gb95 ; gb95 , the artificial protein with gb fold and 95% sequence similarity to ga95 .
the side chain interactions visible in the experimental structures of ga95 and gb95 , as well as the simulated mutant models depicted in figure 2 , demonstrate interactions within a relatively stable , folded state , which are not necessarily illustrative of those interactions occurring during the folding process .
even when the structures are known , it is difficult to ascertain exactly what makes the two proteins follow different fold trajectories .
rather , they rely on calculated interactions within simulated mutants of these folded structures to test the accuracy of fit for a possible template ; thus , even with a perfect energy function , mistakes in fold recognition could occur
. a sequence - to - structure cross of ga95 and gb95 is presented to demonstrate determinants of fold recognition from side chain packing of the nonidentical residues ( red ) .
the lack of profound steric clashes created by applying the side chain identities from t0498 to the structure of gb95 ( left ) misleads predictors to identify an incorrect fold topology .
conversely , the clash that occurs between f30 and a20 when applying the side chain identities from t0499 to the structure of ga95 ( right ) illustrates an incorrect fold for predictors .
ga95 , the artificial protein with ga fold and 95% sequence identity to gb95 ; gb95 , the artificial protein with gb fold and 95% sequence similarity to ga95 . in this case ,
a multitude of experimentally derived structures for ga and gb and detectable sequence similarity within this group reasonably limit the fold search to these topologies .
crossing fold assignments for ga95 and gb95 enables interrogation of side chain packing for the three nonidentical residues ( figure 2 ) .
the clash occurring between f30 and a20 when the nonidentities from t0499 are applied to the structure of ga95 ( figure 2 , right ) implicates an incorrect fold to predictors .
conversely , minimal steric clashes emerge when the t0498 sequence is applied to the structure of gb95 ( figure 2 , left ) .
this absence of incriminating evidence for the t0498 gb95 sequence fold pair could mislead predictors to select this fold topology . out of over 150 contributing teams ,
four groups recognized the difference in fold caused by three nonidentical residues in the 56 amino acid proteins : hhpred , foldpro , feig , and coma .
the accurate predictions of these groups demonstrate sensitivity to subtle changes affecting folding not previously demonstrated in a bona fide blind prediction scenario .
the emission sequence of hmms includes position - specific insertion and deletion probabilities along with the sequence distributions found in multiple sequence alignment profiles .
hhsearch specifically includes secondary structures via a substitution matrix derived from comparing measurements on the template to target predictions and to the confidence thereof . to interrogate alignments ,
the hhsearch method maximizes the coemission log - odds probability for the pair of hmms derived for a given protein pair .
hhsearch directs the structural similarity search hierarchically by searching databases of alignments organized by fold family rather than lists of disconnected sequences .
the csi - blast ( context - specific iterative basic local alignment search tool ) sequence similarity search method recently published by the group was likely used to build the profile input to the hmms for each alignment .
the cheng group uses a supervised classification approach previously used for fold classification , invoking support vector machines to combine global profile - profile alignment , secondary structure , solvent accessibility , contact map , and strand hydrogen bond pairing .
the feig group used a very typical set of methods , including fold recognition functions overlapping those in lomets ( including hhsearch / hhpred ) , standard model construction , and a modified cluster calculation using a standard discriminatory potential function . other promising work by this group in the refinement category includes the use of an implicit continuum dielectric solvent based on generalized born theory to drive lattice - based course grain searches , monte carlo molecular dynamics , and restrained normal mode sampling .
the venclovas group invokes a profile comparison method for detection of distant evolutionary relationships across profile databases , adding a modified two - level seg ( segment sequences by local complexity ) algorithm to filter noninformative profile regions , variable gap penalties , and adaptive parameterization .
the underlying sequence similarity search is driven by their psi - blast - iss ( position - specific iterative blast intermediate sequence search ) , which evaluates and refines output profile alignments .
the emission sequence of hmms includes position - specific insertion and deletion probabilities along with the sequence distributions found in multiple sequence alignment profiles .
hhsearch specifically includes secondary structures via a substitution matrix derived from comparing measurements on the template to target predictions and to the confidence thereof . to interrogate alignments
, the hhsearch method maximizes the coemission log - odds probability for the pair of hmms derived for a given protein pair .
hhsearch directs the structural similarity search hierarchically by searching databases of alignments organized by fold family rather than lists of disconnected sequences .
the csi - blast ( context - specific iterative basic local alignment search tool ) sequence similarity search method recently published by the group was likely used to build the profile input to the hmms for each alignment .
the cheng group uses a supervised classification approach previously used for fold classification , invoking support vector machines to combine global profile - profile alignment , secondary structure , solvent accessibility , contact map , and strand hydrogen bond pairing .
the feig group used a very typical set of methods , including fold recognition functions overlapping those in lomets ( including hhsearch / hhpred ) , standard model construction , and a modified cluster calculation using a standard discriminatory potential function . other promising work by this group in the refinement category includes the use of an implicit continuum dielectric solvent based on generalized born theory to drive lattice - based course grain searches , monte carlo molecular dynamics , and restrained normal mode sampling .
the venclovas group invokes a profile comparison method for detection of distant evolutionary relationships across profile databases , adding a modified two - level seg ( segment sequences by local complexity ) algorithm to filter noninformative profile regions , variable gap penalties , and adaptive parameterization .
the underlying sequence similarity search is driven by their psi - blast - iss ( position - specific iterative blast intermediate sequence search ) , which evaluates and refines output profile alignments .
a handful of the automated algorithms were able to recognize the fold switch caused by the three nonidentical residues of ga95 and gb95 ( figure 2 ) . however , the experimentally unobserved 60% of naturally occurring proteins and the prospect of designing new folds heralded by top7 demand more methods sensitive enough to detect subtle triggers in fold switching and predict previously unobserved topologies .
developments in the protein fold prediction field can often be limited to incremental engineering optimizations . in this fold recognition problem ,
the proper application of support vector machines and hmm methods enabled success for two groups .
also , two groups created their own improvements on psi - blast : csi - blast and psi - blast - iss , which both enhance quality and relevance of a search by interrogating low - quality regions in the alignment by context and together comprise the first significant improvements on the enormously popular algorithm in a decade .
the novel algorithmic adjustments in fold recognition used in casp8 demonstrate significant progress amounting to new tools for the field .
future developments are anticipated to include the steady stream of mathematical enhancements observed since the inception of the protein structure prediction field but also include new conceptual paradigms such as functional signatures and the use of template - free modeling to drive the difficult fold recognition problems .
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we examine the ability of current state - of - the - art methods in protein structure prediction to discriminate topologically distant folds encoded by highly similar ( > 90% sequence identity ) designed proteins in blind protein structure prediction experiments .
we detail the corresponding prognosis for the protein fold recognition field and highlight the features of the methodologies that successfully deciphered this folding riddle .
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with no therapy shown to improve clinical outcomes to date , the management of patients suffering from heart failure with preserved ejection fraction ( hfpef ) represents an ongoing challenge .
therapies of proven benefit in heart failure with reduced ejection fraction ( hfref ) have repeatedly been shown to add little if any benefit in hfpef , and the treatment of the latter group remains largely directed towards underlying risk factors and/or comorbidities .
the treatment of preserved cardiac function heart failure with an aldosterone antagonist ( topcat ) trial was designed to test the clinical benefit of spironolactone in patients with hfpef .
topcat was a multicenter , double - blind , randomized , placebo - controlled study designed to evaluate the efficacy of spironolactone relative to placebo on the cumulative incidence of a composite endpoint of cardiovascular death , heart failure hospitalization , or aborted cardiac arrest in patients with hfpef .
secondary endpoints included all - cause mortality , new onset of diabetes mellitus or atrial fibrillation , and quality of life .
hfpef was determined by the presence of lvef > 45% , signs and symptoms of heart failure in conjunction with prior hospital admission for hf within the prior year or elevated natriuretic peptides ( bnp > 100 pg / ml or nt - probnp > 360 pg / ml ) within the 60 days preceding randomization .
importantly , objective evidence of diastolic dysfunction was not required for enrolment in the study .
patients with uncontrolled hypertension , those with infiltrative or hypertrophic cardiomyopathy and patients with elevated baseline serum potassium levels ( > 5.0
a total of 3445 subjects were recruited over a period of 4 years from 270 clinical centers in the united states ( 1151 ) , russia ( 1066 ) , georgia ( 612 ) , canada ( 326 ) , brazil ( 167 ) and argentina ( 123 ) , and were randomized on 1:1 basis to either spironolactone ( target dose of 30 mg daily ) or placebo .
the study was funded by the national heart , lung , and blood institute ( nhlbi ) .
the results were recently presented at the american heart association meeting in dallas and will be published in the next issue of circulation heart failure . after a mean follow - up period of 3.3 years , the primary endpoint occurred in 320 ( 18.4% ) and 351 ( 20.4% ) of patients in the spironolactone and placebo groups respectively [ hr = 0.89 ( 0.771.04 ) , p = 0.138 ] .
patients receiving spironolactone had significantly fewer hospitalizations for heart failure compared to placebo ( 245 ( 14.2% ) vs. 206 ( 12% ) , hr = 0.83 ( 0.690.99 ) , p = 0.042 ) but all - cause hospitalization did not differ between both groups [ hr = 0.94 ; ( 0.851.04 ) ] .
the two other components of the primary endpoint cardiovascular mortality and aborted cardiac arrest did not differ between both groups .
hyperkalemia was more common in patients receiving spironolactone [ 322 ( 18.7% ) vs. 157 ( 9.1% ) for placebo , p < 0.001 ] , but there were no deaths related to hyperkalemia .
patients in the spironolactone group were also almost 50% more likely to experience doubling of creatinine above the upper limit of normal [ hr 1.49 ( 1.181.87 ) , p < 0.001 ] . however , the number of patients with creatinine levels 3 mg / dl and the number of patients who required dialysis did not differ between both groups . amongst 22 pre - specified subgroups ,
an interesting post - hoc analysis showed a striking regional difference in the placebo event rates : 280/881 ( 31.8% ) in the americas vs. 71 ( 8.4% ) in east europe .
aldosterone has several deleterious effects in patients with heart failure including salt and water retention , endothelial dysfunction , ventricular hypertrophy , and myocardial fibrosis .
aldosterone receptor blockers have previously been shown to significantly reduce mortality in a wide spectrum of patients with hfref . in hfpef ,
a number of small studies have previously suggested a beneficial effect with spironolactone , and more recently , the aldo - dhf study showed that spironolactone improved left ventricular diastolic function and remodeling compared to placebo .
topcat is the largest study conducted to date to test the effect of spironolactone on clinical outcomes in hfpef , and is one of the largest studies performed in this patient population in general .
the investigators are commended for successfully conducting such a large study with patients recruited from 270 centers across 6 countries in a disease entity where patient recruitment is notoriously known to be difficult .
the study 's rigorous monitoring system contributed to a low dropout rate of only 3.9% by the end of the study , and more importantly , prevented hyperkalemia - related deaths and/or increased dialysis in the active treatment group .
a number of features in topcat however raise some concern as to whether the study subjects truly represent the real - life hfpef population .
first , the overall event rate was low , with 3-year mortality being 10.2 % .
this is in sharp contrast with the previously reported annual mortality rates of 2229% in large community - based studies .
this concern is further intensified by a primary event rate ( in the placebo group ) of 8.4% in russia and the republic of georgia : a rate which not only is unheard of in heart failure studies , but also one that is remarkably less than that observed in the american arm of the same study ( 31.8% ) .
second , the investigators recently published the findings from the echocardiographic substudy of topcat where the echocardiograms of 935 patients approximately 27% of the topcat population
the echo substudy revealed that 46% of this group had normal left atrial size ( indexed left atrial volume < 29 ml / m2 ) and 17% of patients had normal tissue - doppler derived diastolic velocities .
third , compared to patients qualifying for topcat on the basis of prior hospitalization , those enrolled via the elevated natriuretic peptide route were more likely to be recruited in the americas , had significantly larger indexed left atrial volumes ( median 26.6 vs. 30.3 ml / m2 , p < 0.0001 ) , and were more likely to benefit from spironolactone with the primary endpoint occurring in 15.9% in the spironolactone arm vs. 23.6% with placebo , p = 0.003 ( compared to 19.6% vs 19.1% respectively , p = 0.92 in the heart failure hospitalizations group ) .
fourth , the primary end point hazard ratio for the american group ( almost half of topcat 's population ) was 0.82 ( 95% ci 0.690.98 ) compared to 1.1 ( 95% ci 0.791.51 ) in the eastern european group . collectively , these points raise the possibility that a considerable number of patients qualifying for topcat because of prior hospitalization for heart failure , might not have suffered from hfpef in the first place ; a limitation previously encountered with other major hfpef trials owing to the non - specific signs and symptoms of hfpef .
in fact , recruiting markedly heterogonous patient populations ( including non - cardiac patients ) and non - adherence to the recommended diagnostic criteria ( which require objective evidence of lv diastolic dysfunction ) have previously been suggested as possible explanations for the neutral / negative outcomes of hfpef trials .
whether these limitations might have masked a beneficial effect in the spironolactone group remains subject to further detailed analysis .
one might be tempted to choose focusing on the positive effect seen with spironolactone in the american arm or in those with elevated natriuretic peptides , however , this can be very misleading from a statistical standpoint .
topcat was a negative study ; spironolactone failed to reduce the primary outcome compared to placebo in patients with hfpef .
a signal of benefit was also seen in patients with elevated natriuretic peptides and in a geographical subset of patients . based upon these findings , a mixed response from the medical community
is expected : some clinicians will not prescribe spironolactone for hfpef patients , while others will continue using it especially in patients with elevated natriuretic peptides and/or in those with objective evidence of diastolic dysfunction . careful monitoring of renal function and serum potassium levels is mandatory in the latter situation .
given the lack of interest from the pharmaceutical industry in a cheap generic drug and the finite resources of the nhlbi , it is doubtful that there will be any large spironolactone trials in the near future
. it is therefore unlikely that this therapeutic dilemma will be definitively resolved soon .
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heart failure with preserved ejection fraction ( hfpef ) continues to be a challenging form of heart failure one in which no therapy has yet been proven to improve outcome .
aldosterone antagonists have previously been shown to improve survival in a wide spectrum of patients with heart failure with reduced ejection fraction ( hfref ) , and more recently , small trials suggested that they might have role in hfpef patients .
the effect of spironolactone on clinical outcomes in hfpef was tested in the topcat study .
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nematode larvae of the genus anisakis dujardin ( nematoda : anisakidae ) are common parasites of marine fish with a worldwide distribution .
these nematodes have a complex life cycle involving organisms at various levels of a trophic web in the marine ecosystem .
fish and squid are the intermediate or paratenic hosts of the parasites , whereas marine mammals mainly cetaceans are the definitive ones .
adult forms for these parasites found in the gastrointestinal tract of a specific vertebrate host and the life cycles of numerous taxa involve the development of larval stages in one or more intermediate hosts .
they have a potential risk for human health since these larvae can infect humans after the ingestion of raw or undercooked fish [ 68 ] .
berland classified the larvae of anisakid nematodes into two types , namely , anisakis types i and ii , based on morphological characteristics such as the length of the ventriculus and presence or absence of mucron at the tip of the tail .
controversy regarding the species names of anisakis type i and ii larvae still remains because the larvae lack the detailed morphological characteristics required for precise species identification due to their small size and poor morphological distinctions of these nematodes , particularly their larvae [ 10 , 11 ] . on the other hand
, molecular tools can overcome this limitation and allow the genus- or species - specific identification of anisakids .
recent molecular techniques such as allozyme analysis , polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) , and sequencing of the internal transcribed spacer ( its ) region ( its1 - 5.8s rrna - its2 ) of ribosomal dna revealed that the anisakis species consisted of 8 valid species , 5 species : anisakis simplex , anisakis pegreffi , anisakis typica , anisakis ziphidarum , and anisakis sp . and 3 species : anisakis physeteris , anisakis brevispiculata , and anisakis paggiae in the anisakis type i and anisakis type ii groupings , respectively .
therefore , the purpose of the present study was to report the occurrence of anisakid larvae in the selected fish specimens from egyptian water .
in addition , identification of the isolated larvae conducted by using morphological studies combined with specific identification using molecular analysis in order to clarify the taxonomic position of the present anisakis species .
hurghada is one of the most tourist places located on the red sea governorate and far about 399.84 km from cairo .
it stretches for about 36 km along the seashore at 2715n , 3350e for latitude and longitude , respectively .
sixty specimens of the european hake merluccius merluccius lessepsianus belonged to family merlucciidae were trapped alive during the period of february to november 2014 from the commercial fishermen from the studied area .
the weight and length of the collected samples were ranged between 1728 kg and 60110 cm .
all specimens were transferred to laboratory of parasitology in faculty of science at cairo university , under good aeration .
fish were dissected and then their body cavities and internal organs were examined for the presence of parasitic infections under a stereomicroscope .
parasites were removed , counted , and preserved in 70% ethyl alcohol and then cleared in 5% glycerin for further examination and species identification . for scanning electron microscopy , specimens were fixed at 3% glutaraldehyde , washed in sodium cacodylate buffer , dehydrated in a graded series of ethanol , and then examined and photographed under an etec auto scan at 20 kv in electron microscope unit at faculty of science , ain shams university , egypt .
measurements were taken with an olympus ocular micrometer and expressed in millimeter as range followed by mean sd in parentheses ; otherwise they were stated .
the recovered parasites were removed from ethanol and snap frozen in liquid nitrogen prior to dna extraction by using qiaamp1 dna mini kit ( qiagen , gmbh , germany ) according to the manufacturer 's recommendations .
the reactions were performed with primers nc5 ( forward : 5-taggtgaacctgcggaaggatcatt-3 ) and nc2 ( reverse : 5-ttagtttcttttcctccgct-3 ) as mentioned by zhu et al . .
selected amplicons were extracted and purified directly using qiagen qiaquick ( valencia , ca ) columns and then cycle sequenced using abi bigdye terminator v3.1 cycle sequencing ready reaction kit ( applied biosystems inc . , foster city , ca , usa ) on automated capillary sequencer ( abi prism 3100 , applied biosystems ) using the same primers .
bootstrap mp tree was inferred using 1.000 replicates of branch - and - bound method .
animal use followed a protocol approved and authorized by institutional animal care and use committee ( iacuc ) at zoology department in faculty of science , cairo university , egypt .
twenty - two ( 36.66% ) out of sixty examined specimens of european hake m. merluccius lessepsianus were generally found to be naturally infected with anisakis larvae .
the rate of infection was increased during winter to be 60% ( 18/30 ) and fall to be 13.33% ( 4/30 ) in summer season .
these anisakid nematodes were found usually as encapsulated larvae in visceral organs including the peritoneum . the number of larvae per fish ranged from 5 to 30 . the mean intensity of the nematode larvae depended on the fish length .
the highest intensity was recorded in the hosts with a length > 80 cm , which means that the relationship between host length and intensity of infection was positive .
body of the recovered third - stage larvae was cylindrical in shape , attenuated at both ends , and measured 14.125.6 ( 20.4 1.2 ) mm in length and 0.480.62 ( 0.55 0.02 ) mm in width .
the larvae covered with a rigid cuticle that has an annular transverse striation that start from the cephalic region and extended to the slit - shaped anus .
four small labial papillae ( two dorsolateral and two ventrolateral ) were surrounding the triradiate mouth opening .
longitudinal lateral grooves are extended along the body and starting next to the mouth area and ended before the mucron .
the larvae 's oesophagus had anterior muscular part and measured 1.182.68 ( 2.12 0.2 ) mm long and a glandular ventriculus measured 0.710.92 ( 0.82 0.1 ) mm long with an oblique esophagointestinal junction .
the excretory duct runs from the excretory pore , which is situated ventrally below the larval tooth .
the body of larvae ended at a short mucron which measured 0.0190.032 ( 0.025 0.02 ) mm long .
a total of 295 bp was obtained with 45.1% gc content for ssu rdna gene sequences of the present anisakis species .
calculating the percentage of identity between these novel sequences with others retrieved from genbank demonstrated a high degree of similarity ( > 84% ) . this sequence in conjunction with existing data
comparison of the nucleotide sequences and divergence showed that ssu rdna of this species revealed the highest blast scores with small number of nucleotide differences with four anisakidae species , 98% with jx986995 anisakis simplex ( studied previously ) , 94% with am503954 a. pegreffi , 87% with ab592795 a. paggiae , and 86% with jn968636 a. physeteris ( figure 3 ) .
the phylogenetic tree is represented by two major clades : the major one clusters all anisakidae species that were located on a well - supported branch in the ml bootstrap analysis with sequence similarity ranged from 98% to 84% ( figure 4 ) .
the minor clade contains ab277824 pseudoterranova decipiens as out - group with a high divergence value
the parasite is anisakis simplex third - stage larvae ( f : anisakidae ) .
the host is european hake merluccius merluccius lessepsianus linnaeus ( f : merlucciidae ) .
22 ( 36.66% ) out of 60 specimens of the examined fish were infected .
voucher specimens were deposited in zoology department , faculty of science , cairo university , cairo , egypt .
anisakis species have complex life cycles which pass through a number of hosts through the course of their lives .
eggs hatch in seawater , and larvae are eaten by crustaceans , usually euphausiids [ 24 , 25 ] . the infected crustacean is subsequently eaten by a fish or squid , and the nematode burrows into the wall of the gut and encysts in a protective coat , usually on the outside of the visceral organs , but occasionally in the muscle or beneath the skin .
the life cycle is completed when an infected fish is eaten by a marine mammal , such as a whale , seal , or dolphin .
the nematode excysts in the intestine , feeds , grows , mates , and releases eggs into the seawater in the host 's feces . as the gut of a marine mammal is functionally very similar to that of a human
, anisakis species are able to infect humans who eat raw or undercooked fish and they can cause significant clinical diseases [ 24 , 27 , 28 ] . since the first reports confirming the pathogenic effects of anisakis species in humans [ 29 , 30 ] , there has been increasing awareness of fish - borne parasitic diseases [ 20 , 31 , 32 ] .
anisakidae nematodes have been recorded in the present study with 36.66% as a percentage of infection for the european hake m. merluccius lessepsianus represented as high values for larvae abundance ; this is coincided with purivirojkul who stated that larvae of the genus anisakis were found in four fish species ( muraenesox sp .
, epinephelus areolatus , rachycentron canadum , and trichiurus lepturus ) with an infection rate of 52.07% , followed by pardo - gandarillas et al . who found a similar prevalence in the european hake from the atlantic off northwest africa .
several other studies were shown that anisakid larval stages were mostly found in the musculature of different fish species [ 3436 ] , while , in the present study , anisakid larvae penetrate the gut wall and are generally encapsulated on the visceral organs , and peritoneum .
the increase in the infection level of anisakis ( measured by prevalence , intensity , and abundance ) with the age or length of the host reported has often been explained by the accumulation of the parasite throughout the host life and by the increased amount of food ingested by larger fish [ 3740 ] . in the present study
this relationship between prevalence and host length / weight can be seen in a positive correlation .
the known diversity of the genus has increased greatly over the past 20 years , with the advent of modern genetic techniques in species identification .
each final host species was discovered to have its own biochemically and genetically identifiable sibling species of anisakis , which is reproductively isolated .
this finding has allowed the proportion of different sibling species in a fish to be used as an indicator of population identity in fish stocks . based on
morphological characters , nematodes of the present study characterized by the presence of inconspicuous lips possess a prominent boring tooth on the anterior end , and straight gut structure consisting of oesophagus , ventriculus , and intestine , and were identified as anisakis larvae .
these results are coincided with data obtained by rocka in which the third - stage larvae ( l3 ) of anisakis simplex had the above - mentioned characters .
the present species were compared morphologically and morphometric with other anisakis species as shown in table 1 . according to the ventriculus length , the presence or absence of mucron can distinguish anisakis type i and type ii larvae as mentioned by setyobudi et al . .
our finding showed that the ventriculus length was 0.710.92 ( 0.82 0.1 ) mm and the mucron observed and measured 0.0190.032 ( 0.025 0.02 ) mm long ; therefore , our measurement data were overlapped with other a. simplex described previously by hurst , larizza and vovlas , quiazon et al . , and setyobudi et al . .
morphological differentiation of larval and adult stages of anisakid nematodes is difficult , but using molecular identification methods , such as pcr - coupled mutation scanning combined with selective sequencing of the its region of the rdna , has proved to be reliable techniques for the unequivocal identification and differentiation of anisakid nematodes [ 12 , 44 , 45 ] . in the present study , a nuclear rdna region
was amplified by using nc5/nc2 primers mentioned in previous studies by zhu et al . .
its-1 and its-2 regions representing the anisakis larvae identified herein had the sequence similar to homologous regions within the nuclear ribosomal sequence of a. simplex and a. pegreffi . apparently , the tree estimated in this study strongly supported several of the higher taxonomic groups .
anisakidae species represented by the genus anisakis is monophyletic in origin with 100% bootstrap support ; this result is in accordance with that obtained by fagerholm who stated that the rdna trees ( ml and mp ) , taxa representing the contracaecinae , anisakidae , and raphidascarididae , were each monophyletic and reliably supported by strong bootstrap resampling . on the mp tree ,
genus - specific clusters with moderate and strong support were delineated in the genera anisakis and contracaecum .
the mp tree supported the taxonomic position of the present anisakis species which is deeply embedded in the genus anisakis with a close relationship with a. simplex as a more related sister taxon with 100% bootstrap support .
it could be concluded that the parasite species found in m. merluccius lessepsianus was anisakis type i larvae and termed as anisakis simplex third - stage larvae with a unique genetic sequence and having new locality records in egyptian water .
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the prevalence of infection and the identification of anisakid larvae in european hake merluccius merluccius lessepsianus from hurghada city , red sea governorate , egypt , were investigated .
fish samples were collected during the period of february and november 2014 .
twenty - two ( 36.66% ) out of sixty examined fish specimens were found to be naturally infected with anisakis type i larvae mostly found as encapsulated larvae in visceral organs .
there was a positive relationship between host length / weight and prevalence of infection . based on morphological , morphometric , and molecular analyses ,
these nematodes were identified as third - stage larvae of anisakis simplex .
the present study was considered as the first report of anisakid larvae from european hake in the egyptian water .
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the management of acute diverticulitis complicated by perforation traditionally has been the hartmann 's procedure , which may be associated with significant morbidity and mortality and the unpleasantness of a colostomy .
experience in minimal access surgery has increased , and an established place now exists for laparoscopic management of perforated sigmoid diverticular disease ( pdd ) .
we present a case study of a patient presenting with perforated acute diverticulitis managed with laparoscopic oversewing , present a systematic review of the literature , and suggest a management protocol for the laparoscopic management of this surgical emergency .
a 50-year - old male patient presented with a 24-hour history of central abdominal pain localized to the right iliac fossa .
his vital signs were normal , and his right iliac fossa was tender when palpated .
his white cell count was 8600 ml ( normal range , 4300 to 10 800 ) and serum c - reactive peptide level was 129 mgl ( normal range , < 3 ) . diagnostic laparoscopy was performed at which a localized perforation of a sigmoid diverticulum with surrounding abscess cavity adherent to the anterior abdominal wall was seen ( figure 1 ) .
copious lavage of the peritoneal cavity was carried out followed by laparoscopic suture repair of the perforation and insertion of a peritoneal drain .
his bowels returned to normal function by the fourth postoperative day when the peritoneal drain was removed .
the patient subsequently underwent a barium enema study 6 weeks following his in - patient episode , which confirmed the presence of sigmoid diverticular disease .
a literature search of medline from 1950 to september 2007 using the ovid interface combined with manual cross - referencing was performed by using the following strategy : limit ( sigmoid.mp . or colon , sigmoid/ and intestinal perforation / or perforation.mp . and laparoscopy.mp . or laparoscopy/ ) to ( humans and english language ) all publications pertaining to the laparoscopic management of perforated sigmoid diverticular disease were analyzed and then categorized according to their level of evidence . further manual cross - referencing of cited papers
data were then categorized by management strategy and recommendations were then made on the basis of the available data .
forty - two publications were found , 25 of which were of sufficient quality and relevance to be included in this review . the modified hinchey system is a well - recognized method of classifying acute diverticulitis .
hinchey described 2 distinct evolutionary processes of perforated diverticular disease : first , where the neck of the diverticulum is obliterated and the peritonitis is purulent , and second where the neck fails to seal and feculent perforation occurs .
classification is important to both select appropriate management and also to compare and pool outcome data from the experience of different surgeons .
we suggest that management should be along the lines of a classification based on the hinchey system ( table 1 , figure 2 ) .
modified hinchey classification of acute diverticulitis [ modified from ( 9 ) and ( 26 ) ] proposed laparoscopic management algorithm for perforated sigmoid diverticular disease .
although optimum treatment for perforated diverticular disease is resection , a trend now exists towards down staging
a study from germany suggests that obesity , cardiac risk factors , and most significantly immunosuppression are predictive of morbidity and mortality .
hence , the rationale for a damage limitation approach to the initial operation to pave the way for resection when the patient is systemically improved .
a retrospective review from australia reports on researchers ' experience with 14 patients with pdd managed laparoscopically .
ten patients had hinchey grade 3 purulent peritonitis , 2 had grade 2 contamination , and 2 had grade 4 feculent peritonitis .
eleven patients ( 79% ) improved and were discharged following a median of 6.5 days ( range , 5 to 32 ) , whereas 3 patients did not improve and underwent acute resection . of the 11 that settled ,
8 patients subsequently underwent elective resection ( 7 laparoscopic ) without a stoma at a mean interval of 6 weeks .
faranda et al report on a series of 18 patients with peritonitis secondary to perforated acute diverticulitis successfully managed with emergency laparoscopy .
patients had extensive peritoneal lavage , the sigmoid lesion was stuck with biologic glue , the peritoneum was drained , and antibiotic therapy was given .
no patient had a wound abscess or residual deep collections , and mean postoperative stay was 8 days .
a series of 29 patients with purulent perforated sigmoid diverticulitis successfully initially managed with laparoscopic lavage and peritoneal drain insertion reported no serious complications , and all cases resolved without further intervention .
more recently , in a prospective trial from ireland of 92 patients with perforated diverticular disease ( hinchey grades 2 and 3 ) managed by laparoscopic lavage , peritoneal drainage and antibiotics showed favorable results .
the authors report that 89% of patients made an uneventful recovery ; morbidity and mortality rates were 4% and 3% , respectively .
two instances of persistent pelvic abscesses , one managed by percutaneous drainage and one by an open hartmann 's procedure , were reported ; and 2 patients re - presented with diverticulitis during the mean follow - up period of 36 months . with patients in whom the risk of anesthesia is prohibitively high due to comorbidity , computerized tomography ( ct ) may be performed to confirm the diagnosis of pdd and establish percutaneous peritoneal drainage under ct control . a number of small series of between 10 and 16 patients report on short - term successes of radiological placement of peritoneal drains for pdd .
lovisetto et al report laparoscopic suture repair and creation of diverting loop colostomy of an iatrogenic perforated sigmoid diverticulum following colonoscopy .
other papers reports on a successful laparoscopic suture repair of an iatrogenic postcolonoscopy sigmoid perforation without diverting colostomy . a study of a recent case series of laparoscopic repair of iatrogenic postcolonoscopic sigmoid diverticular perforation reports on 11 attempted ( 6 successful ) laparoscopic repairs , with a mean perforation size of 2.7 cm .
three patients were immediately converted to open repair ; one patient who underwent repair of a 4-cm perforation leaked and had a further open repair .
laparoscopy of one patient who underwent a delayed laparotomy for sepsis had an initially nondiagnostic laparoscopy .
a case report of laparoscopic repair of an iatrogenic sigmoid diverticular perforation in which the endostich device was used following colonoscopy resulted in reoperation at day 8 following repair .
the authors suggest that the nature of the endostich device may have lead to inadequate bites of the bowel being taken in the closure .
a review of 197 laparoscopic colonic wall biopsies for slow motility in children reported 8 patients ( 2 not recognized at index operation ) requiring closure of mucosal perforations using endoloops .
all 8 recovered well with no complications . in a gynecological series of 262 women undergoing pelvic laparoscopy , nezhat
rigid sigmoidoscopy and air insufflation into the colon is performed whilst the pelvis filled with saline is observed for air bubbles .
they report that 4 bowel injuries as well as one incomplete repair were identified using this technique and repaired with laparoscopic suturing with no adverse events .
the successful use of the laparoscopic linear stapler to repair postcolonoscopy diverticular perforation was described by hayashi et al .
yamamoto et al report a series of 5 patients with iatrogenic postcolonoscopy perforated sigmoid diverticula who were successfully treated laparoscopically by application of a linear stapler after the edges of the perforations had been aligned by passing stay sutures through each side of the perforations .
a report of a series of 18 patients from the cleveland clinic undergoing laparoscopic sigmoid colectomy for diverticulitis suggests that resection is feasible for class 1 disease with no morbidity , mortality , or conversion to open surgery necessary . however , half of those operated on for class 2 disease necessitated conversion to open surgery , and the incidence of morbidity was 33.3% .
reports of other single - center series of laparoscopic sigmoid colectomy for class 1 and 2 diverticulitis have reported low conversion and complication rates .
a multicenter study of laparoscopic resections for diverticulitis revealed a high rate of conversion to open surgery and a high rate of complications , 18.2% and 28.9% ( 31.8% following conversion ) , respectively .
this view is further supported by the results of a multi - center study showing that more experienced surgeons performed laparoscopic sigmoid colectomy for more complicated cases and had fewer complications than did less experienced surgeons .
a few reports exist in the literature of favorable results with resection and primary anastomosis for class 4 diverticulitis in open surgery .
the authors stress that absence of comorbidity , good response to resuscitation , and availability of intensive care facilities were key to success in these highly selected cases . in 1999 , a consensus conference held by the european association of endoscopic surgery concluded that after 2 attacks of diverticulitis , sigmoid colectomy should be considered and that laparoscopic sigmoid colectomy should be performed for either uncomplicated cases or after abscess had been drained percutaneously .
however , these and similar north american recommendations have recently been called into question by investigators studying 150 patients with diverticulitis who found that having more than 2 episodes of diverticulitis does not increase risk of presenting with perforated diverticular disease or of surgical morbidity or mortality .
hinchey described 2 distinct evolutionary processes of perforated diverticular disease : first , where the neck of the diverticulum is obliterated and the peritonitis is purulent , and second where the neck fails to seal and feculent perforation occurs .
classification is important to both select appropriate management and also to compare and pool outcome data from the experience of different surgeons .
we suggest that management should be along the lines of a classification based on the hinchey system ( table 1 , figure 2 ) .
modified hinchey classification of acute diverticulitis [ modified from ( 9 ) and ( 26 ) ] proposed laparoscopic management algorithm for perforated sigmoid diverticular disease .
although optimum treatment for perforated diverticular disease is resection , a trend now exists towards down staging
a study from germany suggests that obesity , cardiac risk factors , and most significantly immunosuppression are predictive of morbidity and mortality .
hence , the rationale for a damage limitation approach to the initial operation to pave the way for resection when the patient is systemically improved .
a retrospective review from australia reports on researchers ' experience with 14 patients with pdd managed laparoscopically .
ten patients had hinchey grade 3 purulent peritonitis , 2 had grade 2 contamination , and 2 had grade 4 feculent peritonitis .
eleven patients ( 79% ) improved and were discharged following a median of 6.5 days ( range , 5 to 32 ) , whereas 3 patients did not improve and underwent acute resection .
of the 11 that settled , 8 patients subsequently underwent elective resection ( 7 laparoscopic ) without a stoma at a mean interval of 6 weeks .
faranda et al report on a series of 18 patients with peritonitis secondary to perforated acute diverticulitis successfully managed with emergency laparoscopy .
patients had extensive peritoneal lavage , the sigmoid lesion was stuck with biologic glue , the peritoneum was drained , and antibiotic therapy was given .
no patient had a wound abscess or residual deep collections , and mean postoperative stay was 8 days .
a series of 29 patients with purulent perforated sigmoid diverticulitis successfully initially managed with laparoscopic lavage and peritoneal drain insertion reported no serious complications , and all cases resolved without further intervention .
more recently , in a prospective trial from ireland of 92 patients with perforated diverticular disease ( hinchey grades 2 and 3 ) managed by laparoscopic lavage , peritoneal drainage and antibiotics showed favorable results .
the authors report that 89% of patients made an uneventful recovery ; morbidity and mortality rates were 4% and 3% , respectively .
two instances of persistent pelvic abscesses , one managed by percutaneous drainage and one by an open hartmann 's procedure , were reported ; and 2 patients re - presented with diverticulitis during the mean follow - up period of 36 months .
with patients in whom the risk of anesthesia is prohibitively high due to comorbidity , computerized tomography ( ct ) may be performed to confirm the diagnosis of pdd and establish percutaneous peritoneal drainage under ct control . a number of small series of between 10 and 16 patients report on short - term successes of radiological placement of peritoneal drains for pdd .
lovisetto et al report laparoscopic suture repair and creation of diverting loop colostomy of an iatrogenic perforated sigmoid diverticulum following colonoscopy .
other papers reports on a successful laparoscopic suture repair of an iatrogenic postcolonoscopy sigmoid perforation without diverting colostomy . a study of a recent case series of laparoscopic repair of iatrogenic postcolonoscopic sigmoid diverticular perforation reports on 11 attempted ( 6 successful ) laparoscopic repairs , with a mean perforation size of 2.7 cm .
three patients were immediately converted to open repair ; one patient who underwent repair of a 4-cm perforation leaked and had a further open repair .
laparoscopy of one patient who underwent a delayed laparotomy for sepsis had an initially nondiagnostic laparoscopy .
a case report of laparoscopic repair of an iatrogenic sigmoid diverticular perforation in which the endostich device was used following colonoscopy resulted in reoperation at day 8 following repair .
the authors suggest that the nature of the endostich device may have lead to inadequate bites of the bowel being taken in the closure .
a review of 197 laparoscopic colonic wall biopsies for slow motility in children reported 8 patients ( 2 not recognized at index operation ) requiring closure of mucosal perforations using endoloops .
all 8 recovered well with no complications . in a gynecological series of 262 women undergoing pelvic laparoscopy , nezhat
rigid sigmoidoscopy and air insufflation into the colon is performed whilst the pelvis filled with saline is observed for air bubbles .
they report that 4 bowel injuries as well as one incomplete repair were identified using this technique and repaired with laparoscopic suturing with no adverse events .
the successful use of the laparoscopic linear stapler to repair postcolonoscopy diverticular perforation was described by hayashi et al .
yamamoto et al report a series of 5 patients with iatrogenic postcolonoscopy perforated sigmoid diverticula who were successfully treated laparoscopically by application of a linear stapler after the edges of the perforations had been aligned by passing stay sutures through each side of the perforations .
a report of a series of 18 patients from the cleveland clinic undergoing laparoscopic sigmoid colectomy for diverticulitis suggests that resection is feasible for class 1 disease with no morbidity , mortality , or conversion to open surgery necessary . however , half of those operated on for class 2 disease necessitated conversion to open surgery , and the incidence of morbidity was 33.3% .
reports of other single - center series of laparoscopic sigmoid colectomy for class 1 and 2 diverticulitis have reported low conversion and complication rates .
a multicenter study of laparoscopic resections for diverticulitis revealed a high rate of conversion to open surgery and a high rate of complications , 18.2% and 28.9% ( 31.8% following conversion ) , respectively .
this view is further supported by the results of a multi - center study showing that more experienced surgeons performed laparoscopic sigmoid colectomy for more complicated cases and had fewer complications than did less experienced surgeons .
a few reports exist in the literature of favorable results with resection and primary anastomosis for class 4 diverticulitis in open surgery .
the authors stress that absence of comorbidity , good response to resuscitation , and availability of intensive care facilities were key to success in these highly selected cases .
in 1999 , a consensus conference held by the european association of endoscopic surgery concluded that after 2 attacks of diverticulitis , sigmoid colectomy should be considered and that laparoscopic sigmoid colectomy should be performed for either uncomplicated cases or after abscess had been drained percutaneously .
however , these and similar north american recommendations have recently been called into question by investigators studying 150 patients with diverticulitis who found that having more than 2 episodes of diverticulitis does not increase risk of presenting with perforated diverticular disease or of surgical morbidity or mortality .
current evidence on the 2-staged laparoscopic approach to pdd consists of case series ( level 2 ) .
additional larger case series and controlled trials are required to establish the benefit of laparoscopic management of pdd in the wider surgical community .
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background : to develop an evidence - based protocol for the management of perforated sigmoid diverticular disease.methods:a search of the literature was undertaken .
all publications pertaining to perforated sigmoid diverticular disease were analyzed and then categorized according to their level of evidence .
recommendations were then made on the basis of this.results:multiple case reports suggest that primary closure of perforation of sigmoid diverticula is safe in the absence of peritoneal contamination.conclusions:a 2-stage laparoscopic approach incorporating the principles of damage limitation surgery may be a safe strategy in the management of perforated diverticular disease .
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at neonatal age , all patients were palliated with a waterston shunt to increase pulmonary blood flow . when they were toddlers , the shunt was taken down and total surgical repair was performed with enlargement of the hypoplastic pulmonary trunk using a transannular patch ( except in case c where no transannular patch was used ) .
all patients gradually developed severe pulmonary regurgitation and progressive right ventricular dilatation and dysfunction , as well as a significant stenosis of the right pulmonary artery at the anastomosis site of the former waterston shunt .
case a was a male patient of 39 years who was transferred from a referring hospital for management of severe pulmonary regurgitation and sustained ventricular tachycardia with a left bundle branch morphology and an inferior axis .
case b was a 38-year - old female who had visited our outpatient clinic on a regular basis since early childhood . at the age of 14
the right ventricular pressure at that time was 70/12 mmhg as a consequence of stenoses in both the right and left pulmonary artery branches ( gradient 20 mmhg and 35 mmhg , respectively ) in combination with moderate pulmonary valve stenosis .
she had been completely asymptomatic and was managed conservatively . at the age of 31 ,
pulmonary regurgitation had become severe but there were still no echocardiographic signs of dilatation or dysfunction of the right ventricle . at the age of 39 ,
cardiac mri showed a significant increase of right ventricular end - diastolic and end - systolic volumes within one year and confirmed severe pulmonary regurgitation .
case c was a female patient with tetralogy of fallot who had given birth to twins without any cardiac complications .
she visited the outpatient clinic on a regular basis because of moderate pulmonary regurgitation with normal dimensions and function of the right ventricle .
she gradually developed right ventricular systolic dysfunction due to severe pulmonary regurgitation in combination with pulmonary valve stenosis as well as stenosis of the right and left pulmonary artery branches .
her exercise capacity gradually deteriorated and she developed signs of right - sided heart failure .
case d was a female tetralogy of fallot patient who had undergone total correction at the age of 5 . at the age of 12 ,
she had developed severe stenosis of the right branch of the pulmonary artery ( gradient 75 mmhg ) which was treated with surgical patch - plasty .
she remained asymptomatic until the age of 37 . at the age of 37 her exercise capacity deteriorated .
mri showed progression of right ventricular dilatation , impaired right ventricular systolic function , severe pulmonary regurgitation , an aneurysmatic right ventricular outflow tract ( rvot ) and severe ostial stenosis of the right pulmonary artery branch .
all procedures were performed on cpb . the pulmonary trunk was incised longitudinally and the pulmonary artery branches were visualised directly . the length of the stent was selected on the basis of the vessel diameter as visualised by preoperative imaging ( angiography and mri in particular ) , the location of the stenosis , and peroperative anatomical findings .
the cpz8 stent was hand - crimped on a shytak ii balloon and placed across the lesion by the interventional cardiologist .
positioning of the stent was done under direct vision of the proximal part of the stent and the distal aspect was palpated digitally to ensure that the stent would not compromise the ostium of the right upper lobe branch . in all patients ,
additional reconstruction of the aneurysmatic rvot was performed in cases a , b and d. in case a , the procedure was performed on a beating heart .
predilatation was performed using a 12 mm balloon ( 6 atm ) using an amplatzer stiff guidewire .
the length of the cpz8 stent was 28 mm ; after balloon inflation the diameter of the stent was 15 mm ( 4 atm ) .
b , predilatation was performed using a 12 mm balloon ( 6 atm ) using an amplatzer stiff guidewire .
the length of the cpz8 stent was 22 mm , after balloon inflation the diameter of the stent was 14 mm ( 4 atm ) .
the proximal part of the stent was post - dilated to 18 mm to assure good fit .
the cpb time was 85 min . in case c , a cpz8 stent ( length 34 mm )
after balloon inflation ( 4 atm ) the diameter of the stent was 15 mm .
the position of the stent was good , but waisting of the stent was seen in the middle section of the stent . therefore the stent was post - dilated ( 8 atm ) .
the procedure was complicated by a tear of the right pulmonary artery at the distal end of the stent for which aortic clamping was necessary for 25 min .
d , femoral cannulation was necessary because of severe adhesions as a consequence of previous operations .
aortic clamping of 47 min was necessary because of excessive collateral flow and for optimal exposition of the right pulmonary artery ostium .
the length of the cp stent was 22 mm , after balloon inflation ( 4 atm ) the diameter of the stent was 12 mm .
in all patients , a dramatic improvement of right ventricular systolic function was observed , allowing weaning from inotropic support and artificial ventilation within a few hours after the procedure .
postoperative echocardiography showed mild pulmonary regurgitation and an improvement of right ventricular dimensions and function in all patients .
after 3 years of follow - up , case a , b and c patients are still asymptomatic .
in all cases , the stenosis of the right pulmonary artery was located at the site of the former waterston shunt . soon after the introduction of this surgical technique it was recognised that the waterston shunt was associated with problems such as kinking of the pulmonary artery , pulmonary overflow with congestive heart failure and eventually pulmonary vascular disease .
take - down of the shunt was performed by direct suturing from inside the aorta , simple ligation or detachment and patch - plasty of the right pulmonary artery branch .
irrespective of the surgical technique used , there is a high incidence of severe obstruction of the right pulmonary artery due to scarring in combination with compression by the ascending aorta . due to the short - term and long - term complications associated with the waterston shunt ,
this technique was abandoned and other shunt techniques were introduced , such as the modified blalock - taussig shunt . over the past two decades , further developments in surgical techniques and perioperative care have led to a tendency towards primary correction at young age . nowadays in our centre , primary correction is performed between 3 and 6 months and a shunt is avoided whenever possible . only in case of severe hypoplasia of the pa branches and/or tree or in case of unfavourable rvot anatomy ( a long and severely obstructive infundibulum requiring a long transannular patch ) is an initial modified blalock - taussig shunt still used .
subsequent follow - up studies demonstrated that stents maintain long - term vessel patency and can be dilated further at subsequent catheterisation .
[ 69 ] percutaneous stenting is now an established treatment modality for older children and adults with branch pulmonary artery stenosis [ 10 , 11 ] , although high rates of early complications are reported .
intra - operative placement of stents under direct vision as a hybrid procedure has nowadays completed the spectrum of pulmonary artery stenting . a hybrid procedure shortens cpb time and avoids the need for hypothermia and transsection of the ascending aorta and for patch - plasty of the right pulmonary artery .
furthermore , complications of percutaneous stent placement , such as haemodynamic instability and vascular tear , can be avoided or easily addressed in the operative setting with cpb , thereby making the procedure safe and effective .
[ 1318 ] close collaboration between cardiac surgeons , imaging and interventional cardiologists is essential in the modern management of congenital heart defects .
the hybrid approach described above is an example of how this can be accomplished . in our cahal collaboration between amsterdam medical centre , leiden university medical centre and the vu university medical centre ,
the hybrid procedure was shown to be a safe and effective treatment option for patients with severe pulmonary regurgitation and stenosis of the pulmonary arteries .
long - term follow - up of stented patients will be necessary to establish the long - term impact of this hybrid procedure .
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after having undergone surgical correction at an early age , many patients with tetralogy of fallot develop long - term complications including progressive pulmonary regurgitation and peripheral pulmonary stenosis .
a high percentage of these patients need to undergo a second operation in their adolescence or early adulthood .
if simultaneous treatment of both pulmonary regurgitation and peripheral pulmonary stenosis is warranted , a complete surgical approach has several disadvantages .
we describe four cases of fallot patients with severe pulmonary regurgitation and peripheral pulmonary stenosis who were treated using a hybrid approach involving surgical implantation of a pulmonary homograft and peroperative stenting of the pulmonary artery .
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in the post - genome era , proteomic technology has rapidly developed to be a powerful platform for the research of human physiology .
it can be applied for identifying potential novel biomarkers for prognosis , diagnosis and therapeusis ( 1,2 ) . and
in recent years it is shown that body fluids have become one of the important targets for proteomics research ( 3 ) .
the body fluids include a wide variety of compositions like plasma / serum , urine , cerebrospinal fluid , saliva , bronchoalveolar lavage fluid , synovial fluid , nipple aspirate fluid , tear fluid , amniotic fluid and so on .
analysis of the protein composition in body fluids can help to understand human disease proteomics better .
hu et al.,(3 ) reviewed the body fluids research advances in proteome analysis and focused on its applications to human disease biomarker discovery .
the importance of body fluids has also been appreciated by recent proteomics work ( 4 ) . the database mapu : max - planck unified database of organellar , cellular , tissue and body fluid ( 5 ) published in 2007 exhibit the close attention of the proteome researchers to the body fluids .
the mapu database stores the data from their own lab and contains several kinds of body fluids , such as urine and tear fluid . to collect more curated proteomics data in the related literatures of the body fluids and
provide comprehensive protein annotation , as well as explore the relationships between the different body fluids , we constructed this database sys - bodyfluid . abundant proteomics data and in - depth protein annotation make sys - bodyfluid to be a reference database for body fluid and clinical proteomics research .
the manually curated body fluid protein data in the sys - bodyfluid were imported to mysql database by java program .
the protein annotation data were downloaded from international protein index ( ipi ) database , gene ontology ( 6 ) , goa database ( 7 ) and kegg ( 8) pathway database .
open source java library named as jfreechart ( http://www.jfree.org/jfreechart/ ) distributed under lgpl was adopted to plot the image of the statistics data in the web .
we searched pubmed and manually curated 50 related peer - review publications published online before may 2008 .
the primary sequences of the proteins were retrieved by the original i d from their corresponding databases in these publications . due to the database updates ,
the protein sequences reported in the literatures may have changed or depleted in the current databases .
each protein was mapping to the ipi database to uniform the protein i d in sys - bodyfluid by blasting these protein sequences against the database ( human ipi version 3.44 ) ( the e - value cutoff was set to 10 , the blast - hsp coverage was > 0.9 ) .
thus , each of the protein has a corresponding ipi i d in the sys - bodyfluid database .
the total unique proteins and paper numbers of the 11 kinds of body fluids in our database are summarized in table 1 .
for example , there are 13 papers and 7748 proteins about the plasma / serum research in our database .
users can obtain this statistical information about the sys - bodyfluid database in the database web link in the website http://www.biosino.org/bodyfluid .
table 1.the data summary in sys - bodyfluid databasebody fluid nameprotein numberpaper numberplasma / serum ( 1123)774813saliva ( 2431)21618urine ( 3240)19419cerebrospinal fluid ( 4146)12866seminal fluid ( 47,48)9162amniotic fluid ( 4951)8993tear ( 52,53)5092bronchoalveolar lavage fluid ( 54,55)4112milk ( 56,57)1752synovial fluid ( 58)1141nipple aspiration fluid ( 59,60)842total10 13850 the data summary in sys - bodyfluid database
the sys - bodyfluid is accessed from graphical web interface ( http://www.biosino.org/bodyfluid/ ) and the data are available for download through the download link in the website as a text file .
sys - bodyfluid provides users the current database data statistics of different body fluids through the database link for the paper number and the unique protein number ( database link ) .
as shown in figure 1 , sys - bodyfluid offers users an optimal search function , including searching by protein i d , name and sequence similarity ( search link , figure 1a ) .
the comprehensive browse option allows users to explore comparison analysis between two or more different body fluids data ( browse link , figure 1b ) . for each protein in sys - bodyfluid
, we provide detailed annotation information , including protein description , involved body fluids , paper information , domain , gene ontology , pathway , sequence and so on ( figure 1c ) .
furthermore , the availability of pathway analysis will assist users to investigate the difference between body fluids through involved metabolism and signal transduction pathway ( pathway link , figure 1d ) .
the body fluid number and paper number the proteins involved in are also showed in the web page .
protein existed in two body fluids could also be viewed and multi body fluids can be investigated .
there is detailed information in the database for each protein , including description , domain , gene ontology term , sequence and so on .
the proteins ( colored by red ) in different body fluids and their involved pathway are shown in pathway link .
protein existed in two body fluids could also be viewed and multi body fluids can be investigated .
there is detailed information in the database for each protein , including description , domain , gene ontology term , sequence and so on .
the proteins ( colored by red ) in different body fluids and their involved pathway are shown in pathway link .
to get more comprehensive understanding of the relationship between body fluids , we compared the proteins composition in different body fluids .
there are 2928 proteins presented in at least two body fluids and 1359 proteins exist in at least three body fluids .
only 15 proteins exist in total 11 body fluids . for these 2928 proteins , go annotation information were obtained and enrichment analysis was performed using bingo ( 9 ) and cytoscape ( 10 ) .
the node 's size is scaled by protein number and node 's color shows p - value of the enrichment analysis .
, it is shown that some molecular functions like protein binding and enzyme regulator activity are over - presented in this dataset , as well as the biological process like transport and
there are 2928 proteins presented in at least two body fluids and 1359 proteins existed in at least three body fluids .
( b ) gene ontology annotation statistical analysis for the 2928 proteins existing in at least two body fluids .
there are 2928 proteins presented in at least two body fluids and 1359 proteins existed in at least three body fluids .
( b ) gene ontology annotation statistical analysis for the 2928 proteins existing in at least two body fluids .
human body fluids proteome analysis is still a challenge because dynamic range and the complexity of the body fluids protein composition .
it is important to construct a body fluid reference database dedicated to biomarker discovery research .
previous work like mapu is a great effort to integrate the data from their own lab and aim to provide a
it is still necessary to refer to other proteomic literature data . for this reason ,
our database sys - bodyfluid was build as a complementary database to the mapu and aimed to provide users more information about the body fluids accompanied by protein abundant annotations .
as more and more body fluid proteome data have been produced recently , it is planned to update sys - bodyfluid database every 6 months .
new body fluid proteome data produced during the time will be added to our database .
furthermore , more annotation information like protein interaction data will also be included . in the future
, we will collect more body fluid proteome data in the disease proteomics research , for example , cancer and diabetes proteome data .
if possible , tissue proteomics data will be also included to look into the crosstalk between the tissue protein and the body fluid protein .
basic research foundation ( 2006cb910700 ) ; cas project ( kscx2-yw - r-106 , kscx2-yw - r-112 , kgcx1-yw-13 ) ; high - technology project ( 2007aa02z334 ) . funding for open access charge : cas project kscx2-yw - r-106 .
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recently , body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data .
a database is needed to collect and analyze these proteome data .
thus , we developed this web - based body fluid proteome database sys - bodyfluid .
it contains eleven kinds of body fluid proteomes , including plasma / serum , urine , cerebrospinal fluid , saliva , bronchoalveolar lavage fluid , synovial fluid , nipple aspirate fluid , tear fluid , seminal fluid , human milk and amniotic fluid .
over 10 000 proteins are presented in the sys - bodyfluid .
sys - bodyfluid provides the detailed protein annotations , including protein description , gene ontology , domain information , protein sequence and involved pathways .
these proteome data can be retrieved by using protein name , protein accession number and sequence similarity .
in addition , users can query between these different body fluids to get the different proteins identification information .
sys - bodyfluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database .
it is available at http://www.biosino.org / bodyfluid/.
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the need for a safe and effective human immunodeficiency virus ( hiv ) vaccine has never been greater .
the acquired immune deficiency syndrome ( aids ) epidemic update released by unaids on december 2009 , documented that the overall number of people with hiv has stabilized , albeit at an unacceptably high level of about 33.4 million people living with hiv and 2.7 million individuals being newly infected with the virus in 2008 .
the global aids epidemics killed 2 million people in 2008 , and the number of children orphaned by aids may reach 25 millions by 2010 . about 85% of these new infections occur in developing countries where access to antiretrovirals is still an enormous challenge [ 1 , 2 ] .
there is strong evidence to support a role of cytotoxic t lymphocytes ( ctls ) in the containment of hiv replication [ 36 ] and several vaccine approaches have been pursued to elicit anti - hiv ctl responses .
one promising approach is to use mycobacterium bovis bacillus calmette - gurin ( bcg ) as a live recombinant bacterial vaccine vector .
bcg vaccine has been used to immunize more than two billion individuals against tuberculosis with a long record of effectiveness and safety for use in humans .
ctl induction against hiv-1 and simian immunodeficiency virus ( siv ) gag or env antigens has been described following the immunization of mice or nonhuman primates with recombinant bcg ( rbcg ) expressing these antigens [ 8 , 9 ] .
more recently , recombinant mycobacterium bovis bacillus calmette - gurin ( bcg ) expressing hiva immunogen has been generated and shown to be stable and to induce durable and high - quality hiv-1-specific cd4 + and cd8 + t - cell responses in balb / c mice .
furthermore , when the recombinant bcg vaccine was used in a priming - boosting regimen with heterologous components , the hiv-1-specific responses provided protection against surrogate virus challenge , and the recombinant bcg vaccine alone protected against aerosol challenge with m. tuberculosis .
three issues are critical for engineering a stable and immunogenic mycobacterial - based vaccine vector : ( i ) antigen localization , ( ii ) codon optimization , and ( iii ) in vivo plasmid dna stability and genetic rearrangements .
the antigen secretion and fusion of foreign antigens to mycobacterial surface lipoproteins will provide these antigens with access to the major histocompatibility complex ( mhc ) class i pathway and subsequently enhance the immunogenicity and to prevent foreign proteins from becoming toxic to bcg .
the use of mycobacterial optimal codons will enhance the transcriptional / translational activity of the foreign gene .
homologous recombination , which takes place between repeated dna sequences , is one of the most important mechanisms for bacterial genome rearrangements .
any repeated dna sequences on a chromosome can induce homologous recombination , but insertion sequences ( is ) are among the most abundant of these [ 1315 ] .
mycobacteria have been reported to contain several is . in mycobacterium tuberculosis , it has been suggested that is6110 is an important source of genome variation .
however , the role of such is elements has not been thoroughly explored in heterologous gene expression in mycobacteria [ 1618 ] .
the extent of this burden will determine the degree to which fitness of the recombinant bcg is compromised .
thus , the relative level of fitness of the recombinant and any derived mutants will determine the rate at which the inserted element ( structural instability ) or its expression ( functional instability ) is lost from the bacterial population .
husson et al . constructed a plasmid shuttle vector that allows insertion of foreign dna and stable integration in the mycobacterial genome by homologous recombination .
the structural instability of several recombinant plasmids expressing the cirscumsporozoite protein ( csp aa 18391 ) from plasmodium falciparum was described by haeseleer .
the analysis in m. smegmatis and bcg of numerous rearranged plasmids showed that the presence of a functional specific expression cassette was responsible for the plasmid instability and that the observed deletions seemed to come from recombination between homologous and nonhomologous sequences .
monitored the role of such genetic rearrangement events in the expression of cloned escherichia coli lacz gene in mycobacterium smegmatis host strain using integrative ( pmv361::lacz ) and episomal ( pmv261::lacz ) vectors .
the lacz gene present in both vectors was the mutable target within the vector and simple color detection assay in mycobacteria was used to screen the genetic rearrangements during replication in mycobacterium smegmatis .
they reported that a loss of lacz phenotype was due to the insertion of an is element in the lacz gene of the integrative vector ( frequency 1.7 10 ) , whereas in the case of the replicative vector , the loss of lacz phenotype was due to deletions of different sizes in the lacz gene and the hsp60 promoter region ( frequency 2 10 ) . in this study
we evaluated the expression of disrupted heterologous hiv-1gp120 gene , from shiv - hxbc2p 3.2 clone ( genbank accession number af041850 ) , by genetic rearrangements in mycobacterium bovis bcg host strain using a replicative vector ( pmv261 ) regulated by bcg hsp60 promoter ( strong promoter ) . to compare the hiv-1gp120 gene expression and plasmid dna stability in vivo , the replicative ( pjh222 ) and integrative ( pjh223 ) vectors carrying a wild - type lysine - complementing gene in the lysine auxotroph of bcg host strain
-antigen promoter ) to regulate hiv-1gp120 gene expression and bcg lysine auxotrophs complemented with a lysine gene do , in fact , prevent the disruption of gene expression caused by genetic rearrangements .
escherichia coli cultures were grown in luria - bertani ( lb ) broth ( sigma - aldrich , usa ) or on lb agar plates ( sigma - aldrich , usa ) at 37c .
lb was supplemented with kanamycin ( 40 g / ml ) ( sigma - aldrich , usa ) .
mycobacterial cultures were grown in middlebrook 7h9 broth ( becton - dickinson , usa ) or on middlebrook 7h10 agar medium ( becton - dickinson , usa ) supplemented with 10% albumin - dextrose - catalase ( adc ) ( becton - dickinson , usa ) and containing 0.05% tween 80 ( sigma - aldrich , usa ) and kanamycin ( 25 g / ml ) .
the l - lysine monohydrochloride was purchased from sigma - aldrich ( usa ) , dissolved in distilled water and used at a concentration of 40 g / ml .
tables 2 and 3 listed the parental e. coli / mycobacterial shuttle vectors used for cloning the hiv-1gp120 gene from shiv - hxbc2p 3.2 clone ( genbank accession number af041850 ) and the constructs
. plasmid dna vectors pmv261 ( 4,480 bp ) , pjh222 ( 6,423 bp ) , and pjh223 ( 6,313 bp ) were used as parental plasmids for all plasmid dna constructs obtained as described below .
the entire dna coding sequence of hiv1gp120 ( 1,578 bp ) antigen was synthesized by polymerase chain reaction ( pcr ) , using oligonucleotide primers specific for hiv-1 gp120 gene and cloned into the different e.coli/mycobacterial shuttle vectors .
plasmid dna pmv261 is a replicative vector ( multicopy , extrachromosomal ) that contains a dna cassette encoding kanamycin resistance ( tn903-derived aph gene ) , an e. coli origin of replication ( orie ) , a mycobacterial plasmid dna origin of replication ( orim ) , an expression cassette containing a mycobacterial promoter , a multiple cloning site , and a transcriptional terminator . for pmv261::hivgp120
, the hivgp120 gene was fused to the first six codons of the cytoplasmically expressed bcg hsp60 protein .
for immunodetection purposes , we fused downstream of the hiv-1 gp120 gene with the influenza virus hemagglutinin epitope ( ha ) coding sequence and six residues of histidine .
the primers were designed to incorporate bamhi ( forward 5-caaggatccgaaattgtgggtcacagtc-3 ) and hindiii ( reverse 5-cagaagcttctagtggtggtggtggt-3 ) sites at the 5 and 3 termini of the amplified dna fragment to be cloned into the pmv261 plasmid dna at 4,326 bp - bamhi/4,346 bp - hindiii .
plasmid dna pjh222 , a derivative of pmv261 , contains also the lysa complementing gene , under the regulatory control of bcg hsp60 promoter . for the pjh222::hiv-1 gp120 construct
, the hiv-1 gp120 gene was fused to the 5 region encoding the 19 kda lipoprotein signal sequence from mycobacterium tuberculosis , and the ha epitope and six residues of histidine were fused downstream .
the primers were designed to incorporate hindiii ( forward 5-cagaagcttgggcccgaaaaa-3 ) and psti ( reverse 5-caactgcagctagtggtggt-3 ) sites at the 5 and 3 termini of the amplified dna fragment to be cloned into the pjh222 plasmid dna ( 335 bp - hindiii/341 bp psti ) .
it contains a dna cassette encoding kanamycin resistance ( tn903-derived aph gene ) , an e. coli origin of replication ( orie ) , a dna segment carrying the attachment site ( attp ) and the integrase ( int ) gene from the mycobacteriophage l5 , the lysa complementing gene under the regulatory control of bcg hsp60 promoter , and an expression cassette containing a mycobacterial promoter , a multiple cloning site and a transcriptional terminator .
the pjh223::hiv-1 gp120 plasmid dna was constructed using the same method as for pjh222::hiv-1 gp120 .
the primers were designed to incorporate hindiii sites ( forward 5-cagaagcttgggcccgaaaaa-3 and reverse 5-caaaagcttctgcagctagtggtgg-3 ) at the 5 and 3 termini of the amplified dna fragment to be cloned into the pjh223 plasmid dna ( 2,134 bp - hindiii ) .
the correct cloning of the hiv-1gp120 gene into the plasmid dna and the deletion of the ~900 bp fragment were confirmed by dna sequencing , pcr and enzyme restriction analysis performed following standard protocols .
the bcg wild type host strain was transformed with pmv261::hiv-1 gp120 plasmid dna and lysine auxotroph of bcg was transformed with pjh222::hiv-1gp120 and pjh223::hiv1gp120 plasmid dna by electroporation .
bcg cultures were grown to an od of 0.9 ( 600 nm ) , pelleted at 3,000 rpm , washed twice by resuspension and centrifugation ( 3,000 rpm ) in 10% glycerol at 4c and finally resuspended in 1/20th of the original culture volume with cold 10% glycerol . then 100 l of the cold bcg suspension was mixed with plasmid dna ( 50500 ng ) in a prechilled 0.2 cm electroporation cuvette and transformed using the biorad gene pulser electroporator at 2.5 kv , 25 mf , and 1,000 . after electroporation 1 ml of 7h9 medium , supplemented with adc and containing 0.05% tween 80 , was added and incubated at 37c for 12 hours before plating on middlebrook agar 7h10 medium supplemented with 10% adc and containing 0.05% tween 80 and kanamycin ( 25 g / ml ) .
after transformation by electroporation , the rbcg colonies were used as templates for the pcr analysis .
different rbcg colonies were inoculated into 50 l of distilled water , vortexed and 1 l was used for the pcr analysis .
specific primers for cloning the hiv-1 gp120 gene into pmv261 , pjh222 and pjh223 vectors were used . the plasmid dna before bcg transformation ( pre - bcg ) was used as positive control for the pcr analysis . the qiaprep spin miniprep kit (
qiagen , hilden , germany ) was used for e. coli and mycobacteria plasmid dna purification .
different rbcg colonies that showed the dna fragment deletion by pcr analysis were inoculated in mycobacterial broth culture up to an od of 0.9 ( 600 nm ) .
the standard qiaprep spin miniprep protocol was used with a slight modification : after resuspension in buffer p1 , lysozyme was added at a concentration of 10 mg / ml and samples were incubated at 37c overnight .
for improving the purified plasmid yield , the mycobacterial plasmid dnas from selected rbcg colonies were transformed in e. coli jm109 using 5 l of the isolated plasmid dnas , and purified according to the manufacturer 's instructions for dna sequencing and enzyme restriction mapping . the plasmid dna before bcg transformation ( pre - bcg ) was used as positive control for the enzyme restriction analysis .
the nucleotide sequences were obtained using the bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , usa ) and abi prism 3100 genetic analyzer .
bcg transformants were grown to mid - logarithmic phase in liquid 7h9 medium supplemented with 10% adc and containing 0.05% tween 80 and kanamycin ( 25 g / ml ) .
pellets were washed twice in phosphate buffered saline ( pbs ) plus 0.02% tween 80 and resuspended in 1 ml of extraction buffer ( 50 mm tris - hcl ph 7.5 , 5 mm edta , 0.6% sodium dodecyl sulfate ) , and added with 5 l of 100x protease inhibitor cocktail ( 1 mg / ml aprotinin , 1 mg / ml e-64 , 1 mg / ml leupeptin , 1 mg / ml pepstatin a , 50 mg / ml pefabloc sc , and 10 ml dimethyl sulfoxide ) .
cells were sonicated for 4 minutes on ice with a branson sonicator at output control seven and duty cycle of 50% .
extracts were centrifuged at 13,000 rpm for 10 minutes at 4c and supernatants were collected .
after electroblotting , nitrocellulose membranes were first probed with a 1 : 1000 dilution of mouse monoclonal antibody ha.11 ( covance , usa ) directed to influenza virus hemagglutinin epitope ( ypydvpdya ) followed by a 1 : 2000 dilution of horseradish peroxidase ( hrp)-conjugated antibodies .
the membranes were developed using chemoluminiscent ecl ( ge healthcare , usa ) as hrp substrate .
as shown in figure 1 , pcr analysis of rbcg : hiv-1gp120(pmv261 ) colonies detected a partial deletion of the hiv-1gp120 dna coding sequence using the specific primers that were used for cloning the dna sequence into the pmv261 vector .
this deletion was reproduced in 12 out of 14 rbcg : hiv-1 gp120 ( pmv261 ) colonies analyzed ( data not shown ) .
in addition , this partial deletion was observed only by pcr of plasmid dna from bcg colonies in which the hiv-1gp120 dna fragment was inserted into the pmv261 vector and the gene expression was regulated by the hsp60 promoter from bcg ( figure 1 , lane 2 ) .
the deletion was not detected when the dna fragment encoding the hiv-1gp120 antigen was cloned into pjh222 and pjh223 vectors under mycobacteria spp .
the deletion was not detected in all 10 rbcg : hiv-1gp120 ( pjh222 ) colonies screened by pcr ( data not shown ) .
we also compared the restriction enzyme digestion profile of the pmv261 and pjh222 plasmid dna vectors with and without heterologous hiv-1gp120 dna insert , pre- and post - bcg transformation ( figure 2 ) .
the pmv261 plasmid dna vector without the dna insert corresponding to hiv-1gp120 gene did not show any difference in the enzymatic restriction profiles from pre- and post - bcg transformations ( figure 2 , lanes 1 and 2 ) .
the same result was obtained for the pjh222 plasmid dna vector containing the dna insert ( figure 2 , lanes 5 and 6 ) .
in contrast , the pmv261 containing the hiv-1gp120 insert showed different enzymatic restriction profiles from pre- and post - bcg transformations ( figure 2 , lanes 3 and 4 ) . following sequence analysis and molecular characterization of the deleted dna fragment in the 10 rbcg : hiv-1gp120 ( pmv261 ) mutant colonies
, we demonstrated that the partial deletion ( ~900 bp ) corresponds to gp120 core rich in potential glycosylation sites and the variable regions v3 , v4 , and v5 of hiv-1 env , a key structure involved in the hiv union to its receptor ( cd4 ) and co - receptor on the host cell surface .
strikingly , the deleted dna fragment in the rbcg mutants contained the dna sequence coding for hiv-1 envelope immunodominant ctl , murine h-2d - restricted epitope p18-i10 ( figure 3 ) .
deletion in hiv-1gp120 gene was not observed in any rbcg : hiv-1gp120 ( pjh222 ) and rbcg : hiv1gp120 ( pjh223 ) colonies that carry the pjh222:hiv-1gp120 and pjh223:hiv1gp120 vectors containing the mycobacteria spp .
-antigen promoter to regulate the hiv1gp120 gene expression , indicating that the deletions were not due solely to the toxic effects of hiv-1gp120 expression during growth of the recombinant bcg strains .
expression of hiv-1gp120 protein by rbcg : hiv-1gp120 ( pmv261 and pjh222 ) strains was confirmed by sds - page and western blot analysis of whole - cell bcg lysates . the chimeric 19 kda lipoprotein signal sequence - hiv-1gp120-flu - his recombinant protein , with a relative molecular mass ( mr ) of 67 kilodalton ( kda ) was present in lysates of rbcg : hiv-1gp120 ( pjh222 ) bacterial cells containing the 1.82 kilobase - pair ( kbp ) hiv-1gp120 dna coding sequence in pjh222 vector .
the apparent mr of hiv-1gp120 protein was consistent with that predicted by the gene sequence without post - translation modification .
by contrast , when all rbcg : hiv-1gp120 ( pmv261 ) mutant colonies were analyzed by western blot , we detected a band of 28 kda ( truncated protein ) instead of the expected 67 kda protein ( figure 4 ) .
mycobacterium tuberculosis infection is a major cause of human morbidity and mortality , and vaccine is the most cost - effective intervention to prevent disease .
mycobacterium bovis bcg is a widely used vaccine against tuberculosis , and a single dose given at birth confers long - lasting immunity .
mycobacterium bovis bcg has been suggested as an ideal delivery system for expression of foreign antigens due to the long persistence of bcg in the immunized host .
thus , mycobacterium bovis bcg not only is used as vaccine against tuberculosis , but also offers great potential for innovative approaches for development of polyvalent vaccines [ 25 , 2830 ] . in vivo genetic stability and persistence
a mutant bcg which is rapidly eliminated by the host immune system is unlikely to be an effective vaccine .
the rearrangement of dna is one of the fundamental properties of life , including both prokaryotes and eukaryotes , with great biological significance .
some of these dna rearrangements are important in controlling gene expression in specific cell types ; others may play an evolutionary role by contributing to genetic diversity .
for example , plasmids responsible for expressing m. tuberculosis -antigen in bcg were stable over six consecutive 4-week cultures without selection and changes in the level of expression .
an -antigen - hiv-1 v3j1 chimeric protein was secreted by rbcg for at least 450 passages in vitro . however , in vitro instability of episomal vectors has also been reported regularly .
lim et al . described that expression of siv mac 251 env ( aminoacids , aa , 1 - 245 ) by rbcg was stable in culture ( pblaf promoter ) , but rbcg harboring the same vector containing aa 1 - 521 or aa 215 - 521 were unstable .
chawla and das gupta reported that the disruption of pal5000-derived vectors in m.smegmatis through transposition of an insertion sequence upstream of the kanamycin resistance gene led to structural instability and large deletions .
studied the stability of the expression of escherichia coli -galactosidase under the control of hsp60 promoter in mycobacterium smegmatis using integrative and replicative vectors . in the case of replicative vectors , they detected higher frequencies of lacz expression inactivation as compared with the same lacz cassette , when present in integrated state .
however , springer et al . reported a high - frequency loss of pmv361-based integrating vectors from m. smegmatis and bcg .
al - zarouni and dale investigated the effects of various combinations of posttranslational signals and promoters on heterologous gene expression and stability in different bcg strains .
plasmid dnas were constructed using mycobacterial promoters ( hsp60 , 19-kda antigen , 85a antigen , from mycobacterium tuberculosis complex , and the 18-kda antigen from mycobacterium leprae ) and post - translation signals ( 85a antigen secretion and 19-kda antigen acylation signals ) , coupled with reporter genes .
they provided evidence that the 85a secretion signal markedly enhanced the levels of cell - associated product , while the 19-kda acylation signal had little effect on gene expression .
this group also demonstrated that the hsp60 promoter caused plasmid dna instability and showed that various deletions in the promoter region occurred during or soon after transformation , but not during subsequent growth of the transformants , nor with other promoters .
gross instability was also observed when the episomal vectors were used to express rotavirus vp6 protein under the control of hsp60 promoter .
the possibility of inserting foreign genes into the chromosome at precise positions to ensure the persistence of the heterologous genetic information in the recombinant vaccine strains would represent a crucial step in the development of mycobacterium bovis bcg as a live vaccine vector for expression of heterologous antigens [ 28 , 39 ] .
even though integrative plasmid dnas have been shown to be more stable as they are incorporated into the chromosome via homologous recombination , baulard and colleagues demonstrated that with replicating plasmid dnas , relatively high levels of homologous recombination were obtained in fast- and slow - growing mycobacteria , and 100% of the selected clones underwent homologous recombination .
thus , if bcg is to be used as a live bacterial carrier for generating novel vaccine candidates and as an immunotherapeutic agent , it is essential that a more genetically stable strain be developed .
sander and collaborators demonstrated that mycobacterium bovis bcg reca mutants are a valuable tool for the further development of bcg as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis .
this was confirmed in a study performed by keller and coworkers where it was observed that reca inactivation in bcg russia strain was in part responsible for its high degree of genomic stability , resulting in a substrain that has fewer genetic alterations than other vaccine substrains with respect to m. bovis af2122/07 wild - type .
lack of success in this respect may be due to overexpression of lethality or other forms of protein toxicity .
stover et al . were unable to express hiv-1 gp120 ( hsp60 promoter ) from an episomal vector , but could express it from an integrative vector [ 19 , 25 ] . in our study , we have observed that , in the case of the replicative pmv261 vector , disruption of hiv-1gp120 gene expression was due to a consensus dna fragment deletion in the hiv1gp120 gene detected in all ten different bcg clones . by contrast , we did not detect any hiv-1gp120 gene expression disruption in the replicative ( pjh222 ) and integrative ( pjh223 ) vectors .
sequence analysis indicated that the dna fragment deletion contained several potential glycosylation sites , the variable regions v3 , v4 and v5 and a ctl immunodominant epitope ( p18-i10 peptide ) .
based on this observation , we hypothesized that the loss of expression of this immunodominant epitope could help mycobacteria to escape the host 's immunological response and may represent part of an ongoing adaptation to survival in host environments that are screened by immunological defense mechanisms .
huber and collaborators have described a natural loss of expression of highly immunogenic proteins caused by a variety of genomic changes in mycobacterium ulcerans that may confer a selective advantage to this emerging pathogen . on the other hand ,
the deleted dna sequence in the rbcg : hiv-1gp120(pmv261 ) mutant colonies analyzed in our study ( n = 10 ) was highly conserved , which is in disagreement with dennehy et al . that deletions in the vp6 heterologous gene ( hsp60 promoter ) in rbcg were random and different among clones examined .
-antigen promoter ) to regulate hiv-1gp120 gene expression and bcg lysine auxotrophs complemented with a lysine gene do , in fact , prevent the disruption of gene expression caused by genetic rearrangements .
the results showed that such genetic rearrangements can significantly affect the expression of foreign genes in mycobacteria .
thus , it could result in an rbcg that does not induce optimal immune responses to the hiv antigen .
mycobacterium bovis bacillus calmette - gurin ( bcg ) as a live vector of recombinant bacterial vaccine is a promising system to be used .
however , few promising approaches have emerged in the last five years to overcome the genetic instability in rbcg - based vaccines . to prevent the plasmid instability in vivo and in vitro and the genetic rearrangement by mycobacteria
, different approaches should be considered : ( i ) the use of expression vectors containing small hiv dna coding sequences , ( ii ) dna fragments lacking glycosilation sites , ( iii ) the use of weak promoters , ( iv ) the use of bcg auxotrophic strains ( containing the complementing gene in the expression vectors ) , ( v ) the use of inducible promoters , ( vi ) codon optimization of the recombinant gene , ( vii ) the choice of expression vector backbone and ( viii ) antigen secretion to enhance the immunogenicity and to prevent foreign proteins from becoming toxic to bcg . in five years ' time
we will have a better picture of how the immune responses induced by rbcg could be enhanced , and what sort of route , doses , immunization schedule , timing for immunological assays are likely to induce optimal responses in mice and monkeys .
heterologous prime - boost regimen , different immunization schedule , routes and doses , should be performed to evaluate the potential for enhancing specific immune responses .
bcg has a long record of safe use in humans and is able to induce long - lasting immunity .
in addition , it could be the best hope for protecting newborn infants , reducing the adult burden of hiv infection and protecting neonates against vertical transmission .
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mycobacterium bovis bacillus calmette - gurin ( bcg ) as a live vector of recombinant bacterial vaccine is a promising system to be used . in this study
, we evaluate the disrupted expression of heterologous hiv-1gp120 gene in bcg pasteur host strain using replicative vectors pmv261 and pjh222 .
pjh222 carries a lysine complementing gene in bcg lysine auxotrophs .
the hiv-1 gp120 gene expression was regulated by bcg hsp60 promoter ( in plasmid pmv261 ) and mycobacteria spp .
-antigen promoter ( in plasmid pjh222 ) . among 14 rbcg :
hiv-1gp120 ( pmv261 ) colonies screened , 12 showed a partial deletion and two showed a complete deletion .
however , deletion was not observed in all 10 rbcg : hiv-1gp120 ( pjh222 ) colonies screened . in this study , we demonstrated that e. coli / mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of bcg could prevent genetic rearrangements and disruption of hiv 1gp120 gene expression , a key issue for engineering mycobacterial based vaccine vectors .
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the concept of functional foods has been evolved recently after extensive research to find out the role of nutrients on health .
functional foods contain physiologically active components , which provide health benefits beyond basic nutrition by affecting one or more functions in the body in a targeted way .
the functional component could include essential macronutrient with specific physiological effect and components that have some nutritive value but are not classified as essential ' , such as oligosaccharides , or food components with no nutritive value , such as live microorganisms or plant chemicals .
a dietary supplement is a product intended for ingestion as a supplement to the diet and it can be manufactured as pills , tablets , capsules , gel caps , liquids , and powders .
dietary supplements or functional foods contain vitamins , micronutrients , antioxidants , certain bioactive peptides , herbs , polyunsaturated fatty acids , and so forth .
the concept has now moved markedly towards gastrointestinal function , in particular the impact of gut bacteria .
the most frequently used dietary method of influencing the gut flora composition is that of probiotics .
probiotics are live microorganisms ( in most cases , bacteria ) that are similar to beneficial microorganisms found in the human gut . according to fao / who ( food and agriculture organization and world health organization ) , probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host .
over 100,000 billion bacteria ( more than 500 species ) live in the human gut .
the metabolism products of friendly bacteria ( probiotic ) such as lactic acid and acetic acid can inhibit the growth of harmful bacteria and confer health benefits on the host .
probiotics are ingested for their purported positive advantages in the digestive tract and/or systemic areas like the liver , vagina , or bloodstream , for example , neutralization of toxins , increase of the immune response [ 7 , 8 ] , antimutagenic and anticarcinogenic activities [ 911 ] , reduction of cholesterol levels , control of diarrhea , alleviation of lactose intolerance , and inflammatory bowel diseases .
they are also a source of vitamins , especially of the b group [ 16 , 17 ] , and can also be used as complementary and alternative medicine ( cam ) .
consumers should be provided with an independent assessment of physiological , microbial , and safety aspects of these live microbial products , especially if they can improve health .
the latest trend in the functional food market is to combine probiotics with prebiotics to enhance the effect of probiotics .
nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of probiotic bacteria in the colon .
, they must have a high viability in the product and have robust survival properties in the gut , which is their first point of contact . because of the presence of potentially pathogenic species such as enterococcus faecium and e. faecalis in probiotic products [ 2 , 19 ] , the production and marketing of functional foods should be strictly controlled and carefully monitored .
information on the label of the product , especially regarding the composition and identity of the probiotic strains included , needs to be accurate to guarantee safety and functionality . in most cases , the identity and number of viable strains recovered did not correspond with the information on the label . in view of the above ,
the aims of this study were to complete a market and product assessment of probiotic containing food supplements available in bangladesh .
this involved identifying the range of products available on the bangladesh market and evaluating claims made on the labels .
to conduct the study , different pharmacies , super shops , and medicine corners were surveyed to find out probiotic containing dietary supplements .
four probiotic supplements readily available in pharmacies were selected to determine the viability ( growth ) of the probiotic strains and to compare actual viable cell numbers with the label claim in this regard .
the content of the capsule was resuspended in 10 ml sterile distilled water . after performing serial dilution ,
appropriately diluted solutions were than plated out , in duplicate , onto a de man rogosa sharp ( mrs ) agar .
plates from each dilution were incubated at 37c and colonies were counted after 48 hours . to assess the viability of the probiotics included in the products , probiotic strains isolated from each of the 4 products were screened for inhibitory activity against the following 6 indicator strains : salmonella typhi , salmonella sp .
the probiotic strains were cultured in mr broth ( biolab ) for 18 hours at 37c and 10 l was spotted on mrs agar ( biolab ) .
the plates were incubated for 24 hours at 37c and then lawned with active cells of the indicator strains ( approximately 10 cfu / ml ) , embedded in soft agar ( 0.8% , m / v ) .
the plates were incubated at 37c for 24 hours and the colonies were examined for the formation of zones , which indicates the level of inhibitory activity and therefore viability .
the prebiotic and probiotic containing product market is a fast - growing industry worldwide and the list of available products increases on a daily basis .
although as a developing country , bangladesh has limited scope and facility but is trying to cope up with the health demand of probiotic .
one of the leading pharmaceutical companies has realized the importance of functional food and launched a probiotics product in capsule form . throughout the world prebiotics and probiotics
are available in fermented food like yoghurt and also in baby foods . through the market assessment , different types of food products were found containing probiotics and combination of probiotics and prebiotics . among them
one product is manufactured in bangladesh and two capsules were from india and one sachet ( powder ) was from taiwan .
three samples were of capsule form and only one was in powdered form available in sachet .
all the four samples contained same probiotics like lactobacillus acidophilus , lactobacillus bulgaricus , bifidobacterium bifidum , lactobacillus salivarius , lactobacillus casei , bifidobacterium longum , saccharomyces boulardii , lactobacillus rhamnosus , bifidobacterium infantis , lactococcus lactis , lactobacillus paracasei , and streptococcus thermophilus but the viable cell numbers are different . as a functional food component ,
prebiotics and probiotics are conceptually intermediate between foods and drugs . depending on the jurisdiction ,
they typically receive an intermediate level of regulatory scrutiny , in particular of the health claims made concerning them . for probiotic to exert beneficial properties
, they must have a high viability in the product and have robust survival properties in the gut , which is their first point of contact .
comparison of viable cell numbers stated on the labels of the supplements with the actual viable cell numbers is presented in table 1 and figure 1 . to achieve health benefits ,
probiotic bacteria must be viable and available at a high concentration , typically 1010 cfu / g of product . from this investigation
, it was found that viable cell numbers of all supplements were three to four log cycles lower than the claimed number and three of the four products are marginally potential for their proposed health benefit purpose . in different countries , despite the importance of viability , surveys conducted to validate viability claims have shown low populations of probiotic bacteria in probiotic products .
several factors have been claimed to be responsible for the loss of viability of probiotic organisms : acidity of products , acid produced during refrigerated storage ( after acidification ) , level of oxygen in products , oxygen permeation through the package , and sensitivity to antimicrobial substances produced by bacteria . to determine the inhibitory activity of probiotics , six indicator
the result of the screening of the probiotic strains isolated from the 4 selected supplements against 6 indicator strains is presented in table 2 .
table 2 reveals that the viable probiotic cells showed good inhibitory activity despite their numbers were lower than respective label claims .
therefore , in order to find out their actual efficacy , in vivo assessment should be considered .
this situation indicates that as there is no regulation in bangladesh for these types of products , the consumers are being manipulated by the manufacturers and marketers into buying a product under false claim .
it is important that the health claims stated on the labels of products supply the consumer with reliable information because such claims influence consumer behavior and potentially affect public health .
proper manufacturing practice and correct storage should be maintained by the manufacturers and marketers to ensure cell survival .
different probiotics containing food supplements and varieties of fortified baby foods ( containing probiotics and prebiotics ) are imported in bangladesh .
but in bangladesh there is no regulation to assess the quality of functional food containing products .
that is why marketers of these products are misleading consumers with a number of health claims that are not scientifically sound .
now it becomes a prime concern to produce proper legislation and regulation in the field of probiotics and prebiotics containing food supplement and also it should be revised properly , so that quality products of prebiotic - probiotic or functional food can be manufactured and marketed for the benefit of consumers .
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probiotics containing food supplements available in bangladesh market were identified and collected for assessment . to assess their label claim , they were resuspended into sterile distilled water .
then , series dilutions of each sample solution were prepared and immediately plated out , in duplicate , into de man rogosa sharpe ( mrs ) agar .
these plates were then incubated at 37c for 48 hours and colonies were counted .
viable cell numbers stated on the labels were compared with actual viable cell numbers . to assess the viability of the probiotics included in the products , probiotic strains were isolated from each of the four products and screened for inhibitory activity against six indicator strains .
it was surprisingly found that although the viable cell numbers of all supplements were three to four log cycles lower than label claim of the products , however , this problem did not affect the inhibitory activity of the probiotic strains against indicator strains according to in vitro assessment .
legislation and regulation regarding prebiotic - probiotic containing products should be built up in bangladesh to ensure quality products supply to the consumers .
moreover , manufacturers of probiotic containing products should take the responsibility for providing the consumer with scientifically and legally correct information .
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a 68-year - old man was referred to our pain management center from the department of neurology with motor weakness of the left leg following painful herpetic rash on his left lower extremity .
ten days before presentation , a herpetic rash occurred in his left l4 dermatome and 2 days after the onset of vesicular rash his diagnosis of hz was made by a neurologist .
he was managed with famciclovir 250 mg three times daily , tramadol 150 mg once daily , and pregabalin 75 mg twice daily . even with these medications ,
his pain was rated as 7 on the visual analogue score ( vas ) from 0 ( no pain ) to 10 ( worst pain imaginable ) .
he refused to take opioids due to side effects associated with opioids such as nausea and vomiting .
nine days after the onset of the rash , he noticed progressive motor weakness of his left lower limb .
an mri scan of his brain and lumbar spine was performed to explore the causes of weakness in his left lower extremity by the neurologist .
the neurological examination showed that the muscle strength of his left knee extension was 3/5 using the medical research council muscle strength grading system .
two month previous , he had received a cardiac stent insertion due to myocardial infarction and he was taking warfarin 2 mg and aspirin 100 mg once a day for prevention of thrombosis .
he suffered from intermittent , spontaneous , sore and throbbing pain over the left l4 dermatome , which was provoked by brushing .
pcb was done with 0.5% lidocaine 15 ml and triamcinolone 40 mg under fluoroscopic guidance at the l4 transverse level ( fig .
in addition , 6 days after the pcb , the motor weakness of his knee extension greatly improved and 12 days after the pcb the motor weakness was completely resolved .
hz most commonly involves the sensory ganglion leading to pain and sensory change in the affected dermatome .
the hz - induced motor paresis is rare but can occur in 0.5% to 5% of patients .
it was reported that hz - induced motor paresis typically lasts at least several months , which is associated with high rates of phn .
the possible pathogenesis of motor weakness in hz includes the extension of inflammation from the dorsal sensory ganglion into the ventral root , plexus , and peripheral nerve .
it has a good prognosis , but some patients suffer from permanent motor weakness . when added to antiviral therapy , administration of a steroid along the spinal root and within the epidural space , via interlaminar or transforaminal epidural block ,
can be effective in reducing acute pain from hz and preventing the occurrence of phn .
conliffe et al . reported in 2009 successful management of zoster motor paresis with l5 distribution with a lumbar transforaminal epidural block .
but one of the fatal side effects associated with these procedures is the epidural hematoma , leading to neurological damage .
it has been reported that in 63% of the patients with zoster motor paresis , it was associated with a lesion on postganglionic structures , such as the plexus or peripheral nerve .
a pcb produces a unilateral segmental sensory and motor block by injecting local anesthetics into the paravertebral space containing the lumbar plexus from the l1l4 roots with a contribution from t12 .
therefore , the pcb is known as a lumbar paravertebral block or lumbar plexus block . a pcb is easier and has less frequent adverse effects with equivalent analgesic efficacy , compared to an epidural block .
it has been reported that a thoracic paravertebral block with triamcinolone 40 mg and local anesthetic was effective to treat abdominal segmental motor paresis and pain following hz .
although since 1992 four cases of retroperitoneal hematoma were reported in patients receiving pcbs and thrombophylaxis , it is suggested that deep and superficial plexus nerve blocks do not increase the risk of major bleeding in patients taking thrombophylaxis .
it was reported that a deep cervical plexus block with local anesthetic plus methylprednisolone 30 - 40 mg was effective to treat herpetic pain in patients receiving anticoagulants .
kim and chung reported that administration of triamcinolone 40 mg around the cervical roots and brachial plexus was effective in alleviating pain and improving motor weakness in a patient with brachial plexitis following hz . in the present case ,
therefore , we performed a pcb with local anesthetics plus triamcinolone 40 mg , which could hasten rapid resolution of pain and motor weakness from hz .
the steroid has a strong anti - inflammatory effect , which can decrease inflammatory response in the involved nerve , resulting in hastening recovery of the damaged nerves . in conclusion
, hz - induced motor paresis rarely occurs with good prognosis , but some patients have permanent motor dysfunction . in the present case ,
a pcb hastened a rapid resolution of pain and recovery from motor weakness in a hz patient receiving anticoagulant medication .
therefore , pcb can be a safe and effective option for treatment of hz patients who are treated with anticoagulants .
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reactivation of the latent varicella zoster virus in the sensory ganglion causes herpes zoster ( hz ) .
its characteristic symptom is a painful rash in the involved dermatome .
hz - induced motor weakness is rare and is usually resolved within one year of the onset , but some patients permanently experience motor dysfunction .
epidural steroid administration , with antiviral therapy , can be effective in treating pain from hz and preventing postherpetic neuralgia .
but an epidural block is contraindicated in patients receiving thromboprophylaxis .
a psoas compartment block ( pcb ) provides equivalent analgesic efficacy with significantly low incidence of complication , compared to an epidural block . a 68 year
old male patient recieving thromboprophylaxis presented with motor weakness following painful rash in his left l4 dermatome .
ten days before presentation , herpetic rash occurred on his left leg .
we performed pcb with a steroid and local anesthetic , which successfully and safely alleviated the pain and motor weakness from hz .
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the allergic inflammatory response is characterized by a predominant th2-cell pathway , which is initiated by the uptake of allergens by professional antigen presenting cells ( apcs ) that present selected peptides on mhc class ii molecules to naive t cells , together with isotype switching of b cells to generate ige antibodies specific for common environmental allergens .
the cytokines associated with th2 response are il-4 , il-5 , il-9 , il-13 , and il-33 [ 2 , 3 ] .
thymic stromal lymphopoietin ( tslp ) was first identified as a growth - promoting factor produced by mouse thymic stromal cells that supported the development of immature b cells to the b220/igm stage .
tslp is a type i cytokine that acts via the heteromeric receptor consisting of il-7r and a tslp - specific subunit , tslp receptor ( tslpr ) [ 5 , 6 ] signaling via jak1 and jak2 to mediate the activation of stat5a and stat5b .
tslpr has homology to the common cytokine receptor -chain , c , a component of the receptors for il-2 , il-4 , il-7 , il-9 , il-15 , and il-21 .
tslp is expressed by a range of cell types , including epithelial cells , fibroblasts , keratinocytes , mast cells , protease - activated basophils , human cd68 macrophages , and myeloid dcs ( mdcs ) whereas it is not produced by other lympho - hematopoietic cells , including neutrophils , b cells , t cells , monocytes , plasmacytoid dcs ( pdcs ) , and endothelial cells [ 9 , 10 ] .
tslp acts on many cell types including dendritic cells ( dcs ) , t cells [ 11 , 12 ] , mast cells , nkt cells and eosinophils .
furthermore , tslp may act via dcs to regulate the activation , differentiation , and homeostasis of t cells , but it also has direct effects on t cells , promoting their survival and proliferation in response to tcr activation .
tslp has been implicated in the development of asthma [ 11 , 18 ] , atopic dermatitis , inflammatory arthritis , and other inflammatory disease conditions [ 16 , 19 ] .
interestingly , tslpr knockout ( ko ) mice have a defective allergic inflammatory response to ova in the lung , but this can be reversed by adoptive transfer of wild - type ( wt ) cd4 t cells , underscoring a key role for the action of tslp on these cells .
moreover , tslp induces th-2 attracting chemokines and primes naives th-2 cells to produce il-4 , il-5 and il-13 , and tnf and inhibits th-1 differentiation .
it was demonstrated recently that papain activated basophils or hdm activated airways stromal cells also produce tslp and thus may be important in the initiation of th2 responses [ 20 , 21 ] .
moreover , when lung cells were sorted into epithelial cells or dcs , tslp mrna was expressed by the epithelial cells and by the dcs .
here we extended our investigation on the role of tslp in allergic asthma using clinically relevant protease type allergens , house dust mite ( hdm ) extract and papain .
we demonstrate defective dc help for t cells and diminished th2 and th22 and enhanced th17 responses with diminished allergic airways inflammation in tslpr deficient mice .
o - phenylenediamine , 3-amino-1,2,4-triazole , horseradish peroxidase , and bsa grade v were obtained from sigma chemical company ( st .
the antibodies used for facs analysis , fitc - anti cd3e ( clone 145 - 2c11 ) , pe - anti - il-17a ( clone tc11 - 18h10 ) , percp - anti - cd4 ( clone rma-5 ) , biotin - anti cd8 ( clone 53 - 6.7 ) , biotin - anti tcr ( clone h57 - 597 ) and isotype - matched controls were purchased from pharmingen ( san diego , ca ) .
apc - anti - il-17f ( clone ebio18f10 ) , fitc - anti tcr ( clone gl3 ) and percpef710-anti - il-22 ( clone 1h8pwsr ) antibody were purchased from ebioscience .
c57bl/6 wild type mice and tslp - r were bred in our specific pathogen free animal facility at cnrs ( orleans , france ) .
tslpr mice ( on c57bl/6 genetic background ) were from the laboratory of molecular immunology , national heart , lung and blood institute ( dr w. leonard , bethesda , usa ) .
mice were maintained in a temperature - controlled ( 23c ) facility with a strict 12 h light / dark cycle and were given free access to food and water .
all protocols complied with the french government 's ethical and animal experiment regulations . for hdm model , mice were immunized by intranasal route at days 0 and 7 with 25 g of hdm extracts ( alk abello , danemark ) . on day 14 , 15 and 16
, mice were challenged by intranasal route with 5 g of hdm extracts . for ova model ,
mice were sensitized subcutaneously twice at days 0 and 7 with 200 l saline containing 10 g ovalbumin ( ova , grade v , sigma ) without aluminum adjuvant .
one week after the second sensitization , mice were challenged 3 times by intranasal routes ( on day 14 , 15 and 16 ) with 40 l of saline containing 10 g ova .
mice were killed with co2 inhalation after the last challenge via a tracheal canula , lungs were washed 4 times with 0.5 ml of saline solution ( see below bronchoalveolar lavage ) . for protease allergen papain model ,
mice were anesthetized by isoflurane inhalation , followed by intranasal administration of papain ( 25 g , calbiochem ) in 40 l of saline on days 02 as described .
after bronchoalveolar lavage , lungs were perfused via heart puncture with isoton ii acid free balanced electrolyte solution ( beckman coulter , krefeld , germany ) .
half of the lung was stored at 80c for epo enzyme , cytokines , and chemokines analysis and the other half was fixed overnight in buffered 4% formaldehyde solution for histology analysis .
bronchoalveolar lavages ( bal ) were performed by washing the lungs 4 times with 0.5 ml of saline solution at room temperature .
bal cells were sedimented by centrifugation at 400 g for 10 min at 4c .
the supernatant ( cell - free bal fluid ) was stored at 20c for cytokine analysis .
an aliquot of the cell pellets was stained with trypan blue solution , counted , and 100,000 cells centrifuged on microscopic slides ( cytospin at 1000 rpm for 10 min , at rt ) .
air - dried preparations were fixed and stained with diff - quik ( merz & dade a.g . , dudingen , switzerland ) .
one hundred cells were counted twice for the determination of the relative percentage of each cell type present in the bal .
lung sections of 3 m were stained with periodic acid schiff reagent ( pas ) and examined with a leica microscope ( 20 magnification ) .
peribronchial infiltrates and mucus hypersecretion were assessed by a semi - quantitative score ( 03 ) by two observers independently .
epo activity was determined in order to estimate the recruitment of eosinophils to the lung parenchyma .
after bal and perfusion , lungs were excised , stored frozen at 80c or directly homogenized for 30 seconds in 1 ml of 0.05 m tris / hcl buffer ph 8.0 using a polytron ( kinematic ag , luzern , switzerland ) .
epo activity in the supernatant was determined as estimated from the oxidation of o - phenylenediamine ( opd ) by epo in the presence of hydrogen peroxide ( h2o2 ) using the protocol by van oosterhout and colleagues .
the substrate solution consisted of 10 mm opd in 0.05 m tris / hcl - buffer ( ph = 8) and 4 mm h2o2 ( bdh , poole , uk ) .
substrate solution was added to samples in a 96-wells microplate ( greiner ) and incubated at 37c for 30 min .
duplicate incubations were carried out in the absence and presence of the epo inhibitor 3-amino-1,2,4-triazole ( amt , 2 mmol / l ) .
the absorbance was then measured at 490 nm ( flow labs , irvine , uk ) .
results are expressed as od 490 nm and were corrected for the activity of other peroxidases , which were not inhibited by amt .
the lungs were homogenized for 30 s using a polytron ( kinematic ag , luzern , switzerland ) and the cell debris were eliminated by centrifugation at 10,000 g for 15 min .
il-1 , il-13 , il-33 , tslp , ccl11 , ccl17 ccl22 , and ccl24 concentrations in bal or lung homogenate supernatants were determined by enzyme - linked immunosorbent assay ( elisa ) , using commercial kits from r&d ( abingdon , uk ) .
il-10 , il-12p40 , il-17a , and ifn were determined by bio - plex mouse cytokine group i 23-plex on magpix ( luminex , bio rad ) according to the manufacturers ' instructions .
murine bone marrow cells were isolated from femurs of wild type and tslpr mice and differentiated into myeloid dendritic cells ( dcs ) by culturing at 1 10 cells / ml for 10 days in rpmi medium supplemented with 10% fcs ( hyclone ) , non - essential amino - acids , 0.05 g / ml asparagine , mem vitamins , sodium pyruvate , gentamycin ( 2 m , invitrogen ) , penicillin ( 100 u / ml , gibco , invitrogen ) , 10 mg / ml streptomycin , 2-mercaptoethanol 50 m and 4% j558l cell - conditioned medium as a source of gm - csf ( change medium on days 3 , 6 , and 8) .
dendritic cells were treated with 100 g / ml ova - fitc ( molecular probes , france ) for 2 h and analyzed by facs .
lymph node cd4 t cells were purified from ot2 mice by magnetic cell sorting ( dynal , invitrogen ) .
cd4 t cells ( 10 cells ) were co - cultured with 10 wt or tslpr dendritic cells preloaded with ova peptide ( 10 g / ml , 2 h ) .
t cell proliferation was assessed by cfse staining ( 0.5 m ; molecular probes , invitrogen ) .
lung mononuclear cells were isolated from mice 24 h after the last challenge as described .
briefly the aorta and the inferior vena cava were sectioned and the lungs were perfused with saline .
the lobes of the lungs were sliced into small cubes and then incubated for 20 min in 1 ml rpmi 1640 solution containing dnase i ( 1 mg / ml ) and collagenase iv ( 2 mg / ml ) ( sigma - aldrich ) .
lung mononuclear cells were separated by centrifugation on percoll ( amersham biosciences ) gradients ( 37% ) . isolated lung
mononuclear single cells were plated in round bottom 96-well plates ( 2 10/ml ) and restimulated 4 h in vitro with phorbol 12-myristate 13-acetate ( pma ) ( 50 ng / ml ) and ionomycin ( 750 ng / ml ; both from sigma - aldrich ) in complete medium ( imdm supplemented with 5% ( vol / vol ) fcs , l - glutamine ( 2 m ) , penicillin ( 100 u / ml ) , streptomycine ( 100 g / ml ) , and -mercaptoethanol ( 50 nm ) all from invitrogen ) .
cell suspensions from lung were restimulated in vitro for 4 h in complete medium with pma ( 50 ng / ml ) and ionomycin ( 750 ng / ml ; both from sigma - aldrich ) in presence of brefeldine a ( golgiplug , bd biosciences , france ) . to prevent a specific binding to fcr , 2.4.g2 blocking purified antibody was used .
after 4 h , cells were stained with the following monoclonal antibodies , fitc - labeled tcr , biotin - labeled tcr,-v450-labeled cd4 , and apc - cy7-labeled cd8. after washing , cells were permeabilized for 20 min with cytofix / cytoperm kit ( bd biosciences , france ) and stained with apc labeled il-5 , pe - labeled il-17a and percpefluor710labeled il-22 .
fluorescence data were acquired by using diva software ( bd bioscience , france ) and analyzed using flowjo software ( treestar ) .
the data are presented as the mean sem with n = 68 animals per condition .
the significance of differences between two groups was determined by one way anova ( non parametric test ) using prism software .
statistical significance was reported if p < 0.05 was achieved , * p 0.05 ; * * p 0.01 ; * * * p 0.001 .
hdm is a major source of allergens in allergic patients and cause allergic airway inflammation resembling human asthma in mice by facilitating barrier disruption , inflammation , and allergen sensitization of the airways through tlr4-dependent innate and acquired immunity [ 20 , 25 , 26 ] .
previous studies demonstrated reduced allergic response to ovalbumin in tslpr mice , here it was asked whether the inflammatory response to the clinically relevant allergen hdm is dependent on tslpr signaling .
mice were immunized twice and challenged on days 14 , 15 and 16 by intranasal instillation of hdm and the bal fluid and lung tissues were analyzed on day 17 .
eosinophil , neutrophil and lymphocyte influx in the alveolar space were significantly reduced in tslpr mice while macrophages were unchanged ( figures 1(a)1(d ) ) .
furthermore eosinophil peroxidase activity ( epo ) was also reduced in the lung tissue ( figure 1(e ) ) .
il-13 and il-33 are known to drive eosinophil maturation and infiltration , mucus production and bronchial hyperreactivity . to investigate whether tslp signaling disruption could affect cytokine production , we induced local airway allergic inflammation with hdm on tslpr mice .
the analysis of cytokines in lung homogenate revealed a drastic reduction of il-1 , il-13 , and il-33 in the absence of tslpr signaling suggesting a significant reduction of th2 associated local response to hdm ( figures 2(a)2(c ) ) .
furthermore the chemokines ccl11 , ccl17 , ccl22 , and ccl24 were significantly reduced upon hdm allergen exposure ( figures 2(e)2(g ) ) underscoring a defect of eosinophil recruitment in the absence of tslpr .
the absence of tslp upregulation in tslpr mice suggests an autocrine loop of tslp production in the lung ( figure 2(d ) ) .
thus , the data extend the notion of a critical role of tslpr to generate a th2 cytokine / chemokine milieu . in view of an important role of tslpr in hdm induced allergic inflammation we examined the lung tissue at day 17 .
hdm immunized and challenged wt mice developed a robust inflammation with abundant eosinophils and mucus production in the bronchial epithelial cells ( figures 3(a ) and 3(b ) ) .
by contrast eosinophilic inflammation and mucus production was largely abrogated in the absence of tslpr .
papain , a cysteine protease , was shown to preferentially induce an igg1 response and results in mast cell degranulation , both features typical of an allergic reaction .
it has recently been shown that the protease papain could induce asthma like symptoms in rag - deficient mice [ 22 , 28 ] .
this effect is mediated by innate lymphocytes also known as natural helper cells or nuocytes cells .
intranasal administration of papain into tslpr mice showed a dramatic decrease of eosinophils in bal fluid and eosinophil peroxidase activity in the lung , while lymphocyte , macrophage and neutrophil recruitment into balf was not affected ( figures 4(a)4(e ) ) .
histological examination revealed that lung inflammation in papain treated tslpr mice was substantially reduced than in wt mice ( figures 4(f ) and 4(g ) ) .
interestingly , papain induced th2 and inflammatory cytokines such as il-1 , il-13 , il-33 , and tslp ( figures 5(a)5(d ) ) were profoundly impaired in tslpr mice as well as the chemokines ccl11 , ccl17 , ccl22 , and ccl24 ( figures 5(e)5(h ) ) . therefore our data demonstrate that tslp is required in papain induced eosinophil recruitment , pulmonary inflammation and th2 cytokine production . in view of the data suggesting a critical and autocrine effect of tslp via tslpr expressing dc , we investigated antigen uptake by dc in presence or absence of tslpr and the subsequent t cell response . for this investigation we used ova as antigen in order to use peptide specific ot2 t cells since hdm tcr transgenic
we found reduced uptake of fitc - labeled ova by tslpr dc ( figure 6(a ) ) .
furthermore the proliferation of the ova peptide specific ot2 t cells in response to ova peptide pulsed tslpr dc was reduced as compared to wt dc ( figure 6(b ) ) .
finally we verified the previous data that eosinophil recruitment in the bronchoalveolar space in ova immunized and challenged tslpr mice ( figure 6(c ) ) .
therefore , tslpr signaling in dcs is required for antigen uptake and presentation to activate cd4 t cells , consistent with a recent report demonstrating tslp production and response by dc .
since th17 cell differentiation and allergic lung inflammation [ 30 , 31 ] contribute to allergic inflammation , we asked whether tslpr signaling may contribute to th17 cell response .
we reported that il-17a is required [ 32 , 33 ] and il-22 reduces the allergic responses [ 34 , 35 ] .
therefore , we asked whether il-17a expression in t cells from the lung of ova or hdm immunized and challenged mice is altered .
pulmonary il-17atcr and il-17atcr cells were significantly increased in wt mice , but the recruitment of the tcril-17a cells augmented much more in the absence of tslpr ( figures 7(a)7(h ) ) . to address the mechanisms underlying increased il-17a level and diminished th2 response in tslpr mice , we examined the levels of cytokines shown to promote il-17a level and regulatory t cells in the airways .
analysis of il-12p40 , ifn , and il-10 in the lung homogenate revealed that il-12p40 and il-10 levels were increased in tslpr mice treated with hdm , while ifn level was not detectable ( figures 7(i)7(h ) ) .
since we previously reported a cross - regulation of il-22 and il-17 , we investigated the expression of il-22 a pulmonary t cells in the absence of tslpr .
we found a significant reduction of total cd4il-22 t cells from the lung of tslpr mice , while the total lung cd4il-5 t cells were not significantly reduced ( figures 8(a ) and 8(b ) ) .
therefore , tslpr signaling is involved in the balance of th17/th22 , in favor of the development of the th22 subset , suggesting that physiologically tslp dampens il-17a and enhances il-22 production .
based on our previous work showing a reciprocal role of il-17a and il-22 , the altered balance of il-17a and il-22 may contribute to the diminished allergic lung response .
several studies linked tslp to lung inflammation and helminth infection [ 18 , 19 , 36 ] , although the role of tslp in airway inflammation using clinically relevant protease allergens hdm and papain have not been yet addressed . here
we demonstrate that the allergic inflammatory response to protease allergens hdm or papain is dependent on tslpr signaling .
proteases are important components of many allergens and thought not only to disrupt mucosa integrity but also activate airway epithelial cells .
our results demonstrate impaired allergic lung inflammation and th2 response with lower eosinophil influx and reduced il-1 , il-13 and il-33 levels in the airways of tslpr deficient mice .
these findings were consistent with previous studies which demonstrate that tslp may recruit eosinophils to sites of th2 cytokine - associated inflammation by upregulating the common myeloid marker cd11b and the integrin l2 ligand icam-1 on eosinophils .
dendritic cells are known to play a crucial role in allergic lung inflammation and are essential for t cell activation and th2 cell differentiation and recruitment into the airways and trigger local th2 cytokine production [ 37 , 38 ] .
we demonstrate reduced antigen uptake by myeloid tslpr deficient dc and defective help for t cells measured by diminished t cell proliferation .
therefore , the defects in dendritic cell functions may affect th2 cells differentiation , cytokines and chemokines productions in the lung of tslpr - deficient mice .
while the role of tslp on th2 response is established , its effect on il-17a and il-22 cell response is novel .
we has established a regulatory role for il-17a and il-22 in allergic asthma [ 32 , 34 , 35 ] .
our study in tslpr deficient mice suggests that tslpr signalling inhibits il-17a expressing t cells and enhances the il-22 t cell response in the lung .
these findings are novel and consistent with previous data demonstrating that il-22 inhibits allergic lung inflammation by regulating il-17a expression [ 34 , 35 ] .
il-10 has broad immunosuppressive and anti - inflammatory actions relevant to the inhibition of asthma pathology .
il-10 has been found to be essential for effective suppression of allergic responses in the lung [ 39 , 40 ] .
il-10 is a potent inhibitor of proinflammatory cytokine and acts on antigen - presenting cells to dampen t cell activation , including th2 cells [ 41 , 42 ] .
our results demonstrate increase il-10 levels in the lung supernatant of tslpr mice treated with hdm compared to wt mice .
therefore the data suggest tslp modulates il-10 and this might contribute to the inhibition of allergic inflammation in tslpr mice .
these findings add to the complexity of the regulation of an allergic response where tslpr signaling plays an important part [ 18 , 36 , 43 , 44 ] .
furthermore , tslpr dependent regulation of innate lymphoid cells producing il-22 may contribute to the inflammatory response in the lung and intestinal tract .
therefore our data support the notion that tslpr signaling in myeloid dc is required for t cell differentiation into th2 and th22 cells , which may control the il-17a response .
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background . thymic stromal lymphopoietin ( tslp )
is induced in allergic skin and lung inflammation in man and mice .
methods . allergic lung inflammation induced by two proteases
allergens hdm and papain and a classical allergen ovalbumin was evaluated in vivo in mice deficient for tslpr .
eosinophil recruitment , th2 and th17 cytokine and chemokine levels were determined in bronchoalveolar lavage fluid , lung homogenates and lung mononuclear cells ex vivo .
results . here
we report that mice challenged with house dust mite extract or papain in the absence of tslpr have a drastic reduction of allergic inflammation with diminished eosinophil recruitment in bal and lung and reduced mucus overproduction .
tslpr deficient dcs displayed diminished ova antigen uptake and reduced capacity to activate antigen specific t cells .
tslpr deficient mice had diminished proinflammatory il-1 , il-13 , and il-33 chemokines production , while il-17a , il-12p40 and il-10 were increased . together with impaired th2 cytokines , il-17a expressing tcr+ t cells were increased , while il-22 expressing cd4 + t cells were diminished in the lung . conclusion .
therefore , tslpr signaling is required for the development of both th2 and th22 responses and may restrain il-17a .
tslp may mediate its effects in part by increasing allergen uptake and processing by dcs resulting in an exacerbated asthma .
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cornus mas l. is commonly known as european cornelian cherry and belongs to the cornaceae family .
it is a tall deciduous shrub or small tree ( 36 m in height ) that is indigenous to europe and parts of asia [ 1 , 2 ] .
traditionally , c. mas has been used for improving health conditions , such as bowel complaints , fever , and diarrhea [ 36 ] .
fresh european cornelian cherry fruits are often processed to produce drinks , syrups , and jams [ 7 , 8 ] . some investigations of the nutritional and phytochemical properties of european cornelian cherry fruit have been reported previously .
the fruit is reported to contain 0.10.3% fat , 0.4% protein , 21.7% carbohydrate , 0.8% ash , 0.5% dietary fiber , 6.615.1% total sugar ( fructose 33.143.1% , glucose 53.663.1% ) , and 4.229.96% reducing sugars [ 3 , 9 ] .
the fruit has ph 2.73.2 and contains at least 15 amino acids , including aspartic acid , glutamic acid , serine , histidine , glycine , threonine , arginine , alanine , tyrosine , valine , phenylalanine , isoleucine , leucine , lysine , and proline .
minerals in c. mas fruit include copper ( 1.2 to 8.1 mg / kg dry weight ) , iron , zinc , phosphorus , potassium , calcium , magnesium , and sulphur .
the vitamin c content of the fruit is reported to be 16.4 to 38.5 mg/100 g .
c. mas fruit has been found to contain a wide range of phytochemicals , including tannins ( 131.51601.2 mg / l ) , phenolics ( 29.7674.83 mg / g dry ) , organic acids ( 4.67.4% ) , anthocyanin , fatty acids , and flavonoids [ 3 , 4 , 1016 ] .
anthocyanins have been found in the range of 1.12 to 2.92 mg / g in different c. mas fruit sources [ 14 , 16 , 17 ] .
two studies from du and francis reported the presence of five anthocyanins in the fruit , namely , delphinidin 3-galactoside , cyanidin 3-galactoside , cyanidin 3-rhamnosylgalactoside , pelargonidin 3-galactoside , and pelargonidin 3-rhamnosylgalactoside [ 18 , 19 ] .
cyanidin 3-glucoside , cyanidin 3-rutinoside , and pelargonidin 3-glucoside were identified by tural and koca , with pelargonidin 3-o - glucoside being the most abundant followed by cyanidin 3-o--d - galactoside .
delphinidin 3-o--galactopyranoside , cyanidin 3-o--galactopyranoside , and pelargonidin 3-o--galactopyranoside were also identified in c. mas fruit .
these include aromadendrin 7-o--d - glucoside , quercetin 3-o--d - xyloside , quercetin 3-o--l - rhamnoside , quercetin 3-o - rutinoside , quercetin 3-o--d galactoside , quercetin 3-o--d - glucose , quercetin 3-o--d - glucoside , and kaempferol 3-o - galactoside .
fatty acids identified in the fruit include lauric acid , myristic acid , pentadecenoic acid , palmitic acid , palmitoleic acid , stearic acid , oleic acid , vaccenic acid , linoleic acid , linolenic acid , and cis-10,12-octadecadienoic acid .
however , there is very little information regarding the occurrence of iridoids in c. mas .
only two iridoids , secologanin and loganic acid , have been previously reported to occur in c. mas , with only one being reported for the fruit [ 2022 ] .
the current investigation was prompted by a lack of information on taxonomically critical and biologically active iridoids in c. mas fruit .
the aim of this study is to identify more phytochemical compounds in the c. mas fruit , particularly bioactive iridoids , by using ultra performance liquid chromatography ( uplc ) coupled with photodiode array spectrophotometry ( pda ) and electrospray time - of - flight mass spectrometry ( esi - tof - ms ) .
124876 ) , and formic acid of lc - ms grade were purchased from fisher scientific co. ( fair lawn , nj , usa ) .
shbb9224v ) of lc - ms grade was purchased from sigma - aldrich ( st . louis , mo , usa ) .
tartaric acid , malic acid , chlorogenic acid , gallic acid , and rutin standards were purchased from sigma - aldrich ( st . louis , mo , usa ) .
citric acid was purchased from fisher scientific co. loganic acid was obtained from chromadex ( irvine , ca , usa ) .
loganin , sweroside , and cornuside were purchased from chengdu biopurify phytochemicals ltd ( sichuan , china ) .
the standards were accurately weighed and then dissolved in an appropriate volume of meoh to produce corresponding stock standard solutions . working standard solutions were prepared by diluting the stock solutions with meoh at different concentrations .
all stock and working solutions were maintained at 0c in a freezer .
c. mas fruit samples were collected from wild trees in the mountains near kastamonu , turkey , in 2012 .
one gram of mashed fruit puree was weighed accurately and 9 ml of 50% meoh in h2o was added .
the supernatant was transferred into a volumetric flask and volume brought to 10 ml with 50% meoh in h2o .
all samples were filtered through a nylon microfilter ( 0.45 m pore size ) before uplc analysis .
analyses were performed with an agilent 1260 infinity lc system coupled to an agilent 6230 time - of - flight ( tof ) lc / ms system ( agilent technologies , santa clara , ca ) .
the agilent 1260 lc module was coupled with a photodiode array ( pda ) detector and a 6230 time - of - flight ms detector , along with a binary solvent pump and an autosampler .
chromatographic separations were performed with an atlantis reverse phase c18 column ( 4.6 mm 250 mm ; 5 m , waters corporation , milford , ma , usa ) .
the pump was connected to a gradient binary solvent system : a , 0.1% formic acid in h2o ( v / v ) and b , 0.1% formic acid in mecn .
the mobile phase was programmed consecutively in linear gradients as follows : 05 min , 98% a and 2% b ; 40 min , 70% a and 30% b ; 4652 min , 2% a and 98% b ; and 5355 min , 98% a and 2% b. the ionization source was agilent jet stream , with electrospray ionization ( esi ) negative mode employed for acquisition of mass spectra .
uv spectra were monitored in the range of 200 nm and 400 nm .
injection volume was 2 l for each of the sample solutions , followed by needle wash .
nm32la , peak scientific instruments ltd , scotland , uk ) , was used as the drying and nebulizer gas .
other ms instrumental conditions are summarized as follows : drying gas temperature and flow rate were 350c and 11.0 l / min , respectively ; nebulizer pressure was 50 psi ; sheath gas temperature and flow rate were 350c and 12.0 l / min , respectively ; capillary , nozzle , and fragmentor voltages were 3500 , 500 , and 100 v , respectively ; skimmer was 65.0 ; oct 1 rf vpp was 750 .
. data collection and integration were performed using masshunter workstation software ( version b.05.00 ) .
the data was collected in the range of 100 and 1700 m / z .
two independent reference lock - mass ions , purine ( m / z 119.03632 ) and hp-0921 ( m / z 966.000725 ) , were employed to ensure mass accuracy and reproducibility .
compound identities were then confirmed by comparing the uplc retention times and uv spectra of target peaks with those of reference compounds .
the sos chromotest in e. coli pq37 was used to determine the potential for loganic acid , loganin , sweroside , and cornuside to induce primary dna damage .
e. coli pq37 was incubated in lb medium in a 96-well plate at 37c in the presence of the iridoids for 2 hours .
the concentrations tested were 7.81 , 15.6 , 31.2 , 62.5 , 125 , 250 , 500 , and 1000 g ml .
following incubation with replicate samples , 5-bromo-4-chloro-3-indolyl--d - galactopyranoside was added to the wells to detect -galactosidase enzyme activity , which is induced during sos repair of damaged dna .
nitrophenyl phosphate is also added to the wells to measure alkaline phosphatase activity , an indicator of cell viability .
the samples were again incubated and the absorbance of each sample , blanks , and controls was measured at 410 and 620 nm with a microplate reader .
vehicle blanks and positive controls , 1.25 g ml 4-nitroquinoline 1-oxide ( 4nqo ) , were included in this test .
the induction factor of each material was calculated by dividing the absorbance of the sample at 620 nm by that of the blank , while also correcting for cell viability .
the primary dna damage test was performed again , similar to the method described above .
however , the method was modified to include incubation of e. coli pq37 in the presence of 1.25 g ml 4nqo with 250 g ml loganic acid , loganin , sweroside , or cornuside .
induction factors were calculated in the same manner as described above . the percent reduction in genotoxicity
was determined by dividing the difference between the induction factor of 4nqo and the blank ( induction factor of 1 ) by the difference between the induction factor of 4nqo plus iridoid sample and the blank .
although the application of lc - ms may minimize the need for chromatographic separation , a good lc separation may prevent ion suppression and isobaric interferences in the ms analysis .
the uplc system delivered a more rapid and effective separation than hplc , especially for the wide range of phytochemicals identified .
the flow rate , solvent system , and column were optimized for the uplc system relative to the analysis of phytochemicals in c. mas fruit samples
. a moderate flow rate of 0.8 ml / min accommodated electrospray source , column separation and optimized sensitivity and resolution .
better peak shape and resolution for each target peaks , as well as optimal sensitivity for ion detection , were obtained by adding 0.1% formic acid .
the column temperature was kept at 40c to minimize column pressure due to flow rate .
a gradient solvent system of mecn - h2o ensured complete separation of target peaks within a limited time frame .
the time - of - flight mass spectral detector is known for providing accurate and precise mass information .
it can accurately measure mass values with a mass error less than 5 ppm .
the generation of empirical formulae results in possible identification of phytochemicals by means of elemental composition analysis .
our experiments used electrospray ionization and were operated in negative mode as the interface since it generated higher signal information for the compounds with less interruption compared to positive mode .
all other mass spectral parameters , including drying gas flow and temperature , nebulizer pressure , and sheath gas flow and temperature , were also optimized in order to achieve good sensitivity and resolution .
the total ion chromatogram ( tic ) of c. mas fruit is shown in figure 1 .
negative ion electrospray lc - ms chromatograms of the fruit puree resulted in the identification of 10 peaks .
deprotonated molecular ions [ m h ] , chlorinated ions [ m + cl ] , and/or formic acid adducts [ m + hcoo ] of the compounds were identified with the tof detector . as a result , accurate molecular weights were determined . since peaks 6 , 7 , and 9 were not clearly visible on the tic chromatogram due to their low concentration ,
accurate mass spectra ( mass - to - charge , m / z ) of the phytochemical peaks identified are shown in figure 2 .
peaks 1 , 3 , and 4 had deprotonated molecular ions [ m h ] with m / z values of 149.0099 , 191.0201 , and 169.0146 , respectively , corresponding to the molecular formula of c4h6o6 , c6h8o7 , and c7h6o5 .
peak 2 had [ m h ] and [ m + cl ] with m / z values of 133.0145 and 168.9898 , respectively , matching the molecular formula c4h6o5 .
compound 6 had [ m h ] and [ m + hcoo ] at m / z 353.0884 and 399.0936 , corresponding to c16h24o10 .
comparisons against reference compound retention revealed that peaks 14 and 6 were organic acids , namely , tartaric acid ( 1 ) , malic acid ( 2 ) , citric acid ( 3 ) , gallic acid ( 4 ) , and chlorogenic acid ( 6 ) . by the same methods ,
peak 9 was determined to be rutin ( 9 ) , a flavonoid glycoside .
peak 5 is a major peak on the chromatogram , eluting at 22.345 min and displaying both [ m h ] and [ m + cl ] at m / z 375.1319 and 411.1069 .
peak 7 showed [ m h ] and [ m + hcoo ] at m / z 389.1455 and 435.1512 .
peak 8 exhibited a deprotonated m / z , a chlorinated m / z , and a formic acid adduct of 357.1196 , 393.0964 , and 403.1252 , respectively .
peak 10 had deprotonated ions with m / z value of 541.1569 . by comparing these pieces of information with uv and mass spectra , as well as retention times of reference compounds , peaks 5 , 7 , 8 , and 10
were identified as the iridoids loganic acid ( 5 ) , loganin ( 7 ) , sweroside ( 8) , and cornuside ( 10 ) .
a summary of the chromatographic and mass spectral data of the phytochemical compounds identified in c. mas fruit is given in table 1 .
loganin and sweroside were observed to have strong antibacterial , antifungal , and antispasmodic activities .
cornuside has remarkable antioxidant activity and may provide protection against acute myocardial ischemia and reperfusion injury . in the primary dna damage test in e. coli pq37 ( table 2 ) ,
the mean induction factors for loganic acid , loganin , sweroside , and cornuside , at 1000 g ml , were 1.095 , 0.995 , 1.028 , and 1.068 , respectively .
at all concentrations tested , these iridoids did not induce any sos repair at a frequency significantly above that of the blank . statistically , induction factors were not different from that of the blank , and all results remained well below the twofold criteria for genotoxicity .
sos chromotest results have a high level of agreement ( 86% ) with those from the reverse mutation assay .
therefore , the sos chromotest has some utility in predicting potential mutagenicity , in addition to primary dna damage .
these results are consistent with previously published genotoxicity tests of other food derived iridoids . in the antigenotoxicity test
, 4nqo exhibited obvious genotoxicity , inducing sos repair more than 3-fold above that of the vehicle blank .
however , loganin , sweroside , and cornuside apparently reduced the amount of 4nqo that caused dna damage , as mean induction factors were lowered to below 3 in cells incubated in the presence of these iridoids ( table 3 ) .
the uplc - tof - ms method reported has been shown to be useful for secondary metabolite profiling of cornus mas fruit . with this method ,
a total of ten phytochemicals were identified , including five organic acids and four iridoids . to our knowledge , this is the first time that 14 and 610 have been identified in c. mas fruit .
loganin , sweroside , cornuside , and loganic acid will provide valuable information for chemical taxonomy .
iridoids , together with other characteristic components , can be used for identification , authentication , and standardization of c. mas fruit raw materials and commercial products .
the iridoids in the fruit did not display any toxicity . on the other hand ,
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cornus mas l. is indigenous to europe and parts of asia .
although cornus is widely considered to be an iridoid rich genera , only two iridoids have been previously found in this plant .
the lack of information on taxonomically and biologically active iridoids prompted us to develop and optimize an analytical method for characterization of additional phytochemicals in c. mas fruit .
an ultra performance liquid chromatography ( uplc ) coupled with photodiode array spectrophotometry ( pda ) and electrospray time - of - flight mass spectrometry ( esi - tof - ms ) was employed and mass parameters were optimized .
identification was made by elucidating the mass spectral data and further confirmed by comparing retention times and uv spectra of target peaks with those of reference compounds .
primary dna damage and antigenotoxicity tests in e. coli pq37 were used to screen the iridoids for biological activity . as a result ,
ten phytochemicals were identified , including iridoids loganic acid , loganin , sweroside , and cornuside .
nine of these were reported for the first time from c. mas fruit .
the iridoids did not induce sos repair of dna , indicating a lack of genotoxic activity in e. coli pq37 .
however , loganin , sweroside , and cornuside did reduce the amount of dna damage caused by 4-nitroquinoline 1-oxide , suggesting potential antigenotoxic activity .
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the percutaneous transluminal angioplasty ( pta ) and carotid artery stenting ( cas ) have largely emerged as the treatment option over the past decade .
bilateral simultaneous carotid artery stenting done in the same session had been reported in the literature . in our present study , we want to discuss the safety and efficacy of the simultaneous bilateral carotid artery stenting ( sbcas ) in a series of 9 patients .
there are limited number of series in the literature that have evaluated the details of the sbcas.[13 ] we present the first case series of sbcas from india in our study .
we aim to highlight the selection criteria , clinical presentation , the treatment strategy , and the clinical outcome in these patients .
a total of 308 patients underwent carotid artery stenting in our institution from january 2005 to december 2011 . among them , 32 ( 10.3% ) patients had significant bilateral carotid artery disease .
inclusion criteria of the present study were patients who on digital subtraction angiography ( dsa ) showed bilateral carotid artery stenosis > 50% in the symptomatic side and > 60% in the asymptomatic side .
the criteria were based on the measurement using nascet ( north american society carotid endarterectomy trial ) criteria , where smallest luminal diameter at the level of stenosis was compared to the normal arterial diameter distal to the stenosis on dsa .
the inclusion criteria were similar to the eligibility criteria of the crest trial . according to our inclusion criteria ,
simultaneous bilateral carotid artery stenting was performed on 9 patients ( 2.9% of total cases ) .
the study included 8 males and 1 female ( ages range : 50 - 75 years ; average age : 63 years ) .
patient symptoms ranged from recurrent tias lasting for a few minutes to hemiparesis [ table 1 ] .
all the patients in the series had a history of at least one significant medical illness ( diabetes mellitus ( dm ) , hypertension ( ht ) , ischemic heart disease ( ihd ) ) .
magnetic resonance imaging ( mri ) with magnetic resonance angiography ( mra ) was done in all the patients .
since time of flight mra overestimates the stenosis , the inclusion criteria were only based on the dsa findings .
the exclusion criteria of our present study , in spite of presence of significant bilateral carotid artery disease , included deranged renal function ( serum creatinine > 2 mg% ) and compromised cardiac reserve ( ejection fraction of < 40% on 2d - echocardiogram ) , and severely tortuous anatomy of the vessels .
however , in 1 patient among the 9 cases selected , we went ahead with the procedure in spite of morbid obesity , orthopnea , and deranged cardiac function ( ejection fraction of 30% ) since the patient presented with severe triple vessel disease requiring early coronary artery bypass graft ( cabg ) and had an increasing frequency of recurrent bilateral transient ischemic attacks . high - risk consent forms with explanation of benefits versus risks were signed by the patient .
all the patients in the series underwent treatment within 1 week of the onset of the most recent episode of tia or stroke .
symptoms and imaging findings of the patients included in the study all the patients in the present series had symptomatic lesion at least on one side .
five patients among the 9 presented with bilateral recurrent anterior circulation transient ischemic attacks ( tias ) from a period ranging from 1 week to 6 months .
had recurrent right - sided upper limb paresthesias and 2 others had recurrent left - side weakness ( patient no . 2 and 8) .
mri ( t1-weighted sequence , t2-weighted sequence , diffusion - weighted imaging ( dwi ) with apparent diffusion coefficient ( adc ) and gradient echo ) and mra with 3d time of flight was done in all the patients .
small foci of diffusion restriction suggesting acute infarcts in bilateral watershed territories were seen in 2 patients .
tiny acute infarcts were seen in left mca territory in 2 patients , and similar foci were seen on the right side in 2 others .
a chronic right mca infarct ( < 1/3 of the territory ) with no acute foci on dwi was seen in 1 patient ( patient no . 7 ) .
mri of the brain parenchyma was normal in 2 patients who presented with recurrent right - sided tias .
pre - treatment standard double anti - platelet regimen was given in all the cases .
all the patients were on clopidogrel 75 mg / day and enteric - coated aspirin 150 mg / day for at least 10 days prior to the procedure .
anti - hypertensive medications , if any , were not stopped on the morning of the procedure .
all the procedures were done under local anesthesia with careful hemodynamic monitoring except in 1 patient , in whom the procedure was done under general anesthesia .
this patient had severe orthopnea due to morbid obesity and low ejection fraction ( 30% ) .
an 8 f right femoral access was secured , and guiding catheter ( vistabrite mps ) was placed in the common carotid artery ( cca ) .
baseline angiography was done , and stenoses were measured on both the sides by nascet criteria , and intracranial circulation was evaluated .
predilation of the lesion prior to carotid stenting was based on the operator 's discretion and was done in 4 out of 18 lesions .
subsequently , 8 6 40 mm self - expanding stent stent ( protege , ev3 ) was deployed across the lesion .
stenting was first done on the dominant side , which was the symptomatic side at the time of presentation . in case of bilateral tias ,
post - dilation was strictly avoided after the placement of the first stent in all the cases , in order to minimize the baroreceptor response .
post - dilation after the placement of the second carotid stent was done in 3 of the 9 patients where the residual lesion was > 30% .
post - procedure angiogram was done to evaluate the flow across the stented segment . increased antegrade cerebral circulation was documented on the cerebral angiogram [ figures 13 ] .
60-year - old male patient with recurrent - sided tias ( patient 4 in the table ) ( a ) dsa shows 85% narrowing of left ica with ( b ) decreased antegrade cerebral circulation .
( c ) dsa of right cca shows critical stenosis of the right ica with ( d ) reduced antegrade flow in the cerebral circulation .
( e ) post - procedure right cca angiogram shows good glow across the stented segment with ( f ) increased antegrade circulation .
( g ) post - procedure right cca angiogram shows good flow across the stented segment with ( h ) improved intracranial circulation , and ( i ) ap view shows stents on both the sides 55-year - old man who presented with recurrent right sided tias ( patient 1 in the table ) ( a ) mr dwi shows acute infarcts in the left mca terrritory .
( b ) dsa of left cca shows critical stenosis of left ica and ( c ) poor antegrade flow in the cerebral circulation ( d ) post - procedure left cca angiogram reveals the patent stented segment ( e ) with good antegrade flow in cerebral circulation .
( a ) pre - procedure right cca angiogram of the same patient ( patient 1 in the table ) shows severe narrowing of the left ica , and ( b ) right cerebral angiogram shows decreased flow .
( c ) post - procedure left cca angiogram shows good flow across the stented segment and ( d ) shows increased antegrade flow in the cerebral circulation .
the stent on the left side is also seen ( white arrow in c ) all patients were monitored in the neurointensive care unit after the procedure .
majority of the patients ( 6 out of the 9 ) required infusion of dopamine or noradrenaline to treat hypotension due to baroreceptor response during stent / balloon deployment .
the mean arterial pressure ( map ) was maintained between 80 to 90 mm of hg on the infusion .
the patients were shifted out of neurointensive care unit after the infusion was stopped , which was 24 to 48 hours in all the cases .
patients were also closely monitored for headache , vomiting , seizures , and any fresh neurological deficit suggesting cerebral hyper perfusion .
all the patients in the present series had symptomatic lesion at least on one side .
five patients among the 9 presented with bilateral recurrent anterior circulation transient ischemic attacks ( tias ) from a period ranging from 1 week to 6 months .
had recurrent right - sided upper limb paresthesias and 2 others had recurrent left - side weakness ( patient no . 2 and 8) .
mri ( t1-weighted sequence , t2-weighted sequence , diffusion - weighted imaging ( dwi ) with apparent diffusion coefficient ( adc ) and gradient echo ) and mra with 3d time of flight was done in all the patients .
small foci of diffusion restriction suggesting acute infarcts in bilateral watershed territories were seen in 2 patients .
tiny acute infarcts were seen in left mca territory in 2 patients , and similar foci were seen on the right side in 2 others .
a chronic right mca infarct ( < 1/3 of the territory ) with no acute foci on dwi was seen in 1 patient ( patient no .
mri of the brain parenchyma was normal in 2 patients who presented with recurrent right - sided tias .
pre - treatment standard double anti - platelet regimen was given in all the cases .
all the patients were on clopidogrel 75 mg / day and enteric - coated aspirin 150 mg / day for at least 10 days prior to the procedure .
anti - hypertensive medications , if any , were not stopped on the morning of the procedure .
all the procedures were done under local anesthesia with careful hemodynamic monitoring except in 1 patient , in whom the procedure was done under general anesthesia .
this patient had severe orthopnea due to morbid obesity and low ejection fraction ( 30% ) .
an 8 f right femoral access was secured , and guiding catheter ( vistabrite mps ) was placed in the common carotid artery ( cca ) .
baseline angiography was done , and stenoses were measured on both the sides by nascet criteria , and intracranial circulation was evaluated . the stenosis was crossed with 0.014 wire .
predilation of the lesion prior to carotid stenting was based on the operator 's discretion and was done in 4 out of 18 lesions .
subsequently , 8 6 40 mm self - expanding stent stent ( protege , ev3 ) was deployed across the lesion .
stenting was first done on the dominant side , which was the symptomatic side at the time of presentation . in case of bilateral tias , carotid stent
post - dilation was strictly avoided after the placement of the first stent in all the cases , in order to minimize the baroreceptor response .
post - dilation after the placement of the second carotid stent was done in 3 of the 9 patients where the residual lesion was > 30% .
post - procedure angiogram was done to evaluate the flow across the stented segment . increased antegrade cerebral circulation was documented on the cerebral angiogram [ figures 13 ] .
60-year - old male patient with recurrent - sided tias ( patient 4 in the table ) ( a ) dsa shows 85% narrowing of left ica with ( b ) decreased antegrade cerebral circulation .
( c ) dsa of right cca shows critical stenosis of the right ica with ( d ) reduced antegrade flow in the cerebral circulation .
( e ) post - procedure right cca angiogram shows good glow across the stented segment with ( f ) increased antegrade circulation .
( g ) post - procedure right cca angiogram shows good flow across the stented segment with ( h ) improved intracranial circulation , and ( i ) ap view shows stents on both the sides 55-year - old man who presented with recurrent right sided tias ( patient 1 in the table ) ( a ) mr dwi shows acute infarcts in the left mca terrritory .
( b ) dsa of left cca shows critical stenosis of left ica and ( c ) poor antegrade flow in the cerebral circulation ( d ) post - procedure left cca angiogram reveals the patent stented segment ( e ) with good antegrade flow in cerebral circulation .
( a ) pre - procedure right cca angiogram of the same patient ( patient 1 in the table ) shows severe narrowing of the left ica , and ( b ) right cerebral angiogram shows decreased flow .
( c ) post - procedure left cca angiogram shows good flow across the stented segment and ( d ) shows increased antegrade flow in the cerebral circulation .
the stent on the left side is also seen ( white arrow in c ) all patients were monitored in the neurointensive care unit after the procedure .
majority of the patients ( 6 out of the 9 ) required infusion of dopamine or noradrenaline to treat hypotension due to baroreceptor response during stent / balloon deployment .
the mean arterial pressure ( map ) was maintained between 80 to 90 mm of hg on the infusion .
the patients were shifted out of neurointensive care unit after the infusion was stopped , which was 24 to 48 hours in all the cases .
patients were also closely monitored for headache , vomiting , seizures , and any fresh neurological deficit suggesting cerebral hyper perfusion .
post - procedural transient events in the form of hypotension and bradycardia occurred in 3 patients after the placement of one stent on both the sides , in 2 patients after the placement of first stent , and in 1 patient after the placement of second stent .
this was treated with intra - procedure atropine and post - procedure dopamine ( 5 - 15 g / kg / min ) or noradrenaline ( 2 - 4 g / min ) infusion for 6 - 36 hours to maintain the mean arterial pressure ( map ) at 80 to 90 mm hg . however , none of our patients had any minor or major post - procedural complications , such as minor or major stroke , hyperperfusion , etc .
we did not encounter any cases of hyperperfusion , which was a theoretical concern in these patients .
there were no deaths , major or minor strokes , or myocardial infarction either in the peri - procedural period ( up to 1 month ) , or on clinical follow - up after 3 and 6 months , in our series .
carotid doppler imaging at follow - up after 6 months showed good antegrade flow across the patent stent in all 18 stented segments . out of the 9 patients , 1 patient was lost to follow - up .
all the other 8 patients are being regularly monitored every 6 months , and none of these patients have had recurrent symptoms .
this is the first reported series of simultaneous bilateral carotid artery stenting in a select group of patients from india .
we aim to discuss the importance of selection criteria , meticulous hemodynamic monitoring , and technical modification , such as strict avoidance of post - dilation after the placement of first stent and compulsory use of protection devices , and the technical modifications used to minimize the risk of occurrence of adverse events in these patients that were different from other published studies .
al - mubarak et al . , first reported a series of 5 patients who underwent simultaneous bilateral carotid stenting because of recurrent re - stenosis following carotid endarterectomy .
bilateral carotid artery stenosis is known to be treated by staged stent procedure since it is considered as high risk factor for carotid endarterectomy .
the advantage of simultaneous bilateral carotid artery stenting over staged carotid artery stenting had been described in the literature.[58 ] the theoretical risks concerning sbcas mainly include occurrence of hyper perfusion and excessive hemodynamic depression because of activation of bilateral carotid sinus reflex .
we did not encounter any incidence of hyper perfusion syndrome ( hps ) in our patients , which was mainly due to the meticulous hemodynamic monitoring and excellent post - procedure neurointensive care with careful blood pressure monitoring .
since cerebral auto regulation takes several days to normalize after revascularization , after discharge , patients were counseled regarding immediate reporting of onset of any new symptoms such as headache , vomiting , seizures , etc . , ( suggesting hyperperfusion ) .
careful monitoring of blood pressure is required for at least 1 month after sbcas in order to avoid hps .
we believe that selection criteria of the patients for sbcas among the patients with bilateral cad play an important role in affecting the outcome of the procedure .
the selection criteria were similar to the eligibility criteria , based on which patients were taken in the carotid revascularization endarterectomy vs. stenting trial ( crest ) , which was the recent randomized control trial , credentialing and training process of the same is the most rigorous reported to date and may serve as a model for future trials .
our selection criteria of the lesions were based on the same inclusion criteria as those of the crest trial .
the eligibility criteria in crest trial included patients with > 50% stenosis in symptomatic patients and > 60% stenosis in asymptomatic lesions detected on angiography . in 1 of our patients ( patient no . 3 in table 1 ) , who had triple vessel disease ( ef = 30% ) and increasing frequency of recurrent transient ischemic attacks , decision was taken in favor of sbcas in order to avoid delay of the cabg to a later date .
gabriella visconti et al . , described a case of simultaneous hybrid revascularization by cas followed by immediate cabg in a patient with a severe coronary artery disease and bilateral carotid artery disease .
the major concern of staged cas -cabg is double anti - platelet regimen ( ecospirin 150 mg and clopidogrel 75 mg ) , which may increase the risk of peri - procedural bleeding .
in their series suggested that cabg can be done soon after cas is performed with single anti - platelet therapy ( aspirin ) and with loading of clopidogrel in the intensive care unit , when surgical bleeding had definitely stopped . in our present patient with severe co - existing coronary artery disease , cabg was done 1 week after sbcas with a successful outcome .
liu et al . , in their series of 30 patients of sbcas found higher incidence of hps than in unilateral stent placement group ( p = 0.036 ) .
they described the disadvantages of staged intervention , such as higher cost and delay of life - saving treatment procedures or occurrence of new cerebral infarction in unilateral cas and concluded that outcome in their series after sbcas revealed no significant difference compared with those of unilateral stent placement at 30 days and 6 months follow - up .
we did not encounter any incidence of hps in any of the patients in our series , and we agree with these authors regarding the safety and efficacy of sbcas with good outcome in selected patients .
the second important major concern regarding sbcas is carotid sinus reaction triggering hypotension and bradycardia .
franz leisch et al . , studied in detail the carotid sinus reaction during cas in a study of 108 patients during a 2-year period . in their experience , carotid sinus reaction occurred in 42 ( 40% ) patients who underwent unilateral cas ( hypotension defined as systolic blood pressure < 90 or bradycardia
incidence of csr causing hd ( hemodynamic depression ) in sbcas was higher ( 66% ) than that in the reported series of unilateral cas.[1518 ] but , none of our patients had any adverse effects due to this , probably because post - dilatation was strictly avoided after the first stent and all the 6 patients were aggressively managed with intra - procedure atropine , intra- and post - procedure dopamine infusion .
none of our patients had any post - procedural effects because of the hd , usually considered a common and benign event , which does not increase the procedural risk of cas .
the main limitation of the present study is that it is a retrospective analysis with a small series of patients .
a randomized controlled prospective study is required with large numbers to get more insight about the difference in the outcomes .
additional limitation is that there is no comparison with endarterctomy procedure as was done in crest trial .
simultaneous bilateral carotid artery stenting ( sbcas ) is an elegant method of treatment for bilateral carotid artery disease with acceptable risks in select group of patients .
however , a study with large number of patients is required to prove the same in high - risk patients .
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objectives : simultaneous bilateral carotid artery stenting ( sbcas ) is a challenging procedure , and selection criteria play an important role in determining the final outcome .
the aim of the present study was to determine the efficacy and safety of the sbcas in a series of 9 patients with significant bilateral carotid artery disease ( > 50% on the symptomatic side and > 60% on the asymptomatic side).materials and methods : the present study is a retrospective study of 9 patients from january 2005 to december 2012 in a tertiary care center . there were 8 males and 1 female in the age range 50 to 75 years and an average mean age of 63 years .
inclusion criteria of the present study were patients with bilateral internal carotid artery stenosis > 50% ( 50 - 99% ) in the symptomatic side and > 60% in the asymptomatic side as seen on digital subtraction angiography ( dsa ) .
sbcas with use of distal protection device ( spider device , ev3 ) , to prevent intra - procedural embolic migration , was done in all the patients.results:technical success was achieved in all patients ( 100% ) .
post - procedural events in the form of hypotension and bradycardia occurred in 3 patients after the placement of stent on both the sides , in 2 patients after the placement of the first stent , and in 1 patient after the placement of the second stent .
we did not encounter any cases of hyperperfusion , which was a concern in these patients .
there were no deaths , major or minor strokes , or myocardial infarction either in the post - procedural period ( up to 1 month ) or on clinical follow - up 3 and 6 months post-treatment.conclusion:sbcas was an effective and safe alternative treatment method in a select group of patients with bilateral carotid artery disease .
it can be considered as a feasible treatment option with acceptable risks .
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disorders of sex development ( dsd ) refer to a collection of congenital conditions in which atypical development of sex occurs at one or more levels ( chromosomal , gonadal , anatomic ) .
genetic males with dsd ( i.e. , 46,xy dsd ) can present with an external genital phenotype that is female , ambiguous , or male including a micropenis ( stretched penile length < 2.5 sd for age ) .
studies of 46,xy dsd have focused largely on aspects of gender development such as satisfaction with gender of rearing , sexual orientation , gender identity , and gender role ( gi / r ) .
very few studies have reported on additional aspects of physical or mental health despite the fact that patients and families often inquire about such topics .
furthermore , few studies have addressed the question of if , or how , a dsd diagnosis and related treatment impact quality of life ( qol ) for affected individuals . for the purpose of this review
, qol can be operationally defined as the amount of enjoyment a person experiences in the physical , psychological , social , and spiritual dimensions of their life .
although much remains to be learned concerning psychosexual development as it relates to dsd , the purpose of this paper is to review what is known about aspects of physical and mental health that contribute to overall qol beyond gi / r and sexual orientation .
topics reviewed include qol and psychological well - being , cognition , general health , fertility , and sexual function .
additionally , the focus of this review is on individuals affected by 46,xy dsd due to androgen insensitivity syndromes ( ais ) , 5- reductase-2 deficiency ( 5-rd-2 ) , or 17-hydroxysteroid dehydrogenase-3 deficiency ( 17-hsd-3)reared male or female .
these conditions were chosen because while much has been written about gender development in affected individuals , less emphasis has been placed on other outcomes that may contribute to qol such as genital surgery or hormone therapy to induce and maintain pubertal development .
congenital micropenis was not included in the current review because too little information exists on outcomes aside from gender development in this dsd for meaningful interpretation of the data .
data on individuals with micropenis is further confused by the incorrect practice of some authors of describing individuals with a small phallus including hypospadias as having a micropenis .
a search of the databases medline and psyc info was performed using the terms disorders of sex development , dsd , intersex , hermaphrodite , male pseudohermaphrodite , ambiguous genitalia , androgen insensitivity , 5- reductase-2 deficiency , and 17-hydroxysteroid dehydrogenase-3 deficiency .
peer - reviewed articles published in english since 1955 were considered if they referred to adults with dsd grouped and analyzed according to the above - mentioned etiologies .
the year 1955 was chosen because this was when john money started to publish extensively on dsd in peer - reviewed scientific journals [ 3 , 4 ] . to be considered for inclusion in the current review
, articles must report on mental or physical health outcomes other than , or in addition to , gi / r and sexual orientation .
articles were excluded if they provided outcome information about gi / r or sexual orientation only , or when data from study participants could not be attributed to one of the specific dsd diagnoses referred to earlier .
aiss in either the complete ( cais ) or partial ( pais ) form represent a relatively common presentation of 46,xy dsd [ 57 ] .
cais and pais result in complete or partial end organ insensitivity to androgens , respectively .
the majority of mutations of the human androgen receptor gene are base substitutions , although base and gene deletions , base insertions , and premature terminations occur .
while it is well established that androgens bind to intracellular androgen receptors to alter gene expression in target tissues , evidence for nongenomic actions of androgens is accumulating .
the degree of androgen insensitivity of target tissues is usually inferred by the extent of under - masculinization of the external genitalia at birth coupled with the degree of under - virilization at puberty .
the clinical presentation of cais in adulthood is typically that of a tall woman with a female distribution of adipose tissue , female breasts and external genital development , and little or no sexual hair . infants can present with testes that descend into the inguinal canals or labia , and adolescents
can present with absent menses and sexual hair in conjunction with normal breast development . due to the female external genital phenotype associated with cais , affected individuals are always assigned and reared as girls despite their possession of a 46,xy chromosomal complement and testes .
individuals with pais typically present as newborns with varying degrees of genital ambiguity . thus , male or female sex of rearing occurs , depending on the degree of under - masculinization of the genitalia . while psychosexual evaluations of girls and women affected by cais unequivocally reveal female gi / r [ 911 ] , outcomes of other aspects of mental health vary greatly across studies .
for example , psychological distress as assessed by the brief symptom inventory ( bsi ) , self - harming behavior , and suicidal tendencies are prevalent in some samples of women with cais recruited from physicians or support groups [ 1214 ] . in a study of women with cais recruited from subspecialty clinics at a university hospital ,
participants affected by cais reported better qol scores and fewer depressive symptoms than women with other types of dsd or unaffected control women when assessed with the danish version of the quality of life - assessment of growth hormone deficiency in adults ( qol - aghda ) questionnaire . in yet another study of women with cais recruited from both a clinic sample and a support group , affected
individuals did not differ from controls on measures of self - esteem , assessed by the self - esteem scale , or psychological well - being , measured by responses to the psychological general well - being scale ( pgwbs ) . while it is surprising that some studies of women with cais report equal or better qol than unaffected women
additional qol studies including larger sample sizes and greater participant diversity are needed in this population to better understand the impact of having a 46,xy chromosomal complement and being born with testes on overall psychological well - being in this group of women .
recently , the german network of disorders of sex development published clinical evaluation data on 439 individuals with dsd .
furthermore , a study that assesses potential differences in long - term qol among women with cais whose testes were removed during infancy , compared to those who consented to gonadectomy later in their development , would provide very useful information for optimizing medical and surgical treatment for this group .
mice with androgen receptor gene mutations ( testicular feminization mutant or tfm ) exhibit a more pronounced impairment on spatial recognition and memory retention tasks when considered in conjunction with the epsilon4 allele of the apolipoprotein e gene ( apoe4 ) .
this subclass of apolipoprotein plays an important role in cholesterol metabolism and is associated with alzheimer 's disease , impaired cognition , and reduced neurite growth .
the implication is that androgen insensitivity may impact cognition in women affected by ais who also express the epsilon 4 allele of the apoe gene .
only two studies of cognitive performance in girls and women affected by cais exist in the peer - reviewed literature . the first employed standardized , age - appropriate intelligence testing for participants ranging in age from 5 to 28 years and observed normal scores for all of the girls and women who participated . a second intelligence assessment was performed in women with cais and revealed no group differences in overall iq between cais participants , unaffected men , and unaffected women
however , unaffected women and those with cais performed worse than men on visuospatial components of the iq measure . taken together , these data reveal that girls and women with cais exhibit similar iq scores and cognitive performance patterns as unaffected women who possess a 46,xx chromosomal complement and end organ responsiveness to androgens . whether or not cognitive differences would be detected on measures of spatial recognition and memory for women affected by cais when apoe4 is considered is not known at this time .
future studies should consider the potential interaction of end organ unresponsiveness to androgens with apoe4 in this population .
additionally , more refined neuropsychological testing , regardless of apoe4 status , might reveal important cognitive differences in girls and women affected by this type of dsd .
few long - term outcome studies of general health have been conducted in women with cais .
obesity is commonly reported , however , at rates that mimic the general population of women [ 10 , 21 ] .
decreased bone mineral densities in the lumbar spine and hip regions occur both prior to gonadectomy in women with cais as well as in gonadectomized women receiving daily estrogen therapy [ 10 , 21 , 22 ] .
these data suggest that women affected by cais are at an increased risk for osteoporosis despite exposure to endogenous or exogenous estrogen . whether women with cais require higher doses of estrogen than their unaffected counterparts to protect their bones , or whether androgens play a direct role in bone health for this group , is unknown at this time . concerning cancer occurrence and risk , both germ cell tumor formation and risk factors for developing prostate cancer
prepubertal girls with cais are considered to be at low risk ( 2% ) for germ cell tumor formation , as determined by review of 55 patients .
however , a seminoma has been reported in a 14-year - old with cais , and a malignant teratoma has been reported in a second affected child ( also 14-years - old ) .
in general , the risk for developing testicular tumors including sertoli cell tumors , seminomas , and leydig cell tumors is thought to increase with age in affected women [ 26 , 27 ] .
additionally , women with cais appear to be at low risk for developing prostate cancer as determined by prostate specific antigen ( psa ) testing and digital rectal exam , despite their possession of some prostate tissue .
no studies were located that investigated other types of cancer occurrence or risk in women with cais .
as women with cais possess neither ovaries nor a uterus , fertility is not possible in this group at the present time .
while vaginal lengthening is needed for penile penetration in some women with cais [ 29 , 30 ] , others have a normal vaginal length and report satisfactory intercourse despite never having received dilatation or surgery [ 10 , 31 ] .
three studies of orgasmic function report that women with cais can reach sexual climax [ 10 , 32 , 33 ] , indicating that androgens are not necessary for this aspect of sexuality in this group .
low libido , or hyposexual function , is reported by some women with cais [ 33 , 34 ] .
interestingly , even when women report sexual dysfunction , they also report sexual satisfaction at levels that equal unaffected women .
perhaps hyposexual function associated with cais is secondary to vaginal hypoplasia , the need for androgens to support libido ( but not orgasm ) , or sexual avoidance by women who feel stigmatized by their medical condition .
further research that includes unaffected women as control subjects is needed to understand the extent , and underlying causes , of hyposexual function in women affected by this 46,xy dsd .
additionally , future studies would benefit from considering the impact of the type and timing of vaginoplasty on sexual satisfaction
self - reported psychological distress as determined by responses to the bsi , self - harming behavior , suicidal tendencies , and suicidal attempts is observed in people affected by pais whether reared male or female [ 1214 ] . in a long - term followup study of adults with pais , psychological counseling for problems such as difficulty with family members , depression , and substance abuse
was commonly reported during interviews once again regardless of male or female rearing .
however , some studies of men and women affected by pais fail to observe significant problems with mental health [ 36 , 37 ] .
furthermore , qol as determined by responses to the sf-36 health survey exceeded normative data in one case series of 3 women affected by pais . while it is clear that some individuals with pais experience mental health obstacles , both the extent and type of these obstacles in this patient population remain to be elucidated .
the potential impact of clinical decisions such as the type and timing of genital surgeries on qol in affected people , regardless of their gender assignment , is important information that is missing from our knowledge base at this time . based on studies of 24 patients , people with pais
are thought to be at high risk ( 50% ) for developing germ cell tumors if the testes are not located in the scrotum . for this group ,
46,xy women affected by pais do not appear to be at risk for developing prostate cancer , despite their possession of prostate tissue , as determined by psa levels and digital rectal exam .
presumably this is due to the fact that these women are gonadectomized , and thus protected from androgenic actions on the prostate .
in contrast , men with pais have psa levels comparable to unaffected men matched for age and race . therefore , when individuals with pais are reared male , screening for prostate disease is recommended in a manner that is similar to the general male population .
no other published reports on general health outcome in 46,xy individuals with pais , reared male or female , were located . additionally , no studies on cognition in this group were found . at the present time
fertility is challenging , but not impossible , for individuals with pais raised male [ 39 , 40 ] .
a study of 15 adults reared male found that none had ever engaged in penile - vaginal intercourse and all experienced severe sexual dysfunction . in a second study of 21 men and 18 women with 46,xy dsd due to pais ,
dissatisfaction with sexual function was common but not universal , and this dissatisfaction was similar for those reared male or female .
smaller investigations of sexual function in women with pais reveal that , for those who participated in sexual relations with a partner , those experiences were rated as both satisfactory and unsatisfactory [ 29 , 37 ] .
the great variability in outcome measures across studies indicates the need to study larger sample sizes as well as employ standardized , validated measures of sexual function when examining men and women with pais .
5-reductase-2 ( 5-rd-2 ) deficiency can result in female external genitalia ( with a normal sized or enlarged clitoris ) and male internal sex ducts in people with a 46,xy chromosomal complement .
this occurs because the enzyme 5-rd-2 is needed to convert testosterone to dihydrotestosterone ( dht ) .
dht has a greater affinity for the androgen receptor than testosterone and is required to masculinize the external genitalia , but not the internal male sex ducts , during fetal development .
clinical distress , determined by responses to the bsi and suicidal ideation , is observed in 46,xy women affected by 5-rd-2 deficiency , although studies of larger sample sizes are needed to determine the generalizability of this finding . whether this distress is due to female assignment
a bone health investigation of affected adults ( none of whom had been gonadectomized nor given exogenous hormone replacement ) revealed that bone mineral density did not differ from unaffected men .
a second bone density study concluded that dht is not needed to maintain normal bone health .
no other studies on general health outcomes were located for review for this particular group of dsd . of particular concern
is the lack of knowledge pertaining to germ cell tumor or prostate cancer risk for this group .
both sperm production and paternity have been documented in men affected by 5-rd-2 deficiency [ 43 , 4548 ] .
high - dose androgen therapy improves virilization , erectile response and ejaculatory volume in individuals reared male . for those who identify as women , sexual activity
similar to ais , it is not clear how sexual function in people with 5-rd-2 deficiency , reared male or female , contributes to qol .
the clinical presentation of 17-hsd-3 deficiency can be confused with cais as affected individuals often present with female external genitalia prior to puberty [ 50 , 51 ] .
if the testes remain in situ , virilization at puberty occurs in a manner that is similar to what is observed in 5-rd-2 deficiency [ 50 , 52 ] .
clinical distress as determined by the bsi and suicidal ideation is observed in 46,xy women affected by 17-hsd-3 deficiency , but interpretation of these data is limited by the small sample size studied .
once again , how gender assignment and the medical and surgical treatment that accompanies such assignment impact qol for this group is unknown .
germ cell tumor risk is estimated to be intermediate ( 28% ) [ 23 , 50 ] .
no other studies of general health or cognition conducted in this category of 46,xy dsd were located for review .
case reports of 46,xy women with 17-hsd-3 deficiency indicate satisfactory sexual function [ 44 , 53 ] , but available information is too incomplete to draw conclusions with confidence .
case reports of affected individuals living as men also indicate satisfactory sexual function [ 50 , 53 ] , although dissatisfaction attributed to having a small phallus appears in the literature .
of the 35 studies reviewed that included outcomes information apart from gender , 24 included information about cais , 9 about pais , 11 about 5-rd-2 deficiency , and 5 about 17-hsd-3 deficiency .
possibly , greater emphasis has been placed on studying cais apart from gender because female rearing in this particular dsd is undisputed .
in contrast , male or female rearing occurs in people affected by pais , 5-rd-2 , and 17-hsd-3 deficiencies .
perhaps gi / r and sexual orientation have been the focus of studies in these conditions in response to the clinical challenges of assigning gender in affected individuals .
finally , because cais has an animal model ( tfm ) associated with it , this condition may lend itself more easily to scientific investigation .
results from studies of psychological well - being in the specific 46,xy dsd categories considered here vary greatly across investigations .
this is likely due to the fact that these include only a handful of participants , interviewed only once , in no particular relation to developmental stage or medical / surgical event .
future studies should include larger samples and take into account developmental stages as well as the potential impact of corresponding medical and surgical procedures associated with these stages . additionally , for those who have access to mental health services , it is critical to determine the effectiveness of these services on psychological well - being within specific categories of dsd .
no systematic studies of iq or cognition have been conducted in people affected by 46,xy dsd due pais or androgen biosynthetic defects due to 5-rd-2 or 17-hsd-3 deficiencies . unlike tfm rodent models
, it is unknown if women with cais are more likely to develop spatial memory problems if they also express the epsilon 4 allele of the apoe gene . as individuals with 46,xy dsd
are generally expected to live a full lifespan , a more complete understanding of the potential for their dsd to impact neuropsychological development , particularly later in development , is needed .
general health outcome studies in people affected by ais , 5-rd-2 deficiency , and 17-hsd-3 deficiency are few and have been limited to gonadal tumors , and bone and prostate health .
further health outcome studies , particularly in conditions that exhibit clear sex differences or influences of sex steroid action , are needed .
for example , whether or not possession of a y chromosome places 46,xy women at risk for developing a male - typical presentation and course of cardiovascular disease , independent of androgen action , is not known .
alternatively , it is currently unknown if under - masculinized genetic males have an elevated risk for developing female - typical medical conditions such as autoimmune diseases or depression . as our understanding of dsd broadens to include physical and mental health outcomes apart from gender development , it is anticipated that qol will improve for affected individuals
. fertility and sexual function can be important components to living a fulfilled life and maintaining relationships ; yet we know very little about such topics .
we do know from patient populations affected by conditions other than dsd that infertility sexual dysfunction and surgeries to the reproductive organs negatively impact health - related qol .
such information , as it pertains specifically to dsd , is crucial in optimizing treatment and supporting affected individuals .
knowledge about the physical and mental health outcomes in dsd throughout the life cycle is important for improving or maintaining health in affected individuals and the population overall .
for example , the few studies of bone health that have been conducted in ais and 5-rd-2 deficiency imply that testosterone , but not dht , is necessary for optimizing bone density .
such information may be useful for developing treatments for conditions such as osteopenia and osteoporosis .
it is important to focus on qol factors for persons with a dsd diagnosis because parents ask questions such as how will my child perform in school ?
will my child have a career ? will my child fall in love ? parents also want to know how their children will feel about their dsd diagnosis
. in short , parents want information about their affected child 's future qol . only through systematic investigation of such questions
we could find no data pertaining to individual adjustment to the categories of dsd discussed in the current review .
additionally , very little is known about how family members respond to a child 's diagnosis of dsd . while we know that parents of children affected by cais or pais report feelings of shock , grief , anger , and shame when they learn of their child 's condition , more studies of parents are needed as their attitudes surely exert a significant impact on subsequent child health and well - being .
finally , the definition for qol presented earlier includes social and spiritual components neither of which were investigated systematically in any of the papers reviewed . in conditions in which gi / r does not always develop in concordance with sex of rearing such as pais , 5-rd-2 deficiency , and 17-hsd-3 deficiency a better understanding of factors that influence qol may help to explain the developmental trajectory of gi / r in people for whom gi / r development does not match their initial gender assignment .
we still have a long way to go in understanding why some people with these conditions change gender while others do not .
importantly , approximately a third of people with dsd other than ais , 5-rd-2 deficiency , or 17-hsd-3 deficiency are born with malformations that likely affect qol .
future studies must consider those individuals as well . this review has attempted to show that we also have a long way to go before we have a full appreciation of the mental , physical , social , and spiritual domains that contribute to qol of people affected by 46,xy dsd who are born with either female or ambiguous external genitalia .
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disorders of sex development refer to a collection of congenital conditions in which atypical development of chromosomal , gonadal , or anatomic sex occurs .
studies of 46,xy dsd have focused largely on gender identity , gender role , and sexual orientation .
few studies have focused on other domains , such as physical and mental health , that may contribute to a person 's quality of life .
the current review focuses on information published since 1955 pertaining to psychological well - being , cognition , general health , fertility , and sexual function in people affected by androgen insensitivity syndromes , 5- reductase-2 deficiency , or 17-hydroxysteroid dehydrogenase-3 deficiency reared male or female .
the complete form of androgen insensitivity syndrome has been the focus of the largest number of investigations in domains other than gender . despite this , all of the conditions included in the current review are under - studied .
realms identified for further study include psychological well - being , cognitive abilities , general health , fertility , and sexual function .
such investigations would not only improve the quality of life for those affected by dsd but may also provide information for improving physical and mental health in the general population .
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preeclampsia , which affects 3% to 5% of pregnancies ( 1 ) , is a pregnancy - specific disorder characterized by hypertension and proteinuria .
the etiology of the condition is unknown , but placental disorders are probably involved in the pathophysiologic mechanism ( 2 ) .
therefore , a reliable and early placental marker could be extremely beneficial in detecting pregnant women at high - risk for preeclampsia .
recently , inhibin a , a glycoprotein mainly produced by the syncytiotrophoblast of the human placenta during pregnancy has been evaluated both for the prediction of preeclampsia ( 3 - 5 ) as well as assessment of severity ( 6 - 8 ) .
( 9 ) reported previously that inhibin a levels might increase as early as 10 to 14 weeks of gestation , based on the observation of 7 patients who subsequently had preeclampsia .
it has been shown that maternal serum levels of inhibin a are 10-fold higher in women with severe preeclampsia compared to gestational age matched controls ( 8) .
10 ) also reported that inhibin a levels are markedly elevated in women at the time they experience preeclampsia . in women who subsequently developed preeclampsia ,
inhibin a levels were found to be elevated at 13 - 18 weeks in a retrospective analysis of a down 's screening programme ( 5 ) .
previous study has demonstrated that inhibin a levels in maternal serum and amniotic fluid are relatively different in normal pregnant women ( 11 ) .
other studies have reported amniotic fluid levels of inhibin a in chromosomally normal and down 's syndrome pregnancies ( 12,13 ) .
however , maternal blood and amniotic fluid obtained at the time of second trimester genetic amniocentesis are rarely used to assess the risk of preeclampsia .
the aim of this study was to investigate the inhibin a level in maternal serum and amniotic fluid at the time of second trimester genetic amniocentesis in patients who subsequently develop severe preeclampsia .
in addition , we compared the correlation between maternal serum and amniotic fluid inhibin a levels in both normal pregnant women and subsequently severe preeclampsia .
a case - control study was desgined with stored maternal serum and amniotic fluid obtained from women who underwent second trimester genetic amniocentesis between octerber 2001 and december 2003 at samsung cheil hospital in seoul , korea .
the study groups consisted of pregnant women who subsequently developed severe preeclampsia and normotentive women who had a normal pregnancy outcome ( term gestation with a neonate with adequate weight for gestational age ) .
forty patients who developed severe preeclampsia were matched for maternal age and gestational age at sampling with 80 normotentive women who had a normal pregnancy outcome .
preeclampsia was defined as hypertension ( systolic blood pressure 140 mmhg and diastolic blood pressure 90 mmhg after 20 weeks ' gestation ) and proteinuria ( 300 mg in a 24 hr urine collection or one dipstick measurement of 1 + ) according to the committee of terminology of american college of obstetricians and gynecologists ( acog ) definition ( 14 ) .
severe preeclampsia was diagnosed on the basis of diastolic blood pressure 110 mmhg or significant proteinuria ( dipstick measurement of 2 + ) or the presence of severity evidences such as headache , visual disturbances , upper abdominal pain , oliguria , convulsion , elevated serum creatinine , thrombocytopenia , marked liver enzyme elevation , and pulmonary edema .
cases with an abnormal fetal karyotype , chromosomal abnormalities , chronic hypertension , diabetes , or renal disease at the time of amniocentesis were excluded .
the control group consisted of patients who had a second trimester amniocentesis who delivered a normal neonate at term without significant medical or obstetric complications .
the ethics committee of samsung cheil hospital approved the collection of these samples and the clinical information and samples for research puroposes .
amniotic fluid was obtained by transabdominal amniocentesis and an aliquot of amniotic fluid was centrifuged and stored at -70 until assay .
maternal blood was drawn at the time of second trimester genetic amniocentesis , collected into plain serum - gel tubes ( becton dickinson , u.s.a . ) , centrifuged , and stored at -70. inhibin a levels in amniotic fluid and maternal serum were measured with a commercially available enzyme - linked immunosorbent assay ( elisa , diagnostic systems laboratories , inc . ,
statistical analysis was performed using the statistical package for social sciences version 10.0 ( spss inc . ,
kolmogorov - smirnov tests were used to test for normal distribution of the data . as inhibin a levels in this study
were not normally distributed , a mann - whitney u tests were used for comparison of continuous variables , and proportions were compared with the and fisher 's exact tests .
we used the receiver operator characteristic ( roc ) analysis to determine the best cut - off value of inhibin a level for predicting severe preeclampsia and calculated the odds ratio ( or ) and 95% confidence interval ( ci ) . a p value < 0.05 was considered statistical significance .
a case - control study was desgined with stored maternal serum and amniotic fluid obtained from women who underwent second trimester genetic amniocentesis between octerber 2001 and december 2003 at samsung cheil hospital in seoul , korea .
the study groups consisted of pregnant women who subsequently developed severe preeclampsia and normotentive women who had a normal pregnancy outcome ( term gestation with a neonate with adequate weight for gestational age ) .
forty patients who developed severe preeclampsia were matched for maternal age and gestational age at sampling with 80 normotentive women who had a normal pregnancy outcome .
preeclampsia was defined as hypertension ( systolic blood pressure 140 mmhg and diastolic blood pressure 90 mmhg after 20 weeks ' gestation ) and proteinuria ( 300 mg in a 24 hr urine collection or one dipstick measurement of 1 + ) according to the committee of terminology of american college of obstetricians and gynecologists ( acog ) definition ( 14 ) .
severe preeclampsia was diagnosed on the basis of diastolic blood pressure 110 mmhg or significant proteinuria ( dipstick measurement of 2 + ) or the presence of severity evidences such as headache , visual disturbances , upper abdominal pain , oliguria , convulsion , elevated serum creatinine , thrombocytopenia , marked liver enzyme elevation , and pulmonary edema .
cases with an abnormal fetal karyotype , chromosomal abnormalities , chronic hypertension , diabetes , or renal disease at the time of amniocentesis were excluded .
the control group consisted of patients who had a second trimester amniocentesis who delivered a normal neonate at term without significant medical or obstetric complications .
the ethics committee of samsung cheil hospital approved the collection of these samples and the clinical information and samples for research puroposes .
amniotic fluid was obtained by transabdominal amniocentesis and an aliquot of amniotic fluid was centrifuged and stored at -70 until assay .
maternal blood was drawn at the time of second trimester genetic amniocentesis , collected into plain serum - gel tubes ( becton dickinson , u.s.a . ) , centrifuged , and stored at -70. inhibin a levels in amniotic fluid and maternal serum were measured with a commercially available enzyme - linked immunosorbent assay ( elisa , diagnostic systems laboratories , inc . ,
statistical analysis was performed using the statistical package for social sciences version 10.0 ( spss inc . ,
kolmogorov - smirnov tests were used to test for normal distribution of the data . as inhibin a levels in this study were not normally distributed , a mann - whitney u tests were used for comparison of continuous variables , and proportions were compared with the and fisher 's exact tests .
we used the receiver operator characteristic ( roc ) analysis to determine the best cut - off value of inhibin a level for predicting severe preeclampsia and calculated the odds ratio ( or ) and 95% confidence interval ( ci ) .
there were statistical differences in nulliparity , gestational age at delivery , and birth weight between patients who developed severe preeclampsia and those in the control group .
as expected , the blood pressures of patients who developed severe preeclampsia were significantly higher than those in the control group .
in contrast , the groups were similar in terms of maternal age , indication for amniocentesis , gestational age at amniocentesis and blood sampling , and platelet count .
. the median maternal serum and amniotic fluid levels of inhibin a were significantly higher in patients who developed severe preeclampsia than those in the control group ( maternal serum : median 414 pg / ml , range 168 - 1,039 pg / ml vs. median 280 pg / ml , range 106 - 522 pg / ml , p<0.001 ; amniotic fluid : median 525 pg / ml , range 142 - 1,305 pg / ml vs. median 275 pg / ml , range 103 - 882 pg / ml , p<0.001 ) . in patients who developed severe preeclampsia , there was a positive correlation between maternal serum and amniotic fluid inhibin a levels ( r=0.394 , p=0.011 ) ( fig .
in contrast , there was no correlation between amniotic fluid and maternal serum inhibin a levels in the control group ( r=0.185 , p=0.126 ) .
roc curves were constructed to select cut - off values at which to dichotomize the level of maternal serum inhibin a or amniotic fluid inhibin a to identify the subsequent development of severe preeclampsia .
the best cut - off values of each maternal serum and amniotic fluid inhibin a level for the prediction of severe preeclampsia were 427 pg / ml and 599 pg / ml ; the estimated ors that were associated with these cut - off values were 9.95 ( 95% ci 3.8 - 25.9 , p<0.001 ) and 6.0 ( 95% ci 2.3 - 15.8 , p<0.001 ) .
we confirmed that the median second trimester maternal serum inhibin a levels in patients who subsequently developed severe preeclampsia was significantly higher than those in normal pregnant women .
our data are consistent with those of previous studies that reported an increase in serum inhibin a concentration before the onset of preeclampsia .
( 15 ) reported an increase in maternal serum inhibin a concentration at 13 to 18 weeks of gestation in 28 patients who subsequently had preeclampsia .
( 16 ) evaluated the screening efficacy of serum inhibin a determination combined with uterine artery doppler studies at 15 to 19 weeks of gestation in 37 women who subsequently had preeclampsia .
king et al . ( 17 ) reported that preeclamptic women had second trimester serum inhibin a levels 1.3-fold higher than the control mean .
in contrast to these reports , some studies found no difference in second trimester serum inhibin a levels between healthy pregnant women and women who later developed preeclampsia ( 18,19 ) . in preeclampsia , there is partial or complete failure of trophoblastic invasion of the myometrial segments of the spiral arteries ( 2 ) , a process that normally has taken place by 20 weeks of gestation ( 20 ) .
the failure of trophoblastic invasion is associated with ischaemic damage to the syncytiotrophoblast causing functional alteration of the surface layer of the syncytiotrophoblast ( 21 ) .
this alteration in the surface layer of the syncytiotrophoblast has been postulated as a contributory factor for the increased ' leakage ' of inhibin a into the maternal circulation ( 22 ) , which might explain the increase in concentration of maternal serum inhibin a in preeclampsia .
this is the first report for inhibin a levels in the second trimester amniotic fluid of the patients who subsequently developed severe preeclampsia .
our results also indicated the amniotic fluid inhibin a levels were significantly higher in patients who subsequently developed severe preeclampsia than those in normal pregnant women .
moreover , inhibin a levels in amniotic fluid were significantly higher than those in maternal serum for this disease .
we speculate that increased inhibin a levels in amniotic fluid may enter maternal circulation , leading to higher detectable levels in maternal blood of patients who subsequently developed severe preeclampsia .
further investigations for the mechanism of inhibin a through the amniotic fluid to maternal blood are necessary .
our data suggest that amniotic fluid may be another significant source of inhibin a for the prediction of severe preeclampsia . during pregnancy ,
the placenta produces and secretes inhibin a , which then enters both the maternal and the fetal circulation ( 11,12,23 ) .
the placental inhibin a that enters maternal and the fetal circulation may cause an elevation in maternal serum and amniotic fluid inhibin a levels . we observed that the amniotic fluid inhibin a levels were positively correlated with the maternal serum inhibin a levels in the patients who subsequently developed severe preeclampsia .
this correlation may be associated with increased amniotic membrane permeability in patient with severe preeclampsia . in conclusion ,
the second trimester maternal serum and amniotic fluid inhibin a levels in pregnant women who subsequently developed severe preeclampsia were significantly higher than those in normal pregnant women .
there is a positive correlation between elevated maternal serum and amniotic fluid inhibin a levels in patients who subsequently developed severe preeclampsia . at the time of genetic amniocentesis
, we suggest that the elevated level of inhibin a in maternal serum and amniotic fluid may be a risk factor for the subsequent development of severe preeclampsia , although larger studies are needed to confirm this point .
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the purpose of this study was to evaluate whether maternal serum ( ms ) and amniotic fluid ( af ) inhibin a levels are elevated in patients who subsequently develop severe preecalmpsia , and to investigate the correlation between ms and af inhibin a levels in the second trimester .
the study included 40 patients who subsequently developed severe preecalmpsia and 80 normal pregnant women .
inhibin a levels in ms and af were measured with enzyme - linked immunosorbent assay ( elisa ) .
the ms and af inhibin a levels in patients who developed severe preeclampsia were significantly higher than those in the control group ( both for p<0.001 ) .
there was a positive correlation between ms and af inhibin a levels in patients who developed severe preeclampsia ( r=0.397 , p=0.011 ) , but not in the control group ( r=0.185 , p=0.126 ) .
the best cutoff values of ms and af inhibin a levels for the prediction of severe preeclampsia were 427 pg / ml and 599 pg / ml , respectively ; the estimated ors that were associated with these cut - off values were 9.95 ( 95% ci 3.8 - 25.9 , p<0.001 ) and 6.0 ( 95% ci 2.3 - 15.8 , p<0.001 ) . an elevated level of inhibin a in ms and af at the time of second trimester amniocentesis may be a risk factor for the subsequent development of severe preeclampsia .
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lung cancer still remains one of the deadliest cancer types in the world . both the incidence and mortality are still high , equally among male and female patients , in practically all parts of the world .
lung cancer is still a leading cause of cancer related mortality , with an overall 5-year survival < 20% in europe and usa .
the estimated number of new cases of lung cancer in usa for 2009 is 219,440 .
the estimated number of deaths in usa for 2009 is even more discouraging : 159,390 [ 1 - 4 ] .
many prevention and political measures , such as prohibition of smoking in public places or the banning of cigarette commercials , might contribute to a decrease in morbidity and mortality .
however , these measures need time to show their true potential . in the past decade , we have observed a drop in morbidity ( mostly in squamous cell lung cancer ) due to the public measures and increased public awareness , but mortality still remains alarmingly high [ 5 - 8 ] .
the introduction of novel chemotherapeutic agents , the development of targeted therapy , the combination of standard chemotherapy with molecular targeted therapy , and combinations with various radiotherapy regimens , might result in a better survival of lung cancer patients . indeed , some clinical trials with novel targeted therapy agents , or their combinations with chemotherapy , have shown a significant improvement in the disease free survival , or even the overall survival .
on the other hand , we should always be cautious about the results of clinical studies and wait for more data , or meta - analyses before implementing these results in the clinical practice [ 4,9 - 12 ] .
nevertheless , in the light of the current situation regarding lung cancer mortality , it is true that the most appropriate treatment for a patient is his / her inclusion in a clinical trial .
practically all cancer societies , dealing with the problem of lung cancer , recommend treatment in a clinical trial setting [ 12 - 16 ] .
however , in the hunt for a better survival and better results , with a substantial number of patients in clinical trials , what do we actually know about our patients ' thoughts and feelings ? what do they themselves know about their own condition and the treatment itself ?
the regulations and rules for the clinical trials are strict and defined , and all physicians investigating the treatment of lung cancer adhere to these rules and regulations [ 14 - 18 ] .
many patients receive therapeutic benefits from participating in clinical trials , even moreso than they are aware , and in some cases these benefits exceed those that standard care could provide . however , the patients participating in clinical trials contribute not only to their own and future patients ' treatment benefits , but also to the benefits of medicine and to science itself .
the physicians are aware of this fact , but are the patients aware of the same fact , too ?
the major aim of the study we conducted was to investigate what patients knew and thought about their disease , about the options for their current and future treatment , as well as about the clinical trial they participated in .
we wanted to investigate how satisfied they were with the treatment in the clinical trial ( this is why we included only previously treated patients ) , and what they thought and knew about the novel chemotherapy they were treated with .
we designed a short questionnaire in order to obtain the answers to some of these questions .
the secondary aim of the study was to investigate the influence of age , gender , education , lung cancer type and stage , chemotherapy duration and chemotherapy line on the patients ' opinion about the disease , treatment , and clinical trial .
this was a prospective trial conducted at the clinic for pulmonary oncology of the institute for pulmonary diseases of vojvodina , serbia .
the study was carried out over a 1-year period , from july 2008 to july 2009 , and it was approved by the institutional review and ethical board .
all the patients who agreed to participate in the study and answer the questionnaire were informed about the study and signed the informed consent form . of 86 patients who at the time participated in clinical trials on administration of novel chemotherapeutic agents , 59 met all the inclusion criteria and were eligible for the study .
the inclusion criteria were : current participation in a phase ii or iii clinical trial on non - small cell lung cancer ( nsclc ) or small cell lung cancer ( sclc ) , the treatment including targeted molecular therapy , chemotherapy or their combination , advanced stage ( iiib , iv ) nsclc or extensive sclc , second or third line chemotherapy , good performance status graded by eastern cooperative oncology group ( ecog ) 0 - 2 , and willingness to participate in the study .
the exclusion criteria were : the first line chemotherapy , stage i - iiia nsclc , limited sclc , current or concurrent radiotherapy , ecog 3 , inability or refusal to participate in the study .
the patients were asked to answer the questionnaire after the third course of the second or third line chemotherapy regimen .
all of the patients had sufficient time to answer the questions and to place the questionnaires in the " answer box " .
the " answer box " was a sealed carton container placed in the main hall of the clinic , enabling the patients to keep their privacy and anonymity after completing the questionnaire .
it was explained to the patients that they should write their initials and date of birth in order to enable obtaining other relevant data from their medical charts needed for evaluation of the results to be performed later .
ad - hoc questionnaire to investigate patients ' impressions about participation in a clinical trial .
descriptive statistics were generated for all study variables , including the mean and standard deviation for continuous variables and relative frequencies for categorical variables .
the variables concerning ' the patients ' satisfaction ' were treated as the data in nominal category . in order to compare these answers according to gender , cancer type , cancer stage , education level of patients and line of chemotherapy , we used pearson 's test with p < 0.05 as a significant level of probability .
all statistical analyses were performed using the spss for windows , version 11.5 ( spss inc . ,
the average age of the patients was 56 10 years ( range 36 - 73 ) .
the average chemotherapy duration was 11 4 months ( range 3 - 19 ) .
there were 48 ( 81.4% ) male patients and 11 ( 18.6% ) females included in this study .
most patients ( n = 32 , 54.2% ) had squamous cell lung cancer diagnosed ; small cell lung cancer was diagnosed in 16 ( 27.1% ) patients , and adenocarcinoma in 11 ( 18.6% ) patients .
the majority of the patients ( 30/59 , 50.8% ) had an advanced stage iv nsclc .
there were 17 patients ( 28.8% ) enrolled in phase ii clinical trials versus 42 ( 71.2% ) in phase iii clinical trials .
the majority of the patients ( 32 or 54.2% ) were high school graduates ; 16 ( 27.1% ) patients had a university degree , while 11 patients ( 18.6% ) had elementary school education .
an overview of the questions , and frequency and percentage of the answers is presented in table 1 .
it is clear from the table that all the patients who participated in both clinical trials and our study were familiar with the nature of their disease .
there was also a significant number of patients who knew what kind of chemotherapy they received prior to the enrollment in the clinical trial .
most patients were previously treated in our institution ( 44/59 or 74.6% ) ; 14 ( 23.7% ) patients were treated in the regional hospital , while one patient did not know where he was treated .
almost all patients ( 58/59 or 98.3% ) knew that they were participating in a clinical trial ; the remaining patient responded that he did not know he was participating in a clinical study .
the large majority of patients ( 47/59 or 79.7% ) said they understood what " a clinical trial " was , but 12 patients ( 20.3% ) were not sure what " a clinical trial " means .
one of the most interesting results shows that 5 patients ( 8.5% ) did not read the entire informed consent form ( icf ) .
however , a significant number of patients ( 54/59 or 91.5% ) had read the entire icf .
question 9 addressed the level of satisfaction with the information about the treatment within the clinical trial .
forty - three patients ( 72.9% ) were very satisfied with the information given by the investigators or provided in the icf and 16 patients were satisfied .
there were no patients who were not satisfied or who were unable to answer question 9 .
practically equal numbers of patients thought they would / would not require additional information during the clinical trial period , i.e. 47.5% and 49.2% of the patients , respectively .
patients ' responses per response category ( number and % ) to each item of the questionnaire answers on questions marked with asterisk are explained in the text .
one of the most important findings obtained in the study was the opinion of patients that the chemotherapy given in the clinical trial would give them a better chance for survival .
a significant number of patients ( 93.2% ) thought they had a better chance of survival in the clinical trial , and 6.8% of the patients were not sure about it .
there was also a large number of patients who answered that chemotherapy given in the clinical trial was better than the therapy used previously .
forty - eight patients ( 81.4% ) thought that chemotherapy in the clinical trial was better than the " usual " chemotherapy , 2 patients thought it was not better , while 9 patients ( 15.3% ) were not sure .
a majority of patients ( 98.3% ) understood that during the clinical trial they might not receive the investigational product .
there were 38 patients ( 64.4% ) who thought that chemotherapy given in the clinical trial had fewer side effects , 16 who thought the contrary ( that chemotherapy given in the clinical trial did not have fewer side effects ) , and 5 ( 8.5% ) who were not sure .
there were 57/59 ( 96.6% ) patients who thought that the medical personnel conducting the clinical trial had sufficient knowledge and skills .
this shows that a majority of patients believe that their doctors and nurses are adequately trained .
all the patients enrolled in the study expressed the opinion that the physicians and nurses were more easily accessible to them because they participated in the study .
a significant number of patients ( 57/59 or 96.6% ) believed the treatment within a clinical trial was better than the treatment given to patients not included in trials .
a majority of the patients ( 89.8% ) graded their overall treatment in the clinical trial as excellent .
five patients ( 8.5% ) assessed their treatment as very good , while one patient assessed it as good .
there were no patients who assessed their treatment as " neither good nor bad " or " bad " .
interestingly , 88.1% of the patients answered that they would participate in future clinical trials when they finished the treatment in the current one .
this means that a significant number of patients would like to continue the treatment within the clinical trial . on the other hand , two patients ( 3.4% )
would not any more participate in clinical trials , while 5 patients ( 8.5% ) were not sure about it .
the majority of patients ( 56/59 or 94.9% ) would recommend the clinical trial treatment to other patients .
one of the patients would not recommend it , while two were not sure about it .
several factors that could potentially influence the patients ' answers were evaluated in the multivariate analysis , including age , gender , lung cancer type , current lung cancer stage , chemotherapy line , patients ' education level , and duration of chemotherapy treatment .
however , only one factor proved influential on the patient 's answers on specific questions : and that factor was the patients ' education level .
a statistically significant higher number of patients ( p = 0.008 ) with university education knew the exact type of lung cancer they suffered from compared to patients with high or elementary school level of education .
there was also a statistically significant number of patients with a university degree ( p < 0.001 ) who knew the exact stage of their disease , a significant number of them ( p = 0.004 ) understood what the " clinical trial " was .
there was also a significant number of patients with university degree who had read the entire informed consent form ( p = 0.02 ) and a significant number of them thought that they would require additional information regarding the trial ( p = 0.001 ) . on the other hand , there was a statistically significant number of patients with high school education level who believed that the investigational product given in the chemotherapy trial caused less side effects ( p = 0.04 ) .
there was also a statistically significant number of patients with a lower education level than university degree ( high school ) who would join another clinical trial after completion of the current one ( p = 0.01 ) .
the findings of this study reveal that the majority of the patients included in the study had an advanced stage nsclc , primarily the squamous cell lung cancer .
this is due to the fact that squamous cell lung cancer is the most prevalent type of lung cancer in our country , but selection bias also played a significant role : most of the studies from which our patients were drawn were examining chemotherapy in squamous cell lung cancer .
that is the main reason why the distribution of lung cancer types does not correlate with the general distribution of lung cancer types in most published studies .
lung cancer is usually diagnosed in its late or advanced stage ; in our country almost 80% of patients with nsclc are diagnosed with an advanced stage lung cancer .
the majority of patients with sclc are also diagnosed in an extensive disease stage . in most countries
the same is true for serbia , but here we are also facing low public health awareness .
the political measures for prevention of lung cancer are still in their infancy in serbia , as in many developing countries , and it is too early to expect the results until in the next few years .
our patients were asked to answer the questionnaire after the third cycle of the clinical trial chemotherapy regimen in order to be able to compare their previous experience with the current one .
the patients included in this study were receiving second or third line chemotherapy within the clinical trial in which they were participating , so they already had substantial experience with chemotherapy .
the results show that most patients know the true nature of their disease and even the type of lung cancer they suffer from , suggesting that patients are well informed about their condition and disease . a frank approach to a patient ,
giving him / her a thorough explanation of the disease characteristics and the treatment , is a cornerstone for gaining the patient 's trust .
it can also create a relationship in which it is easier to explain to the patient the significance of participation in the clinical trial , if the patient qualifies for it .
this is particularly important in low income countries where clinical trial therapy , even for control groups , is a big step forward with respect to the therapy patients would usually get .
we often have the situation that the physicians are highly interested in the clinical trial , not from the financial standpoint , but because it is the only way for patients to be treated as recommended by the guidelines .
this is probably one of the reasons why we are often among the sites with the highest enrollment in many clinical trials .
the adequate approach to the patient with lung cancer can also be observed through a significant number of the patients who knew what type of chemotherapy they received prior to the enrollment in the clinical trial .
however , this may be related to the fact that the majority of the patients received that therapy in our institution where the institutional policy is to explain to patients what kind of therapy they will receive .
one of the most important results showed that the majority of the patients knew that they were participating in the clinical trial , except for one patient .
one of the reasons might be insufficient engagement of the physician or inability to completely understand the informed consent form ( icf ) .
the icf is usually too robust for a patient to read it completely , and without significant help from the physician the patient can hardly understand all that is written in the document
. whatever the reason for that one negative answer is , it stands as a warning .
we must pay due attention to all patients in clinical trials all the time they are under our treatment .
the fact that 8.5% of the patients did not read the entire icf represents also an alarming finding . as stated before , icfs usually provide more detailed information about the trial than an investigator can give during the interview with a patient .
the multivariate analysis showed that a significant number of the patients with a university degree read the entire icf .
this suggests we should pay more attention to less educated patients , especially when providing information and icf documentation . on the other hand ,
the scientific management and study protocol teams should pay more attention to the concept of the icf , taking into consideration its length and the patients ' ability to understand what is written .
too extensive icfs exceeding a certain number of pages , with complicated explanations and medical terms are usually not understandable for the patient . no matter how a patient may be motivated to read the entire document , 20 pages of text are a bit too much .
these translations are usually made by professional translators and not medical professionals , leading to a literary translation which is sometimes hard to understand even for physicians .
the overall satisfaction with the level of information provided was significantly high in our group , suggesting that our study teams provided sufficient and relevant information .
we tried to avoid potential biases by providing a level of anonymity to the patients who dispatched their answers in a sealed carton box placed in the clinic lobby .
we also explained to the patients that the sincerity of their answers was of major importance , and that whatever answers they gave , it would not have any influence on their further treatment .
the level of education influenced the requirement of the patients for additional information although the number of patients who answered that they would require additional information and those who answered that they would n't was practically equal .
this is related to the fact that more educated patients expect a more detailed update on their condition .
the patients ' belief and trust in the treatment is also reflected by a significant number of patients who answered that they believed that chemotherapy given in the clinical trial is better than the " usual " chemotherapy .
the answer might be biased by an excessive explanation of the advantages for participation in the clinical trial given by the physician .
however there were two patients with university degree who gave a negative answer , and 9 patients who were not sure , and they represent almost 20% of the patients included in the study .
these results suggest that the possible bias could be minimal , and that the patients really believed the trial chemotherapy was better .
the important issue is that chemotherapy given in the clinical trial protocol in developing countries is more advanced , from the medical point of view , and patients are able to recognize this fact .
it is very important that a significant majority of the patients know that they might not receive the investigational product during the trial .
that shows that the patients are aware of the study design , its benefits and pitfalls and they still want to participate , even though they have the opportunity to withdraw from the trial whenever they want .
still , they are satisfied with the treatment and consider their physicians as trustworthy , well educated and knowledgeable .
the patients enrolled in the clinical trial think that because of their participation in the trial , nurses and physicians are more easily accessible to them .
this might be true - it is a fact that more time and special care is provided to the patients in the clinical trial .
it is sometimes required by the protocol of the trial itself ( to perform more examinations or more detailed examinations , more lab tests , to check the patients ' condition several times a day ) .
all this creates an impression of special care received , so the patients usually feel more comfortable and more appreciated . this might be also one of the reasons why the majority of the patients expressed the opinion that their treatment was better than the treatment of the patients not included in the trial .
a significant number of our patients qualified their overall treatment in the clinical trial as excellent .
certainly , the quality and engagement of the sponsors who provided travel reimbursement and refreshments could play a significant role in such assessment of the treatment . on the other hand ,
the fact is that the combination of drugs in the clinical trials in advanced stage lung cancer nowadays includes targeted therapy alone or in combination with chemotherapy .
this results in fewer side effects and better clinical improvement compared to standard chemotherapy available in developing countries .
the patients can observe and feel the improvement and they usually feel satisfied with the treatment .
the physicians are eager to monitor the patients more carefully , ready to dedicate more time and attention to them in order to detect a result of the treatment and record the patients ' current condition .
the physician 's interest is generated on one side by scientific and , on the other , by financial reasons .
but , no matter what generates it , a higher physicians ' vigilance results in better care for the patients and this is what matters most .
this of course is the case in developing countries ; the situation in more developed economies could be substantially different .
the overall success of the treatment in the clinical trials in our study is testified by the fact that a statistically significant number of patients would join another trial after they finish the current one , and a significant majority of the patients would recommend the treatment in clinical trials to other patients .
the majority of the published guidelines suggest that the best treatment for patients with advanced stage lung cancer is in a clinical trial .
but if one patient recommends the trial to another , the trial itself gains popularity and importance among the patients .
there is no better motivation for a patient to join a trial than the recommendation of a friend or a fellow patient .
we encountered situations in which a significant number of patients were interested in joining the study because they had heard about it form other patients .
however , this implies a huge responsibility for the physicians : a very selective choice of the trials with special concern for a full ethical discolsure of the study , taking of course into consideration the medical justification .
a major disadvantage of this study is that the questionnaire was developed by our own study team that included medical physicians and non - medical staff ( pulmonologists and medical oncologists , nurses , statisticians and it engineers ) .
the questionnaire did not undergo all the testing phases in concordance with the guidelines for questionnaires in clinical trials . however , our intent was to make a survey of the patients ' opinion , not to measure their satisfaction , because that would require a fully developed and tested questionnaire , which is not available in that form . with the form of the questionnaire we created , we tried to minimize the biases and maximize the quality of the answers used for the analysis
clinical trials in advanced stage lung cancer are currently the best option for the treatment of patients with that condition .
our study showed that the patients included in several clinical trials have a very positive attitude toward the current trial and clinical trials in general .
the overall satisfaction with the treatment was very high , suggesting that the patients think they received the best possible care , and probably they think so because they are enrolled in a clinical trial .
the patients ' confidence and trust in their physicians and nurses is very firm and the majority of patients completely trusted their physicians .
the major conclusion of this study could be that not only do the physicians have high hopes from the trials , but that patients understand what the trial is and have high hopes too .
this study showed that the patients would recommend the trial to other patients , proving that they are aware of the fact that they could , with their opinion and experience , help others , too . the only factor identified in this study that possibly influences the patients ' opinion about
we should adjust our approach to each individual patient with a special concern about the level of education .
our recommendation to study teams and protocol writers would be to pay special attention to the icf , its length and amount of information provided by it . in conclusion
it is difficult to say how much effort we must additionally invest to obtain better results and longer survival of patients with lung cancer
. one thing is sure : we must give our best to justify the confidence and the hope our patients place in us .
none of the authors has any conflict of interest to declare in relation to the subject matter of this manuscript .
the authors would like to express their gratitude for the statistical management of the data to zoran potic and marijela potic .
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backgroundthe major aim of this study was to investigate what patients with advanced stage lung cancer , enrolled in a clinical trial , thought about their treatment .
we also wanted to investigate if there exist any characteristics that could influence patients ' opinion about the clinical trial.patients and methodsover the period from june 2008 to june 2009 , 59 eligible patients were enrolled in this study .
the major inclusion criteria were : participation in a clinical trial , previously treated advanced stage lung cancer , and good performance status ( ecog 0 - 2 ) .
all patients were asked to answer a questionnaire designed to investigate their impressions about participation in a clinical trial .
the questionnaire was deposited in a sealed box which was opened at the end of the study.we investigated a possible influence of age , gender , education , lung cancer stage , chemotherapy line and tumor type on the patients ' opinion about some aspects of the clinical trial.resultsthe majority of the patients were aware they were participating in the clinical trial and a significant number of them were very satisfied with the treatment .
of the investigated factors , only the level of education had a statistically significant influence on some of the questions raised in the questionnaire.conclusionspatients participating in clinical trials are satisfied with their treatment , ready to proceed with it and would recommend it to other patients .
it depends mainly on health professionals to maintain this level of confidence and justify their trust .
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the objectives of therapy for cryptorchidism include preservation of fertility , reduction of the risk of malignancy , and alleviation of physiological stress in human male .
the incidence of cryptorchidism in the newborn is 2 - 6% ; however , with spontaneous descent , this incidence drops to 1% by the age of 3 months .
both the testes and ovaries originate from a common bipotential precursor . in the embryonic level ,
several factors such as sry genes ( sex - determining region on y chromosome ) , sry proteins , anti - mullerian hormone ( amh ) , and mullerian - inhibiting substance ( mis ) helps in the differentiation of the male gonad from their bipotential precursor .
the male gonads undergo sry directed changes in an organization . at that time , male gonad , i.e. the testis remains situates into the abdominal cavity because the genital ridge develops in the upper lumber region at the 8 week of the embryonic life .
therefore , a phenomenon called testicular descendence occurs in different animals at a particular time in their life . in case of humans ,
the testis starts to descend from its retroperitoneal , abdominal position to the pelvic position between 10 and 15 week of gestation . within 35 to 40 week of human pregnancy
, it descends into the scrotum and remains permanently in that position . after the descendence of the testis ,
soon after , sertoli cells develop and secrete mis , the level of which remains high throughout gestation and causes regression of mullerian ducts . at 9 week of gestation ,
prenatal ultrasonography shows no testicular descent before 28 weeks gestation other than transabdominal movement to the internal inguinal ring .
trans - inguinal migration , thought to be under hormonal control , occurs at 28 - 40 weeks gestation , usually resulting in a scrotal testis by the end of a full - term of gestation .
although most mammals have notable scrota , rhinoceros , seal , elephant and whale are notable exceptions . in those vertebrates who do not have the scrota ,
the testis remains in the abdominal cavity , but due to their opened inguinal canal , it comes into the scrotal sac during their breeding season .
however , the chiropteran does not have any scrotal sac and their testis descend in the caudal abdominal wall only during their breeding season .
there are several factors that affects this transabdominal descend of the testis , such as mis that acts on a fibrous structure gubernaculums , which attaches the testis in the posterior abdominal wall ; as the body increases in size , the gubernaculums does not elongate , i.e. it becomes shorter in males .
the passage of the testis through the inguinal canal into the scrotum is probably controlled by the action of androgens . in man and domesticated mammals ,
it is not uncommon for the testis to be retained in the abdominal cavity rather than to descend normally into the scrotum ; this condition is called cryptorchidism or cryptorchism .
thus , there are several types of cryptorchidism found according to its reasons , such as natural cryptorchidism , which may occur due to the obstruction of the inguinal canal or by the inguinal canal dysfunction in human beings ; evolutionary cryptorchidism , which is found in some animals due to the evolutionary effects ( e.g. monotrems , elephants ) .
conversely , for experimental purpose , cryptorchidism may also be induced in animals such as bilateral cryptorchidism ( blc ) , in which both the testis remains within the abdominal cavity .
it ultimately results in complete sterility and unilateral cryptorchidism ( ulc ) , in which one of the testes remains within the abdominal cavity and the other descends into the scrotal sac and remains normal .
the one that remains normal can continue both the spermatogenesis and steroidogenesis in normal manner , but at a reduced rate .
however , in the cryptorchid testes , both these two functions of the gonad become degenerated .
overall , 32 - 79% of undescended testes are associated with some type of epididymal abnormality .
however , abnormalities that inhibit sperm transport ( e.g. complete caput separation , artesia , agenesis ) have been reported in only 8% of patients with cryptorchidism .
in addition , when the processus vaginalis is patent , the epididymis is more likely to be abnormal .
cryptorchidism is a pre - existing factor in 3 - 8% of infertile men and in 20% of men with azoospermia .
it is generally accepted that a relatively low temperature is preferable in spermatogenesis in mammalian species .
actually , several studies indicate that the temperature in the scrotum is 4c to 5c lower than that in the abdomen in most mammals .
waites and moule measured the temperature of the abdominal aorta and testicular artery at various points and found that the temperature of the testicular artery was significantly smaller . a major deleterious effect of high intratesticular temperature on spermatogenesis has been demonstrated .
it is well - known that the function of the cryptorchid testis is impaired by the rise in temperature .
the effect of ulc on ipsilateral testicular function has been well studied in humans and experimental animals .
however , the effect of ulc on contralateral testicular function has not been extensively studied .
ono and sofikitis have held that the endocrine and exocrine functions of the contralateral testis are impaired by a rise in temperature .
the present study focuses on the effects of left cryptorchidism on the right testicular and epididymal function and the overall sperm fertilizing capacity .
male mice ( parkes strain ) weighing 25 - 30 g were used in this study .
the animals were randomly distributed into cages and allowed to acclimatize for 10 days in a well - ventilated room at a room temperature of 25.0 2.0c under natural lighting condition , 12 h light / dark cycles were maintained in the animal house .
the animals were fed with standard laboratory diet and allowed free access to water daily .
all animals used in this study were handled in accordance with the institutional guidelines for care and use of laboratory animals .
each group was further subdivided into 2 groups ( n = 8) , for 15-days study and for 30-days study . to create ulc ( groups b and c ) , left inguinal incision was performed .
the left testis was sutured at the posterior abdominal wall with nylon sutures under ether anesthesia .
the sham operation ( group a ) included a left inguinal incision , placement of the left testis into the abdomen and replacement of the testis into its normal position .
group of ulc mice were weighed and killed by cervical dislocation after 15 and 30 day .
weight of the cryptorchid testis , normal testis , epididymis , and seminal vesicle were measured .
in addition , the control and cryptorchid testes were fixed by immersion in bouin 's solution and processed into paraffin .
sperm samples were collected from the cauda epididymis and counted by a hemocytometer chamber under light microscope . to minimize error ,
one - way analysis of variance ( anova ) test was first carried out to test for any differences between the mean values of all groups .
if differences between groups were established , the values of the treated groups were compared with those of the control group by a modified t - test .
male mice ( parkes strain ) weighing 25 - 30 g were used in this study .
the animals were randomly distributed into cages and allowed to acclimatize for 10 days in a well - ventilated room at a room temperature of 25.0 2.0c under natural lighting condition , 12 h light / dark cycles were maintained in the animal house .
the animals were fed with standard laboratory diet and allowed free access to water daily .
all animals used in this study were handled in accordance with the institutional guidelines for care and use of laboratory animals .
animals were divided into three main groups : ulc , blc , and control . each group was further subdivided into 2 groups ( n = 8) , for 15-days study and for 30-days study . to create ulc ( groups b and c ) , left inguinal incision was performed .
the left testis was sutured at the posterior abdominal wall with nylon sutures under ether anesthesia .
the sham operation ( group a ) included a left inguinal incision , placement of the left testis into the abdomen and replacement of the testis into its normal position .
group of ulc mice were weighed and killed by cervical dislocation after 15 and 30 day .
weight of the cryptorchid testis , normal testis , epididymis , and seminal vesicle were measured .
in addition , the control and cryptorchid testes were fixed by immersion in bouin 's solution and processed into paraffin .
sperm samples were collected from the cauda epididymis and counted by a hemocytometer chamber under light microscope . to minimize error , count was repeated at least five times for each rat .
one - way analysis of variance ( anova ) test was first carried out to test for any differences between the mean values of all groups .
if differences between groups were established , the values of the treated groups were compared with those of the control group by a modified t - test .
as the testes of the mature male mice were surgically confined into the abdominal cavity , a significant decrease in the weight [ table 1 ] , size , and turgidity of these organs were found in comparison between the unilaterally cryptorchid and control mice [ figure 1 ] .
in addition , the accessory sex organs such as the epididymis , seminal vesicle , and vas difference also became significantly reduced in their size , weight , and function as compared to their corresponding control tissues [ table 1 ] .
the experimental animals were also found to have significantly reduced sperm count than the sham - operated mice [ figure 2 ] .
weight of testis and accessory sex organs in control and treated rats reproductive organs of ( a ) sham - operated , ( b ) unilateral cryptochid mice and , ( c ) bilateral cryptorchid mice comparison of sperm count between 15 days and 30 days of ulc and blc histologically severe disorganization of the seminiferous tubules was observed time - dependently .
histopathological examinations showed deterioration in germinal epithelium and seminiferous tubules [ figure 3 ] . in case of the 30-days blc testis
, it was found that all germinal elements of the tubules were lost and little remained except a single layer of sertoli cell - like cells next to the basement membrane of the tubules .
this has been referred to as sertoli cell - only syndrome in the ulc and blc animals within 15-days period .
most highly differentiated cells have disappeared first in order of degeneration were spermatozoa , spermatids , spermatocytes , and spermatogonia .
histological sections of testis ( 400 ) showing , ( a ) the changes in spermatogenic cell count in control , ( b ) unilaterally cryptorchid mice ( 15 days ) , ( c ) unilaterally cryptorchid mice ( 30 days ) , ( d ) bilaterally cryptorchid mice ( 15 days ) , ( e ) bilaterally cryptorchid mice ( 30 days )
in the present experiment , almost no sperm was observed in the caudal part of the epididymis of abdominal testis in ulc and blc mice for both 15- and 30-days study .
however , in the caudal part of the normal non - cryptorchidimized testis ( right ) in the ulc mice , normal sperm was found , though quite lower than that in the control mice [ figure 2 ] . according to the sperm count of that cryptorchid testis , it can be assumed that the spermatogenesis and other testicular functions of experimental animals were totally disrupted , because the temperature of the abdominal cavity is approximately 4c higher than that of the scrotum .
it has been reported that higher temperature causes impairment of the testicular functions . in the scrotum , pampiniform plexus
, a network of veins from the testes and epididymis , keeps the temperature of the testes lower than that of the body cavity .
this plexus is supplied by spermatic artery and the venous blood empties into the spermatic vein . the plexus functions to cool the blood from the body before it enter the testes and also to warm the blood from the testes before it is returned to the systemic circulation .
thus , the pampiniform plexus controls the temperature of the testes and the scrota and keeps these two in a lower temperature of about 36c . in that low temperature of the scrotum , different testicular functions and
the spermatogenesis can occur in their normal manner . as the testis was removed from its scrotal position
decrease in sperm count , testicular , and accessory sex organ weights are relatively higher in blc mice because of impairment of androgen biosynthesis by leydig cells . a time - dependant decrease was also noticed in both cases [ table 1 ] . on the contrary , it was found that the weights of accessory sex glands in ulc mice were not affected , as the remaining testis was capable of producing adequate amount of testosterone to maintain structural integrity of these androgen - dependent glands .
it was also reported that cryptorchid testis is equally capable of producing testosterone in vitro as in the control testis .
cellular degeneration was observed in histological sections of testis , which is also an indicator of low testosterone level .
however , some reports demonstrated a reduced serum testosterone level after 7 days of cryptorchidism in rat , but it came to normal in subsequent days up to day 28 .
measurement of serum testosterone and luteinizing hormone ( lh ) levels demonstrated that the cryptorchid mouse testes are capable of secreting significant level of testosterone to cause a normal regulating function of the hypertrophy .
some investigations reported that follicle stimulating hormone ( fsh ) increases on day 7 of cryptorchidism , which progressively rises with increased duration of cryptorchidism in the mouse .
another report demonstrated extensive damage along with lower level of fsh in comparison to castrated condition , which suggests that blc can exert suppressive feedback activity on fsh reduced activity , possibly due to continued secretion of testosterone by the leydig cells . particularly in the case of ulc , it has been reported that spermatogenesis does not proceed within the clinically unilateral undecended testis during puberty . in the present study , the arrest of spermatogenesis was also observed in both 15- and 30-days ulc , although some androgens were produced from the remaining testis in the ulc mice .
however , the secretion rates of those androgens are considerably lower than that in normal mice .
while correlating this ulc with the unilateral ovariectomy ( ulo ) , it can observed that there is no compensatory stimulation by the increased levels of lh in case of the testis that remains normal in the ulc .
thus , it is reduced in size , weight , and sperm count in a negligible manner than in the control .
the present investigation showed compensatory androgen production in ulc mice , whereas absence of androgen mediated reproductive functions in experimental animals after blc .
thus , the current findings suggested that ulc and blc may cause detrimental effects on male reproductive physiology .
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background : experimental unilateral cryptorchidism ( ulc ) and bilateral cryptorchidism ( blc ) are excellent methods to study undescended testis in relation to spermatogenesis against a temperature gradient.objectives:in case of ulc , it is possible to compare the testicular functions between normal condition and cryptorchidism in the same animal , whereas blc shows the necessity of testicular androgens for proper maintenance of reproductive structures and functions.materials and methods : in the present study , experimental ulc and blc was done on same - aged adult mature male mice and kept for 15 days and 30 days , respectively , to observe the changes due to the induced cryptorchidism on the different reproductive organs , viz . , the testis and accessory sex organs along with epididymal sperm count .
reproductive tissues were collected from individual animals and histopathological studies of testis were done to investigate different cytological changes.results:the size of the testes and accessory sex organs were found to be significantly reduced in blc mice , whereas only testicular weight reduction was observed in ulc mice .
histopathological studies showed degenerative changes throughout the seminiferous tubules.conclusion:thus , the present investigation showed compensatory androgen production in ulc mice , whereas absence of androgen mediated reproductive functions in blc animals .
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assault , though a major cause of maxillofacial injuries in the developed nations , has not been adequately investigated among nigerian population .
this study aimed to analyze the pattern of maxillofacial injuries caused by assault in our institution .
a descriptive clinical survey of patients with assault - induced oral and maxillofacial injuries presenting to our maxillofacial surgery clinic / emergency ward was carried out .
demographic data and pattern of injuries obtained from patients record and department trauma database were analyzed .
thirty - four cases were due to assault and male to female ratio was 1.8:1 .
the mean age of the patients was 21.46.26 years ( age range 248 years ) .
23.6% ( n=8 ) of the injuries were due to domestic violence between spouses while 35.3% ( n=12 ) resulted from fight .
students unrest and armed robbery attack accounted for six cases each ( 17.7% , n=6 ) , while there were two cases due to child battering .
64.3% ( n=22 ) of the injuries sustained involved soft tissues while 35.7% involved hard tissues .
contusion was the most common isolated soft tissue injury accounting for 56% ( n=10 ) while dentoalveolar fracture was the most encountered hard tissue injury ( 62.5% , n=16 ) .
there is need for preventive strategies to reduce the incidence of assault - induced maxillofacial injuries .
the etiology of maxillofacial injuries varies from one country to another and even within the same country depending on the prevailing socioeconomic , cultural , and environmental factors.[14 ] road traffic crashes ( rtc ) , assaults , sports , falls occupational hazards are documented causes of oral and maxillofacial injuries.[148 ] animal attack is another cause of facial injuries that has been reported in the northern part of nigeria .
being the most exposed part of the human body , the maxillofacial region is vulnerable to varying degree of injuries when the individual is subjected to physical trauma .
skeletal and soft tissue injuries of the face constitute quite a significant portion of the workload of the oral and maxillofacial surgeons in nigeria.[47 ] therefore , periodic verification of the etiology of maxillofacial injuries helps to recommend ways in which maxillofacial injuries can be averted . in the past ,
road traffic crashes ( rtc ) used to be the most common cause of facial fractures globally.[811 ] the trend is different in the developed countries where assault appeared to have overtaken rtc as the leading cause of oral and maxillofacial injuries.[1214 ] however , rtc still remain the leading cause of maxillofacial injuries in developing countries , nigeria inclusive.[1519 ] other causes of oral and maxillofacial injuries have received little attention in the nigerian literature probably because of the predominance of rtc in the etiology of such injuries .
assault - induced maxillofacial injuries have been under - reported in the literature despite increase in cases of altercations worldwide and available reports were mostly from foreign literature .
one of the few reports on assault related maxillofacial injuries is the study done by olasoji et al . on prevalence of assault - induced oral and maxillofacial injuries in north - eastern part of nigeria .
he attributed the increasing wave of assault - induced maxillofacial injuries to the nomadic life style of the people of the region .
the present study was conceived because of dearth of studies on assault related maxillofacial injuries among nigerian population .
the aim of the study was to determine the prevalence and pattern of assault - induced oral and maxillofacial injuries in our institution .
the study was carried out by recruiting patients who presented at the accident and emergency ward and maxillofacial surgery clinic of the university teaching hospital , ado - ekiti .
the hospital is a major referral center covering ekiti and adjoining ondo and kogi state in south - western nigeria .
it is a descriptive clinical study including oral and maxillofacial assault cases that presented between october 2009 and december 2010 .
inclusion criteria include history of assault and confirmation of maxillofacial injury on examination and investigation .
age , sex , mechanism of assault , and examination findings concerning injury pattern were documented .
data obtained on the prevalence , pattern of injuries and assault mechanism were summarized in tables and figures .
descriptive analysis of data was done with statistical package for social sciences ( spss ) version 17 .
of the 156 cases of oral and maxillofacial injuries seen between october 2009 and december 2010 , 34 cases were due to assault
the mean age of the patients was 21.46.26 years ( age range 248 years ) and more than 50% of the patients were between 21 and 30 years of age [ figure 1 ]
it can be deduced that most victims of assault induced facial injuries are between 20 to 30 years of age causes of assault - induced maxillofacial injuries in our series include domestic violence , interpersonal altercation ( fight ) , child battering , students unrest , and armed robbery attack .
interpersonal altercation ( fight ) accounted for 35.3% of the injuries while 23.6% of the injuries were due to domestic violence between spouses .
students unrest and armed robbery attack accounted for six cases each ( 17.7% , n=6 ) , while there were two cases due to child battering .
it is worthy to note that the assaulted victims of domestic violence were all females and the most common means of assault is the fist .
apart from the fist , weapons such as broken bottles ( n=3 ) , knife ( n=5 ) , machete ( n=4 ) , and pipes ( n=2 ) were used for assault .
none of the patients in this study had gunshot injury . showed the causes of oral and maxillofacial injuries in the patients studied .
the figure revealed that fight accounted for more cases of injuries ( n=12 ) followed by domestic violence ( n=8 ) .
other causes of injuries are armed robbery attack ( n=6 ) , students unrest ( n=6 ) and child abuse ( n=2 ) injury pattern revealed that 64.3% ( n=22 ) of the injuries sustained involved soft tissues while 35.7% involved hard tissues .
contusion was the most common isolated soft tissue injury accounting for 56% ( n=10 ) of such injuries while dentoalveolar fracture was the most encountered hard tissue injury ( 62.5% , n=16 ) .
there were two cases each of mandibular and nasal bone fractures [ table 2 ] .
etiology and pattern of injury all patients received initial treatments aimed at control of hemorrhage and prevention of infection by antibiotic medication .
nasal fractures were reduced with walsham 's nasal forceps , dentoalveolar fractures were treated by splinting the segments with 0.5 mm soft stainless steel wire while mandibular fractures were immobilized with 2.0 mm titanium locking plates .
all patients were treated under local anesthesia except the mandibular fractures where patients had to be intubated for general anesthesia .
although rtc remain the leading cause of oral and maxillofacial injuries in most developing countries , other causes such as assaults , industrial accident , and sport - related injuries have increased over time.[2227 ] the situation is different in the developed nations where assaults and interpersonal altercations are the leading cause of oral and maxillofacial injuries .
the prevalence of assault in our study was 21.8% this is comparable to previous studies where assault cases constituted less than 20% . on the contrary , olasoji et al .
he attributed this to the nomadic lifestyle of the people in the region where animals are moved over several kilometers of land grazing over farms and destroying cash crops in the process , this frequently leads to fight between farmers and cattlemen . peak age incidence of 2130 years found in our study is in agreement with the literature probably because people in this age group are in the active phase of livelihood and are likely to be involved in assaults more than other age groups .
also , we found a higher male preponderance in agreement with other studies ; however those involved in domestic violence in this study were all female victims of assault by the spouse .
poor socioeconomic condition and stress from unemployment could lead to aggressive tendencies by men and this often leads to assault on wives .
the influence of alcohol consumption on domestic violence has also been reported in the literature , though we could not establish such in this study .
some of the patients in our series were victims of politically motivated violence and students unrest .
similarly , quite a number of maxillofacial injuries are seen during cult clashes in higher institutions .
this is unlike european and american studies where most of the fights occurred in the street , club or pub .
this may be explained by the fact that fist was the commonest means of assault , and it causes subcutaneous tissue injury while preserving the continuity of the overlying skin .
most authors also reported contusion as the most common soft tissue injury to the maxillofacial region .
this finding may be due to the fact that assaults are less likely to cause mandibular ormid - facial fractures which are more commonly related to road traffic crashes . moreover , dentoalveolar injuries related to rtc and other causes are under - reported in most maxillofacial injury surveys . however , gassner andco - workers in a large survey of maxillofacial injuries involving 9543 patients reported an incidence of 49.9% of dentoalveolar injuries .
similar findings were reported in studies where dental oral tissues were included . in conclusion ,
assault - induced oral and maxillofacial injuries constitute a significant workload of maxillofacial surgeons and as such prevention strategies should be embarked on . increasing unemployment rate and the poor per capital income of an average nigerian which
has decreased by more than 75% during the past 25 years are contributory to violent conduct .
assault - induced oral and maxillofacial injuries can be curbed if unemployment is tackled and education of youth is adequately addressed .
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background : assault , though a major cause of maxillofacial injuries in the developed nations , has not been adequately investigated among nigerian population .
this study aimed to analyze the pattern of maxillofacial injuries caused by assault in our institution.methods:a descriptive clinical survey of patients with assault - induced oral and maxillofacial injuries presenting to our maxillofacial surgery clinic / emergency ward was carried out .
demographic data and pattern of injuries obtained from patients record and department trauma database were analyzed.results:156 patients presented with oral and maxillofacial injuries between october 2009 and december 2010 .
thirty - four cases were due to assault and male to female ratio was 1.8:1 .
the mean age of the patients was 21.46.26 years ( age range 248 years ) .
23.6% ( n=8 ) of the injuries were due to domestic violence between spouses while 35.3% ( n=12 ) resulted from fight .
students unrest and armed robbery attack accounted for six cases each ( 17.7% , n=6 ) , while there were two cases due to child battering .
64.3% ( n=22 ) of the injuries sustained involved soft tissues while 35.7% involved hard tissues .
contusion was the most common isolated soft tissue injury accounting for 56% ( n=10 ) while dentoalveolar fracture was the most encountered hard tissue injury ( 62.5% , n=16).conclusion : there is need for preventive strategies to reduce the incidence of assault - induced maxillofacial injuries .
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melanotic neuroectodermal tumor of infancy ( mnti ) is a rare clinical condition , which occurs in infants within the first 6 months of life .
mnti is usually a benign tumor of neural crest origin composed of relatively primitive pigment producing cells .
krompecher made the first description of this tumor in 1918 as a congenital melanocarcinoma . in 1966 ,
borello and gorlin reported a case characterized by a high urinary excretion of vanillylmandelic acid ( vma ) , classically found in a pheochromocytoma as well as in other neuroblastic tumors , suggesting a neuroectodermal origin of the lesion .
alternative terminologies for mnti are pigmented neuroectodermal tumor of infancy , melanotic prognoma , retinal anlage tumor , pigmented epulis of infancy , melanotic adamantinoma , pigmented ameloblastoma , melanotic epithelial odontoma , retinal choristoma , pigmented teratoma , atypical melanoblastoma .
it is well - known to be locally aggressive which may result in tooth displacement as the tumor cells invade the surrounding bone .
this was a case report of a 4-month - old patient who presented with the complaint of swelling in the right back tooth region of the upper jaw reported to the department of pedodontics and preventive dentistry , kamineni institute of dental sciences , narketpally , nalgonda district .
a swelling was observed for the first time when the child was 2 months old which rapidly increased in size , which caused disfigurement of face [ figure 1 ] .
pre - operative extra - oral view on clinical examination , the swelling was diffuse , extending from the maxillary right second primary molar region to the canine region anteroposteriorly and caused expansion of the buccal cortical plate and palate up to the midline , but did not cross the midline .
the rapid increase in the size of the swelling suggested a malignant nature of the enlargement . hence the lymph nodes were checked for any metastasis , but found none .
pre - operative intra - oral view normal radiographic examination ( occlusal radiograph ) showed a diffuse osteolytic radiolucent lesion in the right maxilla and showed displacement and dysmorphic changes in the developing primary tooth buds .
plain and contrast enhanced computerized tomographic analysis of the para nasal sinuses performed on a 64 slice computed tomography ( ct ) scanner , with coronal reconstructions revealed evidence of an expansile lytic lesion arising from right maxillary sinus showing cortical defects with associated soft - tissue components and calcific densities within .
the lesion measured 3.2 cm 2.1 cm 2.3 cm ( anterio - posterior ( ap ) trans height ) , which displaced the primary teeth inferiorly .
pre - operative computed tomography scan image frontal view pre - operative computed tomography scan image lateral view initial wedge biopsy was planned and performed to aid in proper diagnosis .
the histopathological report revealed nests of cells arranged in alveolar pattern and small basophilic neuroblast like cells arranged in clusters .
wide surgical excision with removal of the tooth buds involved in the lesion to avoid local recurrence was performed under general anesthesia .
the histopathological analysis revealed , tumor cells arranged in alveolar pattern within a delicate fibrous connective tissue stroma .
large epithelioid cells containing pigment ( melanocyte like cells ) are arranged peripherally in the clusters enclosing central small basophilic neuroblast like cells .
infiltration of these cells into the surrounding bone and in between developing tooth buds is appreciated .
the biphasic tumor cell population arranged in a background of fibrous connective tissue stroma is suggestive of mnti involving the cancellous bone [ figures 5 and 6 ] .
low power microscopic view showing tumor cells arranged in alveolar pattern high power magnification view showing epithelioid cells containing pigment ( melanocyte like cells )
early diagnosis and treatment of any pathological condition is the key to avoid serious sequelae .
mnti is a rare clinical condition . according to the literature , only around 365 cases
though very rare , good knowledge about lesions like mnti is crucial because of its early age of involvement and rapidity of its spread .
delay in the diagnosis may lead to rapid encroachment of the tumor onto the adjacent vital structures , which may necessitate a radical resection , leading to increased morbidity .
liu et al . in 2004 explained the possibility of obstruction of infant airway by fairly aggressive expansion of the lesion . in 90% of the instances ,
no gender predilection was reported by stowens and lim ( 1974 ) and lopez ( 1976 ) , but a male to female ratio of 1.48 was given by kruse - losler in 2006 after a review of 140 cases .
other sites reported were mandible , brain , skull , epididymis , shoulder , scapula , fontanelle , mediastenum and uterus .
mnti is described in literature as a benign , rapidly growing , non - ulcerated , locally aggressive tumefaction .
though it is a pigmented tumor , the pigmentation can not always be observed through the overlying tissues .
it is considered as a tumor of neural crest origin composed of primitive pigment producing cells .
krompecher suggested that this tumor derives from epithelial islands trapped during embryonic fusion of facial buds .
halpert , was of the opinion that the tumor arises from retinal anlage by a process of pinching - off of the neuroectoblast eye .
similarity in the tissue organization between mnti and fetal pineal gland suggested a pineal origin .
borello and gorlin suggested the term mnti which has now been universally accepted .
they observed increase in urinary vma , which is a major catabolite of the catecholamines .
its free form when found in great quantity is regarded as a marker of the neuroectodermal tumors .
they proved this by showing decreased vma levels after the surgical excision of the tumor .
however , the diagnosis of mnti is not necessarily correlated with the increase in the serum levels of vma .
a significant number of cases of mnti have been reported with normal levels of vma .
dehner et al . in 1979 described various stages of melanocytes and neuroblast like cells within the tumor , suggesting its neuroectodermal origin .
immuno - histochemical markers in different studies suggest that mnti is a primitive neuroectodermal tumor with polyphenotypic expression of neural and epithelial markers , melanin production , occasional rhabdomyoblastic , glial , ganglionic and osseous differentiation and no photoreceptor differentiation .
it probably represents a dysembryogenetic neoplasm that recapitulates the retina at 5 weeks of gestation .
differential diagnosis of mnti includes various pediatric small round cell neoplasms such as neuroblastoma , ewing 's sarcoma , peripheral neuroepithelioma , rhabdomyosarcoma , peripheral primitive neuroectodermal tumor , desmoplastic small round cell tumor , malignant melanoma and lymphoma .
post - operatively the present case was followed - up for 6 months , which revealed no evidence of recurrence and the child was healthy [ figure 7 ] . although the prognosis of benign mnti is excellent after complete surgical excision , long - term follow - up is mandatory as the course of the disease can not be predicted by morphological findings .
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melanotic neuroectodermal tumor of infancy ( mnti ) is a relatively uncommon osteolytic - pigmented neoplasm that primarily affects the jaws of infants .
the early onset and its rapid disfiguring spread necessitate early diagnosis . a 4-month - old male child reported with the complaint of swelling in the right back tooth region of the upper jaw , which rapidly increased in size causing disfigurement of the face .
radiographic examination showed a diffuse osteolytic radiolucent lesion in the right maxilla and displacement and dysmorphic changes in the developing primary tooth buds .
wide surgical excision was performed under general anesthesia .
histopathological report revealed characteristic large pigmented epitheloid cells ( melanocyte like cells ) .
the biphasic tumor cell population arranged in a background of fibrous connective tissue stroma is suggestive of mnti involving the cancellous bone .
early diagnosis and management of such aggressive tumors precludes significant morbidity of the patient .
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it has generally been assumed that in type 1 diabetes ( t1d ) , increase in urinary albumin excretion rate ( aer ) precedes the development of impaired renal function and end - stage renal disease ( esrd ) . in that model
, the onset of microalbuminuria ( ma ) leads to macroalbuminuria , and the latter is followed by progressive glomerular filtration rate ( gfr ) loss , or renal decline , which eventually leads to esrd ( 1 ) . a recently published large follow - up observation of the diabetes control and complications trial ( dcct)/epidemiology of diabetes interventions and complications ( edic ) cohort showed that macroalbuminuria was a strong predictor of the development of chronic impaired renal function ( gfr < 60 ml / min ) ( 2 ) .
this finding , although consistent with the above model of diabetic nephropathy , is open for different interpretations .
the authors assumed a priori that abnormalities in aer would precede significant gfr loss and did not test the alternative possibility that so - called early progressive renal decline when gfr falls within the normal range , i.e. , from 130 to 60 ml / min , might precede the development of macroalbuminuria .
it is also possible that early progressive renal decline and the development of aer abnormalities are two clinical phenotypes that reflect the disease process underlying diabetic nephropathy that eventually results in esrd .
they allow the examination of the above models and , particularly , the establishment of which of the two phenotypes appears first in the clinical manifestation of diabetic nephropathy .
using some of these tools , we already revealed that progressive renal decline commences earlier than previously recognized and is already present in patients with ma ( 5,6 ) . in our earlier follow - up studies of t1d ( the first and second joslin kidney study on the natural history of microalbuminuria ) , progressive renal decline developed in one - third of the patients with ma ( 5 ) .
furthermore , renal decline was strongly associated with serum concentrations of uric acid and concentrations of tumor necrosis factor receptor 1 or 2 ( tnfr-1 or -2 ) ( 6,7 ) .
furthermore , due to limited follow - up data on patients with normoalbuminuria ( na ) , we were not able to establish whether renal decline can begin in patients with na and , if so , whether its determinants are similar as in patients with ma . to answer these questions
, we expanded our previous study in patients with t1d and na as well as with ma who had been followed with serial measurements of serum creatinine and cystatin c. we combined both markers to derive the most precise indirect measurement of the gfr ( egfrcr - cys ) and trace its changes / trajectories during follow - up ( 4 ) .
clinical characteristics and serum concentrations of markers previously reported in cross - sectional analyses of this cohort ( 8) were examined as determinants of progressive renal decline .
the committee on human studies of the joslin diabetes center approved the protocol and informed consent procedures for the second joslin kidney study on the natural history of microalbuminuria , referred to here as the 2nd jks . a description of the joslin clinic population and the design of the study were reported with results of a cross - sectional examination of the cohort ( 8) .
previous reports of the shorter follow - up of this cohort included only patients with high na and ma ( 6,7 ) .
the current study includes all patients with baseline na as well as ma and extends follow - up to median 8 years ( 25th and 75th percentiles accordingly 69 years ) .
participants in the 2nd jks were recruited from among patients attending the joslin clinic , a major center for treatment of patients with diabetes . during the screening period , 1 january 2003 through 31 december 2006 , patients scheduled for an appointment at the joslin clinic were evaluated for eligibility in the clinical data system .
eligibility criteria included residence in new england , t1d diagnosed before age 40 years , current age 1864 years , and diabetes duration 339 years .
the archived clinical laboratory results during the 2-year pre - enrollment interval were searched for measurements of albumin - to - creatinine ratio ( acr ) in urine specimens . in the joslin clinic ,
laboratory albumin concentration in spot urine was measured by immunonephelometry on a bn prospec system nephelometer ( dade behring , inc . , newark , de ) with n albumin kits , with intra - assay and interassay coefficients of variation 4 and 6% , respectively .
creatinine measurements in urine were assayed by jaffe s modified picrate method on a ciba corning express plus chemistry analyzer ( inter- and intra - assay coefficients of variation 3 and 5% , respectively ) .
individual values for acr ( measured in milligrams per gram ) were converted to aers ( in micrograms per minute ) by the previously published formula log(aer ) = 0.44 + ( 0.85)log(acr ) ( 0.13)sex , where sex is assigned a value of 1 for female patients and 0 for male patients ( 9 ) .
patients were not eligible for enrollment if they had macroalbuminuria ( median pre - enrollment aer 300 g / min ) , were on dialysis , had received a renal transplant , or had a history of hiv or hepatitis c infection . among the 4,000 residents of new england with t1d who attended the joslin clinic during the screening period , we identified 2,667 eligible patients : 2,007 with na ( pre - enrollment median aer < 30 g / min ) and 660 with ma ( pre - enrollment median aer 30299 g / min ) .
the study aimed to enroll eligible patients with ma and a similar number of eligible patients from the much larger pool of patients with na .
furthermore , since the number of eligible na patients with a pre - enrollment aer < 15 g / min was much larger than the number with an aer 1529 g / min , we sought to enroll only one patient with an aer < 15 g / min for each patient enrolled with an aer 1529 g / min .
study recruiters approached patients who had been identified as eligible when they arrived for a scheduled appointment at the clinic . after informing patients about the study and obtaining their written consent to participate ,
the recruiter 1 ) administered a structured interview that solicited the history of diabetes and its treatment , other health problems , and medications ; 2 ) measured blood pressure ; and 3 ) obtained specimens of blood and urine for laboratory determinations .
measurements of aer at enrollment examination were combined with aer measurements from the preceding 2-year pre - enrollment interval , and new medians were determined to represent the baseline aer values .
for all other characteristics , baseline values are those obtained at enrollment examination . as of the end of 2006
patients recruited into the 2nd jks during a second phase ( 20072010 ) are not included in this report .
enrolled participants were followed until 2013 , with a goal to obtain blood and urine specimens at least every 2 years .
although most patients had two or more visits per year , the research specimens were collected less frequently , on average 1.5 years apart .
patients with less frequent visits to the clinic or those who stopped coming to the clinic were examined at their homes .
specimens obtained at baseline and during follow - up were stored at 80c for later analysis .
determinations of aer performed during routine clinic visits and for research purposes were combined and used to evaluate changes in aer over time ( all measurements were performed in the joslin clinic laboratory as described above ) .
we considered three 2-year intervals : the baseline interval and the first and second 2-year intervals after enrollment . in patients with na during the baseline interval , a median aer > 30 g / min in either 2-year follow - up interval constituted the onset of ma . in patients with ma during the baseline interval , a halving of baseline aer constituted regression of ma and the development of macroalbuminuria ( > 300 g / min ) in either follow - up interval constituted progression .
protocols for measuring concentration of tnf- ( free ) and its receptors ( tnfr-1 and -2 ) and other markers such as il-6 , il-8 , ip-10 , mcp-1 , vcam , icam , fas , and fasl as well as uric acid were described previously ( 6,8 ) . from 2009 to 2013
, creatinine and cystatin c were assayed in stored baseline and follow - up serum samples in the advanced research and diagnostic laboratory at the university of minnesota .
protocols used for creatinine measurements yielded values traceable to isotope dilution mass spectrometry reference measurement procedures , and , for the cystatin c , values were adjusted to be traceable to erm - da471/ifcc international reference material as described previously ( 10 ) .
the egfrcr - cys was estimated with the recently published combined creatinine - cystatin c chronic kidney disease epidemiology collaboration ( ckd - epi ) formula ( 4 ) .
longitudinal measures of log - transformed egfrcr - cys values were analyzed with linear mixed - effects regression ( proc mixed , sas v 9.3 ; sas institute , cary , nc ) .
this approach takes into account the correlation between follow - up observations from a patient taken at varying intervals .
it yields individual - specific slope coefficients that are re - expressed as percent change per year in egfrcr - cys ( 6 ) .
progressive renal decline is defined as a negative change equal to or steeper than 3.3% per year , and the patient is referred to as a decliner .
less steep slopes are defined as stable , and the patients are referred to as nondecliners .
this criterion , an egfr loss of 3.3% per year or more , has been used in previous reports ( 5,6 ) and corresponds to the 2.5th percentile of the distribution of annual renal function loss in a general population ( 11 ) .
comparisons between those with renal decline and those with stable egfrcr - cys were made with wilcoxon rank sum tests for continuous variables and test for categorical variables . the risk of renal decline ( egfrcr - cys loss 3.3% per year ) according to clinical characteristics or baseline serum marker concentrations categorized into quartiles of their distributions or according to other ordinal variables
a multiple logistic regression model was used to jointly test the predictors of egfr decline identified in univariate analysis .
participants in the 2nd jks were recruited from among patients attending the joslin clinic , a major center for treatment of patients with diabetes . during the screening period , 1 january 2003 through 31 december 2006 , patients scheduled for an appointment at the joslin clinic were evaluated for eligibility in the clinical data system .
eligibility criteria included residence in new england , t1d diagnosed before age 40 years , current age 1864 years , and diabetes duration 339 years .
the archived clinical laboratory results during the 2-year pre - enrollment interval were searched for measurements of albumin - to - creatinine ratio ( acr ) in urine specimens . in the joslin clinic ,
laboratory albumin concentration in spot urine was measured by immunonephelometry on a bn prospec system nephelometer ( dade behring , inc . ,
newark , de ) with n albumin kits , with intra - assay and interassay coefficients of variation 4 and 6% , respectively .
creatinine measurements in urine were assayed by jaffe s modified picrate method on a ciba corning express plus chemistry analyzer ( inter- and intra - assay coefficients of variation 3 and 5% , respectively ) .
individual values for acr ( measured in milligrams per gram ) were converted to aers ( in micrograms per minute ) by the previously published formula log(aer ) = 0.44 + ( 0.85)log(acr ) ( 0.13)sex , where sex is assigned a value of 1 for female patients and 0 for male patients ( 9 ) .
patients were not eligible for enrollment if they had macroalbuminuria ( median pre - enrollment aer 300 g / min ) , were on dialysis , had received a renal transplant , or had a history of hiv or hepatitis c infection . among the 4,000 residents of new england with t1d who attended the joslin clinic during the screening period , we identified 2,667 eligible patients : 2,007 with na ( pre - enrollment median aer < 30 g / min ) and 660 with ma ( pre - enrollment median aer 30299 g / min ) .
the study aimed to enroll eligible patients with ma and a similar number of eligible patients from the much larger pool of patients with na . furthermore , since the number of eligible na patients with a pre - enrollment aer < 15 g / min was much larger than the number with an aer 1529 g / min , we sought to enroll only one patient with an aer < 15 g / min for each patient enrolled with an aer 1529 g / min .
study recruiters approached patients who had been identified as eligible when they arrived for a scheduled appointment at the clinic . after informing patients about the study and obtaining their written consent to participate ,
the recruiter 1 ) administered a structured interview that solicited the history of diabetes and its treatment , other health problems , and medications ; 2 ) measured blood pressure ; and 3 ) obtained specimens of blood and urine for laboratory determinations .
measurements of aer at enrollment examination were combined with aer measurements from the preceding 2-year pre - enrollment interval , and new medians were determined to represent the baseline aer values . for all other characteristics ,
baseline values are those obtained at enrollment examination . as of the end of 2006 ,
patients recruited into the 2nd jks during a second phase ( 20072010 ) are not included in this report .
enrolled participants were followed until 2013 , with a goal to obtain blood and urine specimens at least every 2 years .
although most patients had two or more visits per year , the research specimens were collected less frequently , on average 1.5 years apart .
patients with less frequent visits to the clinic or those who stopped coming to the clinic were examined at their homes .
specimens obtained at baseline and during follow - up were stored at 80c for later analysis .
determinations of aer performed during routine clinic visits and for research purposes were combined and used to evaluate changes in aer over time ( all measurements were performed in the joslin clinic laboratory as described above ) .
we considered three 2-year intervals : the baseline interval and the first and second 2-year intervals after enrollment . in patients with na during the baseline interval , a median aer > 30 g / min in either 2-year follow - up interval constituted the onset of ma . in patients with ma during the baseline interval , a halving of baseline aer constituted regression of ma and the development of macroalbuminuria ( > 300 g / min ) in either follow - up interval constituted progression .
protocols for measuring concentration of tnf- ( free ) and its receptors ( tnfr-1 and -2 ) and other markers such as il-6 , il-8 , ip-10 , mcp-1 , vcam , icam , fas , and fasl as well as uric acid were described previously ( 6,8 ) . from 2009 to 2013 ,
creatinine and cystatin c were assayed in stored baseline and follow - up serum samples in the advanced research and diagnostic laboratory at the university of minnesota .
protocols used for creatinine measurements yielded values traceable to isotope dilution mass spectrometry reference measurement procedures , and , for the cystatin c , values were adjusted to be traceable to erm - da471/ifcc international reference material as described previously ( 10 ) .
the egfrcr - cys was estimated with the recently published combined creatinine - cystatin c chronic kidney disease epidemiology collaboration ( ckd - epi ) formula ( 4 ) .
protocols for measuring concentration of tnf- ( free ) and its receptors ( tnfr-1 and -2 ) and other markers such as il-6 , il-8 , ip-10 , mcp-1 , vcam , icam , fas , and fasl as well as uric acid were described previously ( 6,8 ) .
from 2009 to 2013 , creatinine and cystatin c were assayed in stored baseline and follow - up serum samples in the advanced research and diagnostic laboratory at the university of minnesota .
protocols used for creatinine measurements yielded values traceable to isotope dilution mass spectrometry reference measurement procedures , and , for the cystatin c , values were adjusted to be traceable to erm - da471/ifcc international reference material as described previously ( 10 ) .
the egfrcr - cys was estimated with the recently published combined creatinine - cystatin c chronic kidney disease epidemiology collaboration ( ckd - epi ) formula ( 4 ) .
longitudinal measures of log - transformed egfrcr - cys values were analyzed with linear mixed - effects regression ( proc mixed , sas v 9.3 ; sas institute , cary , nc ) .
this approach takes into account the correlation between follow - up observations from a patient taken at varying intervals .
it yields individual - specific slope coefficients that are re - expressed as percent change per year in egfrcr - cys ( 6 ) .
progressive renal decline is defined as a negative change equal to or steeper than 3.3% per year , and the patient is referred to as a decliner .
less steep slopes are defined as stable , and the patients are referred to as nondecliners . this criterion , an egfr loss of 3.3% per year or more , has been used in previous reports ( 5,6 ) and corresponds to the 2.5th percentile of the distribution of annual renal function loss in a general population ( 11 ) .
comparisons between those with renal decline and those with stable egfrcr - cys were made with wilcoxon rank sum tests for continuous variables and test for categorical variables .
the risk of renal decline ( egfrcr - cys loss 3.3% per year ) according to clinical characteristics or baseline serum marker concentrations categorized into quartiles of their distributions or according to other ordinal variables was tested using the cochran - armitage trend test .
a multiple logistic regression model was used to jointly test the predictors of egfr decline identified in univariate analysis .
there were 304 patients with ma and 364 patients with na enrolled in the 2nd jks .
this study included those with normal renal function at baseline ( egfrcr - cys > 60 ml / min ) who were followed for 410 years : 248 ( 82% ) patients with ma and 286 ( 79% ) with na .
characteristics of the study groups are summarized in table 1 . in comparison with the na group ,
the ma group included more males ; had a longer duration of diabetes , higher hba1c , and higher blood pressure ; and was more frequently treated with renoprotective drugs . whereas the distribution of aer in the na group was uniform across the normal range ( < 30 g / min ) , the bulk of the distribution in the ma group was in the low abnormal range ( 25th , 50th , and 75th percentiles were 44 , 65 , and 116 g / min , respectively ) .
the distributions of baseline concentrations of serum creatinine and cystatin c and the estimates of egfrcr - cys were similar in na and ma .
characteristics of the study groups duration of follow - up and the available number of measurements of serum creatinine and cystatin c were similar in the two study groups
. the annual rate of egfrcr - cys change was estimated from these serial measurements .
the median rate of loss in patients with ma was more rapid than in patients with na .
renal decline ( rate of egfrcr - cys loss 3.3% per year ) was present in 10% of the na group and 35% of the ma group .
impaired renal function ( ckd - epi 3 ) developed in 6% of the na group and 22% of the ma group , and esrd developed in one of the former and four of the latter .
the trajectories of renal function changes in the 28 decliners with na have particular significance because they have not been documented before ( fig .
, the 28 patients are stratified at the median baseline egfrcr - cys ( 105 ml / min in fig .
two features deserve emphasis . first , the majority of trajectories of egfrcr - cys loss were approximately linear .
second , the slopes of egfrcr - cys loss varied tremendously , ranging from a loss of 3.3 to 21% per year . the fastest decliners , including the two already mentioned , will reach esrd within 515 years , and the slowest might take 2530 years .
the pattern of egfrcr - cys trajectories in 86 decliners with ma was similar ; i.e. , the majority of trajectories were linear and four patients rapidly progressed to esrd within the duration of follow - up ( data not shown due to large number of decliners ) .
egfrcr - cys trajectories in t1d patients with na and progressive renal decline ( loss 3.3% per year ) during 410 years of follow - up .
the trajectories are plotted in patients with baseline egfrcr - cyst 105 ml / min ( a ) and in patients with baseline egfrcr - cys < 105 ml / min ( b ) .
, we compared nondecliners with decliners with regard to baseline clinical covariates and serum concentrations of 12 markers separately in patients with na and ma ( table 2 ) .
the values of egfrcr - cys at last follow - up were similar to baseline for nondecliners in both groups . for decliners , however , the last values were lower ( by definition ) than baseline , down to 61 from 104 ml / min in na and to 59 from 99 ml / min in ma . the median rate of egfrcr - cys loss per year in decliners was 5.0% per year in both groups .
renal function at baseline in patients with na was normal in nondecliners but was already lower in decliners , most likely because the onset of renal decline preceded the enrollment in the study .
similarly in patients with ma , renal function at baseline was normal in nondecliners ( despite the presence of ma ) but was already lower in decliners for the same reason .
characteristics of nondecliners and decliners according to study group during the baseline interval , aer was identical in decliners and nondecliners in the na group , but ma developed in three times as many decliners ( 54% ) as nondecliners ( 17% ) during the 4 years of follow - up .
in patients with ma already present during the baseline period , progression to macroalbuminuria was much more common in decliners ( 27% ) than nondecliners ( 4% ) during the 4 years of follow - up .
by contrast , aer regression was twice as common in nondecliners ( 43% ) as decliners ( 23% ) . thus , aer was more likely to progress in patients with renal decline and more likely to be transient in nondecliners
. with regard to other baseline characteristics , decliners and nondecliners did not differ in either group with regard to sex , insulin dose , bmi , serum cholesterol ( total or hdl ) , as well as cigarette smoking ( data not shown ) .
significant differences between decliners and nondecliners are indicated in the bottom part of table 2 . in both study groups ,
age at entry into the study was older and duration of diabetes was longer for decliners than nondecliners .
in addition , the decliners had poorer glycemic control and higher systolic blood pressure and were treated more frequently with renoprotective drugs than nondecliners . in both study groups , decliners had significantly higher serum concentrations of uric acid , tnfr-1 , and tnfr-2 than nondecliners .
serum markers such as free tnf- , il-6 , il-8 , ip-10 , mcp-1 , icam , vcam , fas , and fasl were not different between decliners and nondecliners ( data not shown ) . to visualize these associations , in table 2
we graphed the risk of renal decline separately by study group according to categories of baseline characteristics that were associated with renal decline in the case - control analysis ( fig .
2a , top ) was flat ( 10% ) across quartiles in the na group . in ma ,
the risk of renal function decline was significantly higher than in na and increased from 23% in the lowest quartile to 55% in the highest . for hba1c ,
the increase in risk was moderate in the na group but strong in the ma group ( fig .
2a ) and increased similarly with duration of t1d because of its correlation with age ( data not shown ) .
plots of the risk of renal decline according to three serum markers are shown in fig .
2b . in both study groups , elevated serum uric acid increased the risk of renal decline , and the effect of an increasing serum tnfr-1 or tnfr-2 was even more striking .
risk of progressive renal decline according to categories of baseline clinical characteristics ( a ) and serum markers ( b ) and according to study groups . cut points for 25th , 50th , and 75th percentiles were 1,173 , 1,394 , and 1,685 pg / ml ; cut points for 25th , 50th , and 75th percentiles were 1,810 , 2,186 , and 2,690 pg / ml . determinants of renal decline that were significant in table 2 and fig .
2 were examined with multiple logistic regression analyses . in separate analyses of the na and ma groups , the coefficients for these determinants were similar so we combined the groups and included an indicator variable for ma as one of the determinants ( table 3 ) .
2a ) than with duration , so only age was included in the model . similarly ,
we only included blood pressure in the model because the intensity of treatment was most likely secondary to the blood pressure , not the reverse . in a cross - tabulation of patients according to both serum uric acid and tnfr-1 ,
the risk of renal decline increased with each one alone , but their effects were not additive .
this is reflected in the significant odds ratio ( or ) but smaller than 1.00 for their interaction term in the multiple logistic model ( table 3 ) .
for example , at the 25th percentile of tnfr-1 ( 1,170 pg / ml ) , the or for the 75th vs. 25th percentiles of uric acid is 3.72 , whereas at the 75th percentile of tnfr-1 ( 1690 pg / ml ) , it is 1.58 .
the relationship is symmetric . at the 25th percentile of uric acid ( 3.8 mg / dl ) ,
the or for the 75th vs. 25th percentiles of tnfr-1 is 4.68 , whereas at the 75th percentile of uric acid ( 6.4 mg / dl ) , it is 1.99 .
there were 304 patients with ma and 364 patients with na enrolled in the 2nd jks .
this study included those with normal renal function at baseline ( egfrcr - cys > 60 ml / min ) who were followed for 410 years : 248 ( 82% ) patients with ma and 286 ( 79% ) with na .
characteristics of the study groups are summarized in table 1 . in comparison with the na group ,
the ma group included more males ; had a longer duration of diabetes , higher hba1c , and higher blood pressure ; and was more frequently treated with renoprotective drugs . whereas the distribution of aer in the na group was uniform across the normal range ( < 30 g / min ) , the bulk of the distribution in the ma group was in the low abnormal range ( 25th , 50th , and 75th percentiles were 44 , 65 , and 116 g / min , respectively ) .
the distributions of baseline concentrations of serum creatinine and cystatin c and the estimates of egfrcr - cys were similar in na and ma .
characteristics of the study groups duration of follow - up and the available number of measurements of serum creatinine and cystatin c were similar in the two study groups
. the annual rate of egfrcr - cys change was estimated from these serial measurements .
the median rate of loss in patients with ma was more rapid than in patients with na .
renal decline ( rate of egfrcr - cys loss 3.3% per year ) was present in 10% of the na group and 35% of the ma group .
impaired renal function ( ckd - epi 3 ) developed in 6% of the na group and 22% of the ma group , and esrd developed in one of the former and four of the latter .
the trajectories of renal function changes in the 28 decliners with na have particular significance because they have not been documented before ( fig .
, the 28 patients are stratified at the median baseline egfrcr - cys ( 105 ml / min in fig .
two features deserve emphasis . first , the majority of trajectories of egfrcr - cys loss were approximately linear .
second , the slopes of egfrcr - cys loss varied tremendously , ranging from a loss of 3.3 to 21% per year . the fastest decliners , including the two already mentioned , will reach esrd within 515 years , and the slowest might take 2530 years .
the pattern of egfrcr - cys trajectories in 86 decliners with ma was similar ; i.e. , the majority of trajectories were linear and four patients rapidly progressed to esrd within the duration of follow - up ( data not shown due to large number of decliners ) .
egfrcr - cys trajectories in t1d patients with na and progressive renal decline ( loss 3.3% per year ) during 410 years of follow - up .
the trajectories are plotted in patients with baseline egfrcr - cyst 105 ml / min ( a ) and in patients with baseline egfrcr - cys < 105 ml / min ( b ) .
to find determinants of renal decline , we compared nondecliners with decliners with regard to baseline clinical covariates and serum concentrations of 12 markers separately in patients with na and ma ( table 2 ) .
the values of egfrcr - cys at last follow - up were similar to baseline for nondecliners in both groups . for decliners , however , the last values were lower ( by definition ) than baseline , down to 61 from 104 ml / min in na and to 59 from 99 ml / min in ma . the median rate of egfrcr - cys loss per year in decliners was 5.0% per year in both groups .
renal function at baseline in patients with na was normal in nondecliners but was already lower in decliners , most likely because the onset of renal decline preceded the enrollment in the study . similarly in patients with ma , renal function at baseline was normal in nondecliners ( despite the presence of ma ) but was already lower in decliners for the same reason .
characteristics of nondecliners and decliners according to study group during the baseline interval , aer was identical in decliners and nondecliners in the na group , but ma developed in three times as many decliners ( 54% ) as nondecliners ( 17% ) during the 4 years of follow - up . in patients with ma already present during the baseline period , progression to macroalbuminuria
was much more common in decliners ( 27% ) than nondecliners ( 4% ) during the 4 years of follow - up .
by contrast , aer regression was twice as common in nondecliners ( 43% ) as decliners ( 23% ) .
thus , aer was more likely to progress in patients with renal decline and more likely to be transient in nondecliners .
with regard to other baseline characteristics , decliners and nondecliners did not differ in either group with regard to sex , insulin dose , bmi , serum cholesterol ( total or hdl ) , as well as cigarette smoking ( data not shown ) .
significant differences between decliners and nondecliners are indicated in the bottom part of table 2 . in both study groups ,
age at entry into the study was older and duration of diabetes was longer for decliners than nondecliners .
in addition , the decliners had poorer glycemic control and higher systolic blood pressure and were treated more frequently with renoprotective drugs than nondecliners . in both study groups , decliners had significantly higher serum concentrations of uric acid , tnfr-1 , and tnfr-2 than nondecliners .
serum markers such as free tnf- , il-6 , il-8 , ip-10 , mcp-1 , icam , vcam , fas , and fasl were not different between decliners and nondecliners ( data not shown ) . to visualize these associations , in table 2
we graphed the risk of renal decline separately by study group according to categories of baseline characteristics that were associated with renal decline in the case - control analysis ( fig .
2a , top ) was flat ( 10% ) across quartiles in the na group . in ma ,
the risk of renal function decline was significantly higher than in na and increased from 23% in the lowest quartile to 55% in the highest . for hba1c ,
the increase in risk was moderate in the na group but strong in the ma group ( fig .
2a ) and increased similarly with duration of t1d because of its correlation with age ( data not shown ) .
plots of the risk of renal decline according to three serum markers are shown in fig .
2b . in both study groups , elevated serum uric acid increased the risk of renal decline , and the effect of an increasing serum tnfr-1 or tnfr-2 was even more striking .
risk of progressive renal decline according to categories of baseline clinical characteristics ( a ) and serum markers ( b ) and according to study groups . cut points for 25th , 50th , and 75th percentiles were 1,173 , 1,394 , and 1,685 pg / ml ; cut points for 25th , 50th , and 75th percentiles were 1,810 , 2,186 , and 2,690 pg / ml .
determinants of renal decline that were significant in table 2 and fig . 2 were examined with multiple logistic regression analyses . in separate analyses of the na and ma groups , the coefficients for these determinants were similar so we combined the groups and included an indicator variable for ma as one of the determinants ( table 3 ) .
we only included blood pressure in the model because the intensity of treatment was most likely secondary to the blood pressure , not the reverse . in a cross - tabulation of patients according to both serum uric acid and tnfr-1
, the risk of renal decline increased with each one alone , but their effects were not additive .
this is reflected in the significant odds ratio ( or ) but smaller than 1.00 for their interaction term in the multiple logistic model ( table 3 ) .
for example , at the 25th percentile of tnfr-1 ( 1,170 pg / ml ) , the or for the 75th vs. 25th percentiles of uric acid is 3.72 , whereas at the 75th percentile of tnfr-1 ( 1690 pg / ml ) , it is 1.58 .
the relationship is symmetric . at the 25th percentile of uric acid ( 3.8 mg / dl ) ,
the or for the 75th vs. 25th percentiles of tnfr-1 is 4.68 , whereas at the 75th percentile of uric acid ( 6.4 mg / dl ) , it is 1.99 .
in this large follow - up study , we showed that progressive renal decline in t1d began during na and , similar to ma , it was determined by multiple factors .
we conclude that this early progressive renal decline can be considered the primary clinical manifestation of disease process leading to impaired renal function and eventually to esrd in t1d .
changes in aer observed during follow - up , such as the onset of ma or its progression to macroalbuminuria , were either caused by or developed in parallel to the early renal decline in t1d . in our study of t1d patients with na , progressive renal decline developed in 10% ( decliners ) within 410 years of follow - up , whereas only 2.5% was expected if these patients had been nondiabetics .
the risk of renal decline did not vary within the normal range of aer in na but increased with increasing aer in the ma range .
the rate of egfrcr - cys loss was continuous ( progressive decline ) in the majority of decliners and could be represented as a linear slope .
for example , in the study , egfrcr - cys loss was so rapid in five patients ( one with na and four with ma ) that they progressed from normal renal function to esrd within the 510 years of observation , whereas the egfrcr - cys loss in other decliners was slow so that progression to esrd might take up to 30 years .
these findings are similar to the renal decline patterns we observed in t1d patients with macroalbuminuria ( 12 ) .
progressive renal decline was present in a significant proportion of t1d patients when their urinary albumin excretion was normal , and it increased the risk of onset of ma several fold during follow - up in comparison with nondecliners ( 51 vs. 17% ) .
this finding provides a very strong evidence that early progressive renal decline is the primary event in the development of diabetic nephropathy .
furthermore , once early progressive renal decline and ma were both present , the risk of progression to macroalbuminuria during follow - up was several fold higher in comparison with nondecliners with ma ( 28 vs. 4% ) .
conversely , the frequency of regression of ma in nondecliners was twice that in decliners .
thus , the abnormal aer that occurred in the presence of early progressive renal decline was more likely to be progressive , whereas the abnormal excretion that occurred in nondecliners was more likely to be transient .
these findings are not consistent with the generally assumed model of diabetic nephropathy in which renal decline is a consequence of or follows macroalbuminuria ( 1 ) . instead , our findings indicate that either both are loosely correlated phenotypes of a disease process that underlies diabetic nephropathy and leads to esrd or that early progressive renal decline is one of the causes of albuminuria .
the former interpretation of our findings explains reports that showed that albuminuria and renal decline are uncoupled phenotypes in t1d ( 2,13,14 ) and t2d ( 15 ) .
recognition that multiple clinical factors contribute in a similar way to renal decline in na and ma strengthens our hypothesis that progressive renal decline is the primary clinical abnormality of diabetic nephropathy .
as mentioned earlier , the effect of renal decline on the risk of the onset of ma and its progression to macroalbuminuria may account in large degree for a higher occurrence of renal decline in our collection of patients ascertained for the presence of ma .
in other words , early renal decline leads to ma , so patients with ma should be enriched with those at risk for renal decline .
the effect of hba1c on renal decline was recently recognized ( 16 ) . in our study , the effect of hba1c on risk of early renal decline was strong . however , the mechanisms through which hyperglycemia contributes to early progressive renal decline independently from aer abnormalities are unknown
. the effect of increasing age on renal decline is recognized in nondiabetic individuals > 40 years of age .
the gfr loss of > 3.3% per year ( about 4 ml / min / year ) occurs in < 3% of the healthy population ( 11 ) .
it is unknown why such gfr loss occurred almost 515 times more frequently in t1d patients 40 years of age or more . in part , this effect was due to a long duration of diabetes .
since these two covariates were closely correlated , it was impossible to separate their effects in this study .
the risk of early progressive renal decline increased with increasing systolic blood pressure , a well - documented risk factor for progression to esrd ( 17 ) .
antihypertensive and renoprotective drugs were prescribed in patients with elevated blood pressure and ma . in univariate analysis , both elevated systolic blood pressure and treatment with these drugs increased the risk of early progressive renal decline .
however , in multivariate analysis , the effect of the latter declined significantly , indicating that antihypertensive and renoprotective treatment had an inconsequential effect on the risk of early progressive renal decline .
similar findings were recently reported in the renin - angiotensin system study ( rass ) clinical trial ( 18 ) .
of the 12 serum markers previously examined in cross - sectional analyses of this cohort ( 68 ) , only the concentrations of uric acid , tnfr-1 , and tnfr-2 significantly contributed to renal decline .
the effects of these markers on the risk of renal decline were equally strong in patients with na or ma .
the congruence of these determinants in ma and na is further evidence for our hypothesis that early progressive renal function is the primary disease process underlying the development of renal decline in t1d and leads to esrd .
the mechanisms underlying the association between high normal values of serum uric acid and increased risk of renal decline are unclear at this time .
serum uric acid has proinflammatory properties and may act as either a pro - oxidant or antioxidant molecule depending on the circumstances ( 19 ) . in vitro , exposure to uric acid leads to upregulation of cyclooxygenase-2 , specific mitogen - activated protein kinases , various inflammatory mediators , and the renin - angiotensin system , as well as to inhibition of endothelial nitric oxide production ( 2022 ) , which may all contribute to the development of kidney damage . in vivo , rats rendered hyperuricemic by means of a uricase inhibitor
develop an afferent arteriolopathy that decreases luminal diameter and produces renal ischemia , leading to glomerulosclerosis and tubulointerstitial fibrosis ( 23 ) .
lowering serum uric acid concentration with allopurinol attenuates these histological and functional changes , although this effect may be due in part to reduced oxidative stress resulting from xanthine - oxidase inhibition ( 19 ) .
it is intriguing to postulate that lowering serum uric acid in patients at risk for renal decline may be an effective intervention to reduce the risk of renal decline in t1d ( 25 ) .
our previous studies described the strong effect of serum concentrations of tnfr-1 or tnfr-2 on the risk of advanced stages of renal decline such as ckd - epi 33 or esrd ( 7,27 ) .
this study extends the demonstration of their effects to the onset of the process of renal decline itself .
we do not know how elevated concentrations of tnfrs initiate renal decline and lead to renal failure .
for example we showed that serum tnf- is not involved directly or indirectly , through regulation of serum tnfrs , in the development of renal decline .
furthermore , we excluded the role of several circulating adhesion molecules and chemokines ( vcam , icam , il-6 , il-8 , ip-10 , and mcp-1 ) in the etiology of renal decline as potential downstream effectors of tnfrs ( 8) .
an intriguing finding of the current study is the negative interaction between serum uric acid and tnfr-1 on the risk of renal decline in both na and ma , meaning that the risk of renal decline for patients with elevated serum uric acid and serum tnfr-1 is less than the sum of the individual risks associated with the two predictors .
this suggests that the predisposing effects of serum uric acid and tnfr-1 converge on some common pathway that can not be further activated by one factor if it has already been turned on by the other .
the egfrcr - cys equation has not been validated in t1d , and this type of egfr assessment may significantly underestimate hyperfiltration in t1d , therefore reducing the steepness of egfr slopes and underestimating the true frequency of early progressive renal decline .
second , our study is descriptive and hypothesis generating , and it can not directly shed any light on the mechanisms of early progressive renal decline in t1d .
however , it provides novel data that question many assumptions about the natural history of diabetic nephropathy in t1d .
third , this study was conducted in t1d and the generalizability of the findings to t2d is uncertain .
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objectiveprogressive decrease in the glomerular filtration rate ( gfr ) , or renal decline , in type 1 diabetes ( t1d ) is observed in patients with macroalbuminuria .
however , it is unknown whether this decline begins during microalbuminuria ( ma ) or normoalbuminuria ( na).research design and methodsthe study group ( second joslin kidney study ) comprises patients with t1d and na ( n = 286 ) or ma ( n = 248 ) who were followed for 410 years ( median 8 years ) . serial measurements ( median 6 , range 316 ) of serum creatinine and cystatin c were used jointly to estimate gfr ( egfrcr - cys ) and assess its trajectories during follow-up.resultsrenal decline ( progressive egfrcr - cys loss of at least 3.3% per year ) occurred in 10% of the na and 35% of the ma ( p < 0.001 ) . in both groups , the strongest determinants of renal decline were baseline serum concentrations of uric acid ( p < 0.001 ) and tumor necrosis factor receptor 1 or 2 ( tnfr-1 or -2 , p < 0.001 ) .
other significant risk factors included baseline hba1c , age / diabetes duration , and systolic blood pressure .
relative impacts of these determinants were similar in na and ma .
renal decline was not associated with sex or baseline serum concentration of tnf- , il-6 , il-8 , ip-10 , mcp-1 , vcam , icam , fas , or fasl.conclusionsrenal decline in t1d begins during na and it is determined by multiple factors , similar to ma .
thus , this early decline is the primary disease process leading to impaired renal function in t1d .
changes in albumin excretion rate , such as the onset of ma or its progression to macroalbuminuria , are either caused by or develop in parallel to the early renal decline .
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acute kidney injury ( aki ) is a well - known complication of cancer [ 1 , 2 ] .
etiology of aki in cancer varies from acute tubular necrosis due to medication or sepsis , volume depletion , tumor lysis syndrome , and obstruction or infiltration of the kidneys .
presence of aki leads to longer hospitalization and increased cost [ 3 , 4 ] .
it also increases mortality in patients with cancer [ 1 , 2 ] , delays treatment , and increases toxicity of chemotherapy . like all cancers
lymphoma patients are prone to develop aki due to acute tubular necrosis secondary to sepsis , nephrotoxic medications , and contrast studies .
lymphomatous infiltration of kidneys [ 5 , 6 ] , light chain proximal tubulopathy , membranous nephritis , membranoproliferative glomerulonephritis , fanconi syndrome , and tumor lysis syndrome [ 1113 ] have all been reported in the literature .
this is the first study done on aki in lymphoma patients and aims to determine frequency and predictors of aki in lymphoma and to study its impact on hospital stay and mortality .
this is a retrospective case series study conducted at the aga khan university hospital karachi , pakistan .
aga khan university hospital is a tertiary care health facility located in the largest city of pakistan , karachi , which has a population of around 15 million .
they were identified from a central computerized record for a period of 4 years from january 2008 to december 2011 with the help of the department of health information management system .
records of the cases were retrieved through codes using international classification of diseases 9th revision clinical modification ( icd-9 cm ) 201.90201.98 for hodgkin lymphoma and 202.80202.88 for non - hodgkin lymphoma .
study subjects included patients aged 14 years admitted with the primary diagnosis of lymphoma , irrespective of type or stage of disease . patients with chronic kidney disease were excluded .
patients were divided into two cohorts ( those with and those without aki ) in order to determine independent predictors of aki .
similarly , length of stay and mortality inside hospital were also compared in these two cohorts .
data on demographics , clinical features , laboratory data , length of stay , recovery of renal functions , and mortality were noted .
we used the acute kidney injury network ( akin ) definition for classification of aki stages .
aki stage 1 was defined as an increase in serum creatinine > 26.5 mole / l ( 0.3 mg / dl ) or 1.5- to 2-fold increase from baseline .
aki stage 2 was defined as an increase in serum creatinine of > 2 - 3-fold from baseline .
aki stage 3was defined as an increase in serum creatinine of > 3-fold from baseline or an absolute serum creatinine of > 354 mol / l ( 4 mg / dl ) .
sepsis was defined as suspected or confirmed infection with the presence of two of the following : temperature > 38c or < 36.5c;heart rate > 90 per minute;respiratory rate > 30 per minute;leukocyte count > 11000 or < 4000 .
temperature > 38c or < 36.5c ; heart rate > 90 per minute ; respiratory rate > 30 per minute ; leukocyte count > 11000 or < 4000 . tumor lysis syndrome ( cairo - bishop definition ) was defined as 2 of the following : uric acid > 8 mg / dl or 25% increase from the baseline;potassium > 6 mmol / l or 25 % increase from the base line;phosphate > 4.5 mg / dl;calcium < 7 mg / dl or 25% decrease from the baseline 3 days before or 7 days afterward .
uric acid > 8 mg / dl or 25% increase from the baseline ; potassium > 6 mmol / l or 25 % increase from the base line ; phosphate > 4.5 mg / dl ; calcium < 7 mg / dl or 25% decrease from the baseline 3 days before or 7 days afterward .
quantitative data were expressed as mean , median , interquartile range and standard deviation ( sd ) and number of observations with percentage ( % ) , respectively .
evaluation of association between outcomes ( aki and without aki group , length of hospital stay ) and potential causative factors were used with -test or fisher 's exact tests of independence and student 's t - test .
odds ratios ( or ) and their 95% confidence intervals ( ci ) were estimated using binary logistic regression , with aki and length of hospital stay ( 7 versus > 7 days ) as outcomes .
multivariable models were constructed , including variables that showed an effect on the prediction of aki and length of hospital stay > 7 days in the univariate analysis .
all p values were based on two - sided tests and significance was set at a p value < 0.05 .
one hundred and sixteen ( 31.8% ) patients had aki . among patients with aki , 78 ( 67.2% )
had akin stage 1 , 25 ( 21.6% ) had akin stage 2 , and 13 ( 11.2% ) had akin stage 3 .
sepsis was found in 64 ( 17.5% ) and tumor lysis in 28 ( 7.7% ) of the patients .
furosemide , vancomycin , amphotericin , and aminoglycoside were used in 40 ( 11% ) , 34 ( 9.31% ) , 16 ( 4.4% ) , and 14 ( 3.8% ) patients , respectively .
various chemotherapy regimen used included r - chop ( rituximab - cyclophosphamide , hydroxy daunorubicin , oncovin , and prednisolone ) , chop ( cyclophosphamide , hydroxy daunorubicin , oncovin , prednisolone ) , r - cvp ( rituximab - cyclophosphamide , vincristine , and prednisolone ) , cvp ( cyclophosphamide , vincristine , and prednisolone ) , abvd ( adriamycin , bleomycin , vinblastine , and dacarbazine ) and dhap ( dexamethasone , high dose ara c , and cisplatin ) .
r - chop , chop , and abvd were the main regimens used in 94 , 86 , and 52 patients , respectively .
univariate analysis revealed increased age ( p 0.005 ) , diuretics ( p < 0.001 ) , vancomycin ( p 0.02 ) , aminoglycoside ( p < 0.001 ) , r - cvp ( p 0.004 ) , dhap ( p 0.009 ) , sepsis ( p < 0.001 ) , and tumor lysis ( p <
however , multivariate logistic regression analysis showed that independent predictors for aki were sepsis ( or 3.76 ; 95% ci 1.837.72 ) , aminoglycosides ( or 4.75 ; 95% ci 1.1519.52 ) , tumor lysis syndrome ( or 3.85 ; 95% ci 1.549.59 ) , and r - cvp regimen ( or 4.70 ; 95% ci 1.2018.36 ) ( tables 2 and 3 ) . patients with aki stages 2 and 3 had statistically increased hospital stay of greater than 7 days when compared to those without aki ( or 2.01 ; 95% ci 1.193.40 ) .
mortality was also high in patients with aki ( 14.7% ) when compared to those without aki ( 6.8% ) ( p 0.03 ) ( table 4 ) .
patients with aki who died were significantly associated with tumor lysis ( p 0.001 ) , need of mechanical ventilation ( p < 0.001 ) , diuretics ( p 0.001 ) , and sepsis ( < 0.001 ) ( table 5 ) .
around 40 patients ( 34.48% ) had persisting aki at discharge but did not need dialysis .
however 14 ( 12.06% ) patients who needed dialysis were still requiring dialysis at the time of discharge ( table 6 ) .
aki is a well - known complication of cancer . however , very little is known about aki in lymphoma . to the best of our knowledge ,
the incidence of aki in various cancers has been reported to be between 12 and 49% [ 1519 ] .
our study incidence is higher than that of christiansen et al . , who reported incidence of aki ( defined as 50% elevation of baseline serum creatinine ) in 29.39% lymphoma patients .
the discrepancy could be explained by the noninclusion of milder aki patients in their study .
our multivariate analysis identified sepsis , aminoglycoside usage , diuretics usage , tumor lysis syndrome , and r - cvp usage as potential negative predictors .
cancer patients may be immunocompromised due to multiple factors such as chemotherapy , radiotherapy , impairment of normal leukocyte function , or use of corticosteroids [ 21 , 22 ] .
the dysregulation of their immune system predisposes patients to sepsis which can lead to unregulated cytokine release and haemodynamic disturbances .
aki usually ensues as a result of alterations in renal perfusion resulting from pro - inflammatory insults .
diuretics usage may have a direct role in causing kidney injury by exacerbating underlying hypovolemic and haemodynamic state .
it can also be argued that patients requiring diuretic therapy are likely to have compromised heart or renal function at baseline and , therefore , more likely to develop and sustain kidney injury . on the other hand ,
it is possible that diuretic therapy was initiated to maintain or increase urine output in patients who may already have aki .
this could be related to its direct nephrotoxic effects or through synergistic nephrotoxic activities with chemotherapy .
the presence of sepsis which may have necessitated the usage of aminoglycoside may also have contributed to the risk of aki .
various studies have reported spontaneous tumor lysis syndrome in leukemia and lymphoma [ 11 , 2326 ] .
hande kjellstrand and his colleagues found tumor lysis in 42% of the patients with lymphoma , which is higher than our result .
however , unlike our study hande kjellstrand et al . included only high grade lymphoma which has a greater propensity for the development of tumor lysis syndrome .
high tumor burden in stages iii and iv follicular lymphoma with potential of tumor lysis in combination with cyclophosphamide nephrotoxicity might be a reason for this .
alternatively , combination of cyclophosphamide with vincristine may be more nephrotoxic , which needs further exploration through future studies .
another study reported six - month mortality of 73% in critically ill patients with cancer and aki .
we in our study found mortality of 14.7% in patients with lymphoma complicated with aki as compared to 6.8% in those without aki .
similarly , presence of aki stages 2 and 3 in patients with lymphoma had more propensity for a prolonged hospital stay .
we speculate that tumor lysis syndrome , concurrent sepsis with superimposed use of aminoglycosides , and diuretics use make lymphoma patients prone to develop aki .
therefore , patients with high grade lymphoma should be cautiously watched during their chemotherapy as they can potentially deteriorate by developing tumor lysis and subsequent aki .
similarly , sepsis should be vigorously looked for and treated appropriately . while managing gram negative sepsis broad spectrum , antibiotics other than aminoglycosides should be chosen if possible .
patients with sepsis and tumor lysis are often volume depleted and need aggressive fluid resuscitation .
therefore , diuretics use should be judiciously used only in the presence of evident fluid overload .
this is a retrospective study based on data from a single centre . due to the retrospective nature of this study , etiology of aki
similarly , due to lack of long - term followup of the patients , long - term impact of aki on lymphoma patients could not be determined .
multicentre prospective studies with long - term followup are needed to analyze etiology of aki , its long - term outcome , and impact on mortality and morbidity .
similarly , there is a need to determine least nephrotoxic chemotherapy regimen through prospective analysis .
judging by the results of this study , we propose the following management considerations for high - risk lymphoma patients : careful attention to haemodynamics and volume status;avoidance of specific drugs;avoidance of tumour lysis with prophylactic measures ( e.g. , prophylactic allopurinol / aggressive rehydration , etc.);early identification and treatment of sepsis;choice of chemotherapy;early initiation of rrt ( ?
careful attention to haemodynamics and volume status ; avoidance of specific drugs ; avoidance of tumour lysis with prophylactic measures ( e.g. , prophylactic allopurinol / aggressive rehydration , etc . ) ; early identification and treatment of sepsis ; choice of chemotherapy ; early initiation of rrt ( ?
tumor lysis syndrome , sepsis , and use of aminoglycoside and furosemide were associated with development of aki .
presence of aki stages 2 and 3 resulted in increased mortality and prolonged hospital stay .
we feel that identification of these negative predictors and early intervention can help to prevent or reduce the severity of aki .
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background .
acute kidney injury ( aki ) is a common but least studied complication of lymphoma . objective . to determine the frequency and predictors of aki in lymphoma and to study the impact of aki on hospital stay and mortality
. methods .
retrospective review of medical records of hospitalized lymphoma patients aged 14 years between january 2008 and december 2011 was done . results .
out of 365 patients , aki was present in 31.8% ( 116/365 ) .
multivariate logistic regression analysis showed that independent predictors for aki included sepsis ( odds ratio ( or ) 3.76 ; 95% ci 1.837.72 ) , aminoglycosides ( or 4.75 ; 95% ci 1.1519.52 ) , diuretics ( or 2.96 ; 95% ci 1.316.69 ) , tumor lysis syndrome ( or 3.85 ; 95% ci 1.549.59 ) , and r - cvp regimen ( or 4.70 ; 95% ci 1.2018.36 ) .
aki stages 2 and 3 was associated with increased hospital stay ( or 2.01 ; 95% ci 1.193.40 ) .
conclusion .
aki was significantly associated with sepsis , aminoglycoside , diuretics , presence of tumor lysis syndrome , and use of r - cvp regimen .
presence of akin ( acute kidney injury network ) stages 2 and 3 aki had increased hospital stay .
aki was also associated with increased mortality .
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infectious endophthalmitis is a devastating vision - threatening condition consisting of inflammation of the entire globe and its intraocular contents .
surgical intervention , such as pars plana vitrectomy ( ppv ) , has been used for cases with infectious endophthalmitis whose visual acuity is worse than light perception after cataract surgery .
ppv for infectious endophthalmitis is challenging given the severe inflammation in the anterior chamber which creates significant media opacity and limits visualization of the posterior pole .
limbal based anterior vitrectomy is sometimes used by our anterior segment colleagues in cases of posterior capsule rupture and vitreous prolapse . additionally , pediatric vitreoretinal cases involving vitreous hemorrhages , retina detachment , and opacified vitreous with uveitis benefit from iimbal vitrectomy .
this surgical technique shows good efficacy and safety that is designed to avoid disruption of the vitreous base and the anterior retina . here , we describe a surgical technique combining limbal based vitrectomy and ppv to manage infectious endophthalmitis .
the limbal based vitrectomy is used to initially remove infectious and inflammatory material from the anterior chamber .
it is an ideal technique for visualization of the posterior pole for more complete ppv as an early intervention for infectious endophthalmitis .
our surgical technique includes a five - trocar setup using 3 standard 25-gauge pars plana trocar / cannulas and 2 limbal anterior trocar / cannulas .
undiluted aqueous and vitreous biopsy can be obtained prior to turning the infusion on , given the multiple trocar availability .
the anterior infusion is initially turned on once it is verified in the anterior chamber via the corneal limbus .
the second anterior corneal limbus cannula is used for anterior chamber washout and removal of the fibrin and inflammatory membranes .
both the vitreous cutter and retinal forceps can be used to remove fibrin membranes from the pupil margins in aphakic cases and over the intraocular lens in pseudophakic cases . after all anterior chamber material is cleared and media clarity optimized
the infusion line of a balanced saline solution is moved to the pars plana cannula and a complete posterior vitrectomy is performed . in cases where the media opacity remains significant ,
the posterior vitrectomy can be performed with the anterior infusion line . at the conclusion of the case
, the small gauge limbus wounds can be hydrated or closed with single interrupted 100-nylon sutures .
any leaking sclerotomies should be closed in a similar manner with 70-vicryl suture ( online suppl .
patients with infectious endophthalmitis who undergo early vitrectomy might have superior outcomes with fewer complications [ 3 , 4 , 5 , 6 ] .
however , early vitrectomy for infectious endophthalmitis might be challenging due to difficult visualization secondary to media opacity secondary to severe inflammation in the anterior chamber .
our surgical technique of five - port combined limbal and ppv for infectious endophthalmitis allows for removal of infectious and inflammatory material from both the anterior chamber and vitreous cavity in a safe and efficient manner .
the limbal based approach greatly aids visualization and potentially improves the efficacy of the ppv in cases where media opacity precludes securing a posterior segment infusion .
this is significantly beneficial at removing infectious material and vitreous debris , which is paramount to accelerating infection clearance and optimizing visual outcomes .
the 25-gauge trocar / cannula limbal system provides a small - gauge easy access port in the anterior chamber with good efficacy and safety .
similar to the 23-gauge limbal approach , no intraoperative or postoperative complications were observed , such as corneal wound dehiscence , descemet membrane tears , iris incarceration , epithelial downgrowth , or corneal dellen , retinal tear or dialysis , retinal detachment , suprachoroidal hemorrhage or effusion . the smaller gauge cannulas ( with or without valves ) often allow for the entire procedure to be sutureless .
if the cornea / limbal wounds are not water tight at the end of the case , they can be hydrated with a balanced saline solution on a blunt cannula . in cases where a pars plana infusion still can not be visualized and verified to be in the vitreous cavity , posterior vitrectomy can be performed with the limbal ( anterior ) infusion cannula .
the anterior infusion line provides adequate intraocular pressure control regardless of lens status ; however , it may cause corneal striae that could degrade the view of the posterior pole .
this can be minimized by ensuring that the infusion line is taped in a radial manner ( i.e. perpendicular to the circumferential cornea ) . when the view is sufficient posteriorly , we elect to move the infusion source to the pars plana cannula as soon as able .
collectively , this surgical technique allows for safe , efficient , and complete anterior and posterior vitrectomy for infectious endophthalmitis .
this case report was in full adherence to the declaration of helsinki and all federal and state laws .
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pars plana vitrectomy for acute infectious endophthalmitis can be challenging due to severe inflammation in the anterior chamber creating significant media opacity .
we describe a surgical technique combining limbal based vitrectomy and pars plana vitrectomy to manage acute infectious endophthalmitis .
limbal based vitrectomy facilitates removal of anterior chamber fibrin and inflammatory membranes for safe and optimal posterior pars plana vitrectomy .
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maternal undernutrition around the time of conception in sheep alters fetal metabolic and endocrine function and growth trajectory but does not alter birth weight .
postnatally , growth regulation is altered , and changes in endocrine function become apparent with increasing age , including suppressed hypothalamic - pituitary - adrenal ( hpa ) and leptin - insulin axis function and impaired glucose tolerance .
one possible mechanism that might contribute to this postnatal metabolic risk after periconceptional undernutrition is altered body composition , for example , if the early nutritional insult also resulted in increased relative fat mass and/or decreased lean mass . in humans , normal weight obesity ( increased - percentage body fat despite a normal - range body mass index ) has been associated with a four fold increased risk of developing metabolic disease . however , it is not clear whether increased fat mass is predominantly due to pre- or postnatal events or the interaction between them .
a number of studies suggest that rapid postnatal growth particularly after in utero growth restriction , occurring because of a mismatch of nutrient availability before and after birth , increases relative fat mass thereby conferring an increased risk of later metabolic disease [ 911 ] .
however , periconceptional undernutrition in sheep alters growth regulation and trajectory without overt in utero growth restriction .
further , the normal relationships between nutrient intake , growth factors , and weight gain in the postnatal period are disrupted in offspring of periconceptionally undernourished ewes , but these effects are not associated with increased early growth velocity .
longitudinal experimental animal studies provide one way of determining the relative contributions of periconceptional nutritional insult , and in utero and postnatal growth and development to later metabolic disease risk .
the aim of this study was to determine the independent effects of timing and duration of maternal undernutrition ; growth during different time periods ; offspring sex on later phenotype .
we hypothesised that periconceptional undernutrition would result in sex - specific changes in body composition in adulthood , and that these effects would be mediated through altered postnatal growth patterns .
we measured growth and body composition from birth to midadulthood in male and female singleton offspring of ewes undernourished before , both before and after , or only after conception , and carcass and organ weights at postmortem .
ethical approval for the study was obtained from the university of auckland animal ethics committee .
five - year old romney ewes were randomly allocated to a control group ( n ) or to one of three periconceptionally undernourished groups .
all ewes received the same high - quality complete feed ( camtech nutrition ltd . , hamilton , new zealand ) .
normally nourished controls ( n ) were fed a maintenance ration at 3 - 4% body weight per day .
ewes in the 3 undernourished groups had their feed quantity adjusted on an individual basis to achieve and maintain a weight loss of 1015% body weight , from 61 days before mating to the time of mating ( day 0 ) ( un-61 - 0 ) , from 61 days before to 30 days after mating ( un-61 - 30 ) , or from 2 days before to 30 days after mating ( un-2 - 30 ) ; all were fed thereafter in the same way as the n group .
ewes were housed indoors in a photoperiod - controlled feedlot from 71 days before mating until two weeks after lambing . during the undernutrition period
, they occupied individual pens with individual feed buckets , so that intake could be accurately recorded .
ewes were synchronised for mating using progesterone - releasing devices ( cidr - g , pfizer , auckland , nz ) ; four rams were used across all mating groups .
un ewes were kept in individual pens during the undernutrition period and then returned to group pens with the control animals until three weeks before expected lambing , when all ewes were moved to individual pens , with visual and limited olfactory contact with other ewes at all times .
lambs were weighed and measured weekly to 12-weeks ( weaning ) and then intermittently to 3 - 4 years of age .
growth velocity was calculated using an exponential method . at 3 - 4 years of age , body composition
was assessed by dual - emission x - ray absorptiometry ( dxa , norland xr-800 , cooper surgical ltd . , fort atkinson , wi , usa ) .
scans were performed under sedation using an equivolume mixture of diazepam 5 mgml and ketamine 100 mgml intravenously .
fat and lean mass were calculated using norland software in an area defined by the thoracic inlet proximally and the base of the tail distally , and from the animal 's back superiorly to the base of the humerus and femur inferiorly , and expressed as a percentage of body weight .
postmortem examinations were carried out shortly after dxa scanning ( 3 - 4 years of age ) , after pentobarbitone euthanasia .
body measurements included live and eviscerated weight , measurements of linear growth ( including shoulder height , hind limb length , and body length from the atlas to the base of the tail ) , and chest and abdominal circumferences .
groups and sexes were compared using anova , and relationships between growth parameters ( birth weight , growth velocity between birth and weaning at 12 weeks of age , and current weight ) and body composition ( lean and fat mass , expressed as a percentage of body weight ) , assessed using regression .
postmortem examinations were performed on 76 animals between 3 and 4 years of age ( table 1 ) .
males were heavier overall than females at birth and in adulthood ( table 1 ) . in males ,
offspring of un-61 - 0 and un-61 - 30 ewes had greater birth weight than offspring of n ewes ( p = 0.02 ) , but growth velocity to weaning ( gv0 - 12 ) was greatest in the n group , so that by 12 weeks , weight was not different among groups . in females , there was no effect of nutritional group on birth weight or postnatal growth velocity . at the time of dxa scanning and postmortem examination
, there was no difference among nutritional groups in live weight of animals of either sex .
adult females in all groups had greater % fat mass and lesser % lean mass than adult males ( table 1 , p < 0.0001 for each comparison ) .
males in all un groups had greater % fat mass ( p = 0.003 , all un groups combined ) and tended to have lesser % lean mass ( p = 0.06 ) than male controls ( n ) ( table 1 ) .
they also had increased perirenal fat at postmortem ( p = 0.008 ) . in females
, there was no difference among groups in adult body composition . at postmortem examination ,
males in all un groups had decreased shoulder height compared with n males ( p = 0.04 ) ; males in un-61 - 0 and un-61 - 30 groups also had shorter body length ( table 1 , p = 0.04 ) .
hind limb length tended to be greater in controls ( p = 0.09 ) , with the difference only significant between n and un-61 - 0 groups ( p = 0.03 ) .
there were no differences among the different un groups , or between un and n groups , in abdominal or chest circumference . in females
adult % fat mass was not related to birth weight in males or females of any group . in males , there was no relationship between adult % fat mass and body weight at 12 weeks or at the time of postmortem .
however , in females , adult % fat mass was positively associated with body weight at 12 weeks ( all females r = 0.13 , p = 0.04 ) , with the effect strongest in the n group ( n r = 0.49 , p = 0.02 ) .
the association between % fat mass and body weight was even stronger at the time of postmortem ( all females r = 0.43 , p < 0.001 ) , so that the heavier female sheep in all nutritional groups were also fatter .
adult % fat mass was not related to growth velocity between birth and weaning ( 12 weeks ) in males or females from any group .
adult % lean mass was not related to birth weight , 12 week weight , or early growth velocity in males or females of any group , but in both sexes it was inversely associated with body weight at postmortem , with the strongest relationship in females and un-61 - 30 males ( all males r = 0.1 , p = 0.09 ; un-61 - 30 males r = 0.56 , p = 0.008 ; all females r = 0.25 , p = 0.002 ) . compared with males , females in all groups had lesser weights , both absolute and relative to body weight , for heart , lungs , kidneys , pancreas , and spleen and greater brain : body weight ratio ( table 2 ) .
liver weight was also lower in females than in males , but liver : body weight ratio was not different between sexes . in males , but not females , all un groups had lesser weight relative to body weight for heart , lungs , spleen , and adrenals ( table 2 ) .
liver weight : body weight was decreased in the un-61 - 0 group compared with the n group .
periconceptional undernutrition affected total gonad weight in animals of both sexes , but in opposite ways , with un males having heavier testes and un females lighter ovaries than n animals ( nutrition x sex interaction , p = 0.006 ) .
periconceptional undernutrition in sheep resulted in sex - specific changes in adult body proportions , body composition and organ size , with increased fat mass , decreased growth of several organs , and increased gonad size in male offspring .
body composition changes occurred within a normal weight range and were independent of birth weight and early growth velocity .
intriguingly , only gonad size was affected in female offspring , but in an opposite way to males , with females exposed to periconceptional undernutrition having smaller ovaries than controls .
it is well established that nutrition is a powerful modulator of fetal development , and that the time around conception is crucial for the development and later function of endocrine and metabolic systems regulating weight , stress hormones , and glucose metabolism [ 1618 ] .
periconceptional undernutrition in sheep has been associated with changes in fetal growth trajectory , hypothalamic - pituitary - adrenal ( hpa ) and somatotropic axis function , disordered postnatal growth regulation , and altered glucose - insulin and hpa axis function in adulthood . here
, we report that despite the relatively small effects on size at birth , postnatal growth trajectory , and final body size , periconceptional undernutrition results in significantly altered body composition in a sexually dimorphic manner .
the fact that all time periods of undernutrition were associated with a similar effect on male body composition suggests a direct effect on the blastocyst or early embryo , rather than on the ovum prior to conception or via an indirect effect mediated by the altered adaptation to pregnancy seen in un-61 - 0 and un-61 - 30 groups . increased fat mass despite normal body mass index ( bmi ) , termed normal weight obesity ( nwo ) , has been increasingly recognised in humans as a risk factor for cardiovascular disease , dyslipidaemia , and the metabolic syndrome [ 8 , 22 ] , with some sex differences in specific risk ; for example , women with nwo are more at risk of cardiovascular mortality .
although the adult literature focuses on recognition of the problem and possible lifestyle interventions to improve outcomes , our data suggest that , in male sheep , increased proportional body fat is most likely a result of prenatal events . in humans
, associations have been found between low birth weight ( reflecting poor intrauterine growth ) and increased adult fat mass , and between higher birth weight and increased lean mass in young adults [ 23 , 24 ] .
this would also support an antenatal influence on later body composition , but not necessarily related to a particular period of gestation .
other periconceptional events include the use of artificial reproductive technologies such as in vitro fertilisation ( ivf ) and twin conception . one study of children born after ivf found perturbed body fat distribution in childhood compared with spontaneously conceived controls , although another found no effect of ivf on body composition . a study in sheep investigating the effect of twin conception on growth and metabolism found increased proportional fat mass in young adulthood in both males and females that were conceived as twins , even if one fetus was ablated early in gestation , again suggesting that events around the time of conception are crucial for normal tissue differentiation .
this suggests that although increased fat mass may mediate some of the metabolic risks of in utero nutritional insult , it may not be causal ; rather , it may be another manifestation of the developmental perturbation resulting from periconceptional undernutrition .
this hypothesis is further supported by the findings of epigenetic changes after periconceptional undernutrition in fetal hypothalamic genes involved in feeding regulatory networks , including proopiomelanocortin ( pomc ) and glucocorticoid receptor ( gr ) .
it has been suggested that rapid postnatal growth may be the pathway by which body composition is altered , and the risk of metabolic disease thereby increased after fetal growth restriction .
however , we found no association between birth weight , early growth rates , and adult body composition in our study .
it is interesting that the disruption of postnatal growth regulation previously demonstrated in offspring of periconceptional ewes was seen in lambs of both sexes , whereas the effects on adult body composition were seen only in males .
this also suggests that a mechanism other than disrupted growth regulation or postnatal growth rate has affected adult fat mass .
although duration and severity of undernutrition in animal studies vary considerably in the published literature , sex specificity of metabolic outcomes is a common finding .
found increased insulin secretion in response to a glucose load at 10 weeks of age in male offspring of ewes actively losing weight at the time of conception , whereas female offspring were not different from controls .
in contrast , we have previously reported greater impairment of glucose tolerance in female offspring after pcun than in males . in merino
d'arles sheep , a hardy breed hypothesised to be less affected by undernutrition , male offspring of ewes fed 50% of their dietary requirements before and after mating had lower plasma leptin concentrations at birth than controls , whereas females had higher leptin concentrations than controls at 4 months of age .
we have also reported clear sex - specific differences in hpa function over time in a subset of the cohort described in this study , with onset of adrenal acth resistance in males much earlier than in females , in contrast to the study by smith et al . , in which there was no effect of maternal weight loss around conception on offspring hpa axis prior to weaning .
better understanding of the signals in embryonic life that determine sex - related growth differences may direct further study in this area .
the observed widespread effect on multiple organ weights in males also suggests a global , sex - specific effect in the embryonic stage of development . one can not assume a direct relationship between phenotype and organ function . adrenal size and function
have been investigated in a number of species under different stressors , but the relationship between them , particularly in a basal state , is unclear and may differ between species and under different conditions [ 30 , 31 ] .
we found little difference between nutritional groups in pancreatic weight , despite several studies demonstrating an influence of fetal nutrition on glucose metabolism ; however , the endocrine components of the pancreas comprise only a small proportion of total pancreatic mass .
the gonadal findings are interesting , and particularly the opposite effects of maternal undernutrition on gonad size in males and females .
it has been suggested that an adverse in utero environment leads to prioritising of investment into early growth and reproductive capacity , despite later
trade offs in size and function of other organs , but males and females may be affected differently .
lifelong reproductive capacity is decreased in ewes exposed to undernutrition in late fetal life or during early postnatal life [ 33 , 34 ] .
female lambs of ewes undernourished from mating until midlate pregnancy had a decreased ovulation rate compared with controls , but male reproductive function was not affected . in female rats ,
the timing of puberty and subsequent ovarian function were found to be influenced predominantly by nutritional status in utero , rather than by later weight gain , with the onset of puberty at a lower body weight and lower plasma preoestrus progesterone concentrations after maternal undernutrition . however , ovarian size was not reported .
women born small for gestational age have been shown to have reduced uterine size and ovarian volume in adulthood .
we have no information about the timing of puberty , or the functional relevance of the smaller ovaries in the female offspring in our study as these ewes were not mated , so fertility was not assessed . in men , testicular dimensions and volume
testicular size is highly correlated with sertoli cell mass , and testicular diameter at 150 days of postnatal age is positively associated with postpubertal testicular size and sperm output . in utero testicular development is dependent on circulating fetal gonadotrophin concentrations , but the effect of the nutritional environment before fetal pituitary function is established is less clear .
maternal undernutrition for 50 days after mating in sheep did not affect testicular size , but was associated with increased testosterone production , advanced development of seminiferous tubules , and increased expression of steroidogenic acute regulatory protein ( star ) at day 50 , implying that undernutrition may upregulate fetal testicular steroidogenesis .
we did not assess fertility in the rams in our study ; however , the increased testicular size across all undernourished groups suggests not only sparing of but also possible enhancement of reproductive capacity in male offspring .
while serial assessments of body composition throughout the life course would have been desirable , this was not possible due to logistic constraints .
the effect of ageing on fat mass might be more profound in males , and there may have been changes over time in body composition that were related to other growth parameters , for example , rapid seasonal weight changes or growth velocity in different time periods , or the timing of puberty .
the attrition over time associated with large cohorts of animals over years also meant that numbers in the male control group were lower than ideal . despite this
, the magnitude of the phenotypic differences between controls and undernourished male offspring suggests a profound effect .
brief undernutrition around the time of conception in sheep alters adult phenotype in male offspring , including an increase in proportional body fat .
this is likely to increase the risk of metabolic dysfunction and may contribute to the observed effects of periconceptional undernutrition on endocrine and metabolic regulation
. the increased gonadal size in males , despite decreased growth of several other organs , may reflect prioritising reproductive capacity in response to an adverse periconceptional environment .
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periconceptional undernutrition ( pcun ) in sheep alters fetal growth and metabolism and postnatal growth regulation , but effects on adult body composition are unknown .
we investigated the effects of pcun on adult phenotype .
singleton lambs of ewes fed normally ( n , n = 17 ) or undernourished before ( un-61 - 0 d , n = 23 ) , before and after ( un-61 - 30 d , n = 19 ) , or after ( un-2 - 30d , n = 17 ) mating ( d0 ) were weighed at birth , 12 weeks , and intermittently to adulthood
. at the age of 3 - 4 years , body composition was assessed by dual - emission x - ray absorptiometry followed by postmortem examination .
compared with n animals , male , but not female , offspring of all un groups had greater % fat mass ( all un versus n : 9 1 versus 2 1% , p < 0.001 ) and perirenal fat ( 544 36 versus 222 44 g , p = 0.002 ) , and proportionately smaller hearts ( 4.5 0.1 versus 5.2 0.2 gkg1 ) , lungs ( 9.1 0.2 versus 10.6 0.5 gkg1 ) , and adrenals ( 0.06 0.002 versus 0.08 0.003 gkg1 ) .
un males also had larger testes ( 726 21 versus 545 32 g , p = 0.007 ) , but un females had smaller ovaries ( 2.7 0.08 versus 3.4 0.4 g , p = 0.01 ) .
changes were independent of birth weight or postnatal growth velocity .
brief pcun has sex - specific effects on adult phenotype , predominantly affecting males , which may contribute to adverse metabolic outcomes .
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behet s disease ( bd ) is an inflammatory disease of unknown cause , characterized by a waxing and waning course in which oral and genital ulcers , skin lesions , uveitis , obstructive vasculopathy and other inflammatory manifestations may flare up and then ameliorate .
familial mediterranean fever ( fmf ) is an autosomal recessive systemic autoinflammatory disease presenting episodic attacks of fever , accompanied by abdominal , chest , or joint pain and the gradual development of nephropathic amyloidosis of the aa type .
spondylarthritis as part of the clinical picture in these two diseases has been questioned and probably it is not a prominent characteristic of any of them .
the following patient had both bd and fmf , and human leukocyte antigen ( hla ) b27 positive ankylosing spondylitis .
a 35-year - old tunisian man was referred to our hospital with sudden onset of severe abdominal pain and fever . he had hip and back pain for 10 years and until the last 4 years he did nt notice any physical impairment .
he had accepted his pain as the result of his heavy work . during the previous 2 years
he had noticed oral ulcers recurring with a frequency of about once a month , which would disappear in 47 days .
he had protracted bouts of abdominal pain irregularly recurring three at five times in a year and spontaneously resolving for at least 15 years .
his oral and genital ulcers did not show any temporal relation with his painful attacks .
a younger sister had similar complaints of abdominal pain with fever of about 38c since the age of 22 years .
the patient had generalized abdominal tenderness with rebound , two scrotal scars of old ulcers and fever of 38. bowel sounds were diminished but audible . the patient had a serious kyphotic posture and severe restriction of the spine in all planes .
positive laboratory findings were as follows : leukocytes count 10.610/l , erythrocyte sedimentation rate 64 mm / h , c - reactive protein 23 mg / l , fibrinogen 5.1
tissue typing showed the presence of hla - b27 antigen , hla - b51 typing was negative .
a radiological examination showed characteristic features of ankylosing spondylitis both in cervical and lumbar region of spine and bilateral sacroiliac ankylosis with mild bilateral coxofemoral joint involvement .
the patient has experienced only two attacks of abdominal pain and fever during the follow - up period of 5 years .
our patient was diagnosed as having a complete form of bd based on recurrent oral and genital aphtosis , and positive pathergy skin test , and venous thrombosis .
nevertheless , the patient has since shown periodic fever with recurrent abdominal pain due to peritonitis , which is an atypical symptom in bd , and dna analysis demonstrated a homozygous mutation in the mefv gene , leading to a diagnosis of fmf . in spite of the distinct clinical features between bd and fmf , they share several common characteristics .
the gene responsible for fmf , mefv , is located on chromosome 16p13.3 and consists of 10 exons and 781 codons .
the product of the mefv gene , named pyrin / marenostrin , is expressed in neutrophils , monocytes , dendritic cells , and synovial fibroblasts .
pyrin is cleaved by caspase-1 and the cleaved n - terminal fragment translocates to nucleus and enhances asc - independent nuclear factor ( nf)- b activation through interactions with p65 nf-b and ib- leading to enhanced inflammatory responses .
neutrophils also play an important role in the development of bd ; they are found in the lesions of bd patients without evidence of bacterial infection .
cytokines and chemokines secreted from antigen presenting cells and t cells are suggested to cause neutrophil hyperactivation .
activated neutrophils secrete some cytokines which prime themselves and also stimulate th1 cells . among other functions of the neutrophil ,
colchicine is used as the first line therapy for both diseases , because the agent suppresses neutrophil motility and chemotaxis through inhibiting microtubule functions .
authors have noted two elements acting upon the presence of both diseases , one is geographic and the other is ethnic . as matter of fact , individuals of sephardic jewish , armenian , turkish , and arab descent are more likely to be affected by fmf , whereas people living along the ancient silk road , which extends from the mediterranean to eastern asia , reveal take much more subjects to bd than in the other areas .
such incredible predominance of the disease is also related with hla - b51 which is almost present in 60 to 70% of bd patients from the previous mentioned areas , the frequency of hla - b51 in tunisian patient with bd was 30% .
besides , many non - hla genes along with environmental factors contribute to the increasing risk of the disease .
some researches have revealed that mefv gene is one of vulnerable genes which are liable to yield bd , mainly for patients with vascular involvement , in france and turkey and israel , where bd and fmf are prevalent .
although tunisia is one of the countries where bd and fmf and ankylosing spondylarthritis are prevalent .
chance occurrence of these two rare diseases in the same patient , who also has hla - b27 positive ankylosing spondylitis , had not been reported . to the best of our knowledge
, there is only a unique case report of association of bd , fmf and hla - b27 negative bilateral sacroiliitis .
widely differing frequency of sacroiliitis and ankylosing spondylitis have been found in different series of patients with bd .
some investigators have reported an increased prevalence of sacroiliitis and ankylosing spondylitis in patients with bd , but others have declared prevalence similar to that of the general population .
the reasons for these different results are probably because of high observer variability in reading anteroposterior radiographs of the sacroiliac joints , and different origins of series of bd as well as different diagnostic criteria used .
, few cases of fmf with seronegative spondylarthritis have been reported , while the most of published cases with spondylarthritis in fmf are also hla - b27 negative and have no radiological lumbar spine involvement , a recent study suggests that hla - b27 positivity may play a role in the development of sacroiliitis and the severity of seronegative spondy - loarthropathy . in conclusion , we report an unusual observation of coexistence of bd and fmf and hla - b27 ankylosing spondylitis .
although this coexistence may be due to either chance or geographical distribution patterns of these diseases , common etiopathogenetic characteristics of bd and fmf are identified .
thus , in clinical practice , we propose to look for ankylosing spondylitis in case of association of these diseases .
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behet 's disease ( bd ) and familial mediterranean fever ( fmf ) , which are two separate diseases sharing some clinical features , may also coexist in the same patient .
further investigations are needed to understand whether this coexistence is due to either chance or geographical distribution patterns of these diseases or to common etiopathogenetic characteristics .
spondylarthritis as part of the clinical picture in these two diseases has been questioned and probably it is not a prominent characteristic of any of them .
we report a 35-year - old tunisian man who had an association of bd , fmf and human leukocyte antigen ( hla ) b27 positive ankylosing spondylitis .
although that spondylarthritis is an infrequent joint involvement of fmf and bd , it must be looked for in case of association of these diseases .
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chronic pulmonary regurgitation ( pr ) is frequent sequelae after repair of tetralogy of fallot .
chronic volume overload of the right ventricle , due to pr , causes ventricular dilatation and impairment of systolic and diastolic function . in the long term follow - up
, pr causes atrial and ventricular arrhythmia , heart failure , and increased the risk of sudden death .
an implantation of the right ventricle to pulmonary artery ( pa ) conduit is currently a well - established method of repair of various right ventricular outflow tract ( rvot ) pathologies , including tetralogy of fallot , pulmonary atresia and others .
the inevitable degenerative changes of conduit lead to rvot obstruction , pr and progressive conduit stenosis , due to somatic growth .
since bonhoeffer et al.1 - 3 ) reported the first percutaneous pulmonary valve implantation in humans , many patients received this procedure and has been an attractive alternative method of conduit replacement .
this valve is made of bovine jugular vein sewn within a balloon expandable stent ( cheatham - platinum stent ) and hand - crimped on a balloon in balloon catheter ( fig .
1 ) . there is growing evidence of efficacy and safety for treatment of various congenital heart diseases , including pr and rvot stenosis .
the melody transcatheter pulmonary valve ( medtronic ) received us food and drug administration approval for human device exemption use in january 2010 and the edwards sapien transcatheter heart valve is still in clinical trials in the united states.4 - 6 ) the sapien transcatheter heart valve is larger in size than melody valve .
others , like the shelhigh injectable stented pulmonic valve and edwards - cribier percutaneous heart valve is clinically undergoing investigation.7)8 ) this article reviews the benefits and risks of percutaneous pulmonary valve implantation .
despite numerous studies of clinical outcomes in patients with postoperative rvot dysfunction , the optimal timing of pulmonary valve replacement ( pvr ) and appropriate methods of treatment before irreversible dysfunction of right ventricle has not been determined .
clinical symptoms , including exercise intolerance , ventricular arrhythmia , and echocardiographic findings , such as progressive right ventricular ( rv ) dilatation and dysfunction , have been used as indications for operation .
but there are concerns regarding the time being too late for restoration of the rv function .
therrien et al.9 ) reported that post - operative rv remodeling , after pvr , may not occur in patients with rv end - diastolic volume > 170 ml / m or endsystolic volume > 85 ml / m .
early pvr is safe and may prevent the detrimental complications of severe pr and improve long - term prognosis of the patients with pr .
although there is clear evidence that pr and rv pressure overload are deleterious , the incremental effects of progressive and increasingly chronic rv volume and pressure overload may be difficult to evaluate .
therefore , some investigators recommended early pvr surgery for pr , even absence of rv dilatation or dysfunction .
surgical conduit or pvr is the established standard treatment of care ; nonetheless , patients with pr or rvot conduit stenosis are often managed medically for many years before referral for surgery .
the bovine jugular venous valved conduits have been used for rvot reconstruction , but life span of the prosthetic conduits is limited .
calcification , stenosis , intimal proliferation , and graft degeneration causes progressive stenosis or regurgitation .
further , there still exists the morbidity of reoperations and the need to replace these valves surgically , particularly , when they are implanted at a young age .
the availability of less invasive options for pvr , such as transcatheter valve implantation , may provide a means of limiting the duration and severity of rv volume and/or pressure overload without increasing the lifetime number of open heart operations .
the transcatheter intervention for pvr is less invasive , low risk of bleeding and infection , as well as low costs compared with surgical procedures .
also , there is significant improvement in exercise capacity and gas exchange efficiency during exercise after transcatheter valve implantation.10)11 )
there are many indications by authors ; recent indications were summarized in table 1.12)17)28 ) the patient selection for percutaneous transcatheter pulmonary valve implantation is crucial to the success . the most common cause of pr during late follow - up is tetralogy of fallot and its variants .
when associated with pulmonary atresia , a valved conduit is used for rvot reconstruction . during late follow - up , rvot obstruction may develop for many kind of reason . for relief of rv pressure overload , due to stenosis , balloon dilatation and stent implantation have been successfully used in the conduits.13 ) however , stent implantation alone can not relieve pr and it may result in the exacerbation of the rv dysfunction .
the limiting factor for valve implantation is the nature of rvot and the size of conduit .
khambadkone and bonhoeffer14 ) reported that in patients with tetralogy of fallot , native outflow tract with repair of only valvotomy or valvectomy may not provide secure landing zone . in case of outflow tract patch or transannular patch
, the outflow tract tends to be dilated and may not have any secure implantation point . a large conduit , which is larger than the fully expanded dimension of the valve , is not good for device implantation .
if the rvot diameter is more than 22 mm , the implanting zone is unlikely to hold a stent .
small sized conduit , especially below 16 mm , which can not be dilated to the diameter of the device .
khambadkone15 ) summarized the indications as follows , 1 ) symptomatic patients with severe pr with rv dysfunction and/or dilatation , 2 ) patients with symptomatic arrhythmias and severe pr with rv dysfunction / dilatation , 3 ) severe pr and evidence of rv dysfunction and objective evidence of decreased exercise tolerance in asymptomatic patients , 4 ) patients with moderate or severe pr and additional lesions ( residual ventricular septal defect , branch pa stenosis and tricuspid regurgitation ) needing intervention , with or without symptoms , 5 ) asymptomatic patients with severe rvot obstruction ( rv pressures equal to or more than three - fourth systemic pressure ) with or without pr , and 6 ) symptomatic patient with rvot obstruction ( rv pressure more than half of the systemic pressure ) with or without pr . active infection is an absolute contraindication for this procedure .
care should be taken to get the previous history of conduit endocarditis , because endocarditis , within the conduit , can be underdiagnosed and may remain dormant , despite negative blood cultures and inflammatory markers . because coronary artery abnormalities are not uncommon
patients less than 20 kg pose higher risk of complications and pregnancy is also contraindication .
the conduits sometimes lie behind the sternum and this lead to external compression by the sternum and adhesions to the anterior chest wall .
the stent fractures occur frequently , and require repeated balloon dilatation , another stent implantation or operation ( table 2 ) .
peng et al.13 ) reported stent fracture occurred in 43% of the patients who underwent cardiac catheterization before a conduit replacement surgery .
nordmeyer et al.16 ) reported that stent fractures occur in about 20% at the 2 year follow up , and substantial number of stent fractures occurred during the first 180 days .
associated risk factors for stent fractures were implantation into native rvot , absence of calcification along the rvot and greater recoil of valve after implantation .
zahn et al.17 ) reported stent fracture rate of 28% during the 6 months follow - up , and it resulted from excessive external loading forces .
restenosis of the valve may result from the stent fracture and it can be evaluated by x - rays , echocardiography or ct . to reduce the stent fracture ,
presenting the conduit with bare - metal stents before implanting the valve is now widely accepted.18 - 21 ) the principle of presenting is to reduce the compressive stress on the valve and preparing the landing zone of the valve with strong metal stents .
it is also helpful in successfully relieved stenosis , particularly , where significant recoil is seen after predilation . in patients with conduit stenosis ,
predilation with high pressure balloon can be helpful because it allows for easier implantation of the stent through large sheath.15 ) valve migration is a fearful complication , as well .
some expert opinion suggested that valve - size mismatch , difficulty with catheter manipulation , suboptimal implant location and rvot hypermobility may be great cause of stent migration or embolization.22 ) the management of percutaneous valve migration is variable and dependent on the size and location of the valve .
if the valve migrates distally , it can be solved by expanding the valve within the branch pa .
however proximal migration to the right ventricle requires surgical approach , because reposition of the valve back into the inferior vena cava ( ivc ) is usually not possible , due to large valve size and small ivc diameter .
it occurs immediately after the procedure , some cases occur after a few months later .
meticulous follow - up is important and careful monitoring of sudden symptom of chest discomfort or arrhythmia is required .
it is essential to assess the risk of coronary artery compression when placing a rvot stent or percutaneous valve implantation . in case of rvot reconstruction , such as tetralogy of fallot
, coronary arterial anatomy may be anomalous and care should be taken during the procedure.23 ) if the rvot conduit passes directly over a major coronary branch , the coronary artery can be compressed because of rigid stent expansion .
sridharan et al.24 ) and biermann et al.25 ) reported a significant coronary artery compression , which was demonstrated by balloon inflation within homograft to maximum diameter and simultaneous selective coronary angiography .
if there are suspicions of coronary artery compression , selective coronary angiography with simultaneous ballon inflation in the rvot is the key procedure to assess the spatial relationship between the coronary artery and stent .
endocarditis is a fearful complication , as well ; aseptic environment is important and treat patients with appropriate intravenous antibiotics .
other complications are bleeding , hematomas , fever , infection , right bundle branch block , and etc .
after valve implantation , the rv systolic pressure and outflow gradient fall there is a significant improvement in pr .
the results of the most recent large studies are listed in table 1 . during the follow up
, there is early device failure ( hammock effect , stent fracture , residual stenosis ) , which leads to recurrent rvot dysfunction .
it is clinically important to know the mechanism of rvot dysfunction and to know the treatment method to restore rv dysfunction .
they compare the results of pulmonary valve implantation between the early and late period that freedom form reoperation was significantly longer in the later period .
a postprocedural gradient > 25 mm hg after the procedure was risk factor for reoperation .
the other factor is patient selection criteria , which is stricter than the early period .
they consider careful evaluation of conduit type , rvot morphology , and distensibility , which are very important in patient selection . as such
, they do not attempt in patients with patch reconstruction of the rvot and very distensible homograft .
nordmeyer et al.27 ) reported that in cases of stent fracture , residual stenosis , repeat valve implantation is an effective treatment method for device failure and lengthen the freedom from reintervention .
mcelhinney et al.30 ) reported recent results of us melody valve trial that implant within an existing stent , new prestent , or bioprosthetic valve was associated with longer freedom from stent fracture . and valve compression and apposition to anterior chest wall were associated with short freedom from stent fracture .
medium - term follow - up is good , and regurgitation due to degeneration of valve leaflet is very rare .
calcification of valve leaflets during the late follow - up is also rare . in late explants
freedom from reoperation was 70% at 5 years and freedom from transcatheter reintervention was 73% at 5 years .
survival at 83 months is 96.6%.26 ) in terms of valve failure , more data is needed because follow up period is a little short .
percutaneous pulmonary valve implantation is safe and effective method of treatment in patients with repaired congenital heart disease .
medium follow - up data shows good freedom from reoperation and valve implantation can delay open heart surgery . but care should be taken before procedure to evaluate possible coronary compression by stent or percutaneous valve implantation .
stent fractures were not uncommon after valve implantation and more likely in patients with severely obstructed conduits and located directly behind the anterior chest wall
more careful consideration of rvot morphology and prestent with bare metal stent maybe a good procedure before transcatheter pulmonic valve implantation .
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pulmonary regurgitation ( pr ) is a frequent sequelae after repair of tetralogy of fallot , pulmonary atresia , truncus arteriosus , rastelli and ross operation . due to patient growth and conduit degeneration , these conduits have to be changed frequently due to regurgitation or stenosis .
however , morbidity is significant in these repeated operations . to prolong conduit longevity , bare - metal stenting in the right ventricular outflow tract ( rvot )
obstruction has been performed . stenting the rvot can reduce the right ventricular pressure and symptomatic improvement , but it causes pr with detrimental effects on the right ventricle function and risks of arrhythmia .
percutaneous pulmonary valve implantation has been shown to be a safe and effective treatment for patients with pulmonary valve insufficiency , or stenotic rvots .
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at the end of the last century , in addition to the conventional screening of naturally occurring bioactive compounds , a strategy was developed to construct desired novel hybrid antibiotics by combinatorial biosynthesis , by specifically combining genes from various biosynthetic pathways originating from different antibiotics producing organisms . in streptomyces species ,
the most important producers of bioactive molecules , genetic approaches to improve secondary metabolite production are generally hampered by various phenomena such as restriction barriers , absence of an efficient gene transfer systems , and lack of suitable cloning vectors .
recently , in order to circumvent these problems , there has been considerable interest in the use of intergeneric conjugation as an efficient means of gene transfer and expression in streptomyces species .
this technique allows the construction and the manipulation of recombinant plasmids in e. coli and their subsequent transfer to a recipient of interest .
the initial protocol for intergeneric transfer of plasmids from e. coli to streptomyces species was developed by mazodier et al . .
since then , this method has been successfully applied to several streptomyces species [ 24 ] via methylation - deficient e. coli strains , such as strain et12567 , as a dna donor to avoid methylated dna restriction systems of actinomycetes .
several cloning vectors that could be transferred from e. coli to streptomyces species by conjugation method have been constructed [ 7 , 8 ] . among them , pset152 is a nonreplicative plasmid in streptomyces that carries the attp site and integrase gene of c31 phage and consequently can integrate into the chromosomal attb site of the bacteriophage c31 . like integrases , c31 integrase catalyses the site - specific recombination between the chromosomal attb site and an attp site to form attl and attr .
however , unlike those of integrases , the sites are both very small , with attp and attb being just 39 and 34 pb in size , respectively .
the dna sequences of attb were characterized from various streptomyces species , and the analysis of these sequences revealed the presence of high conserved positions . in this genus of bacteria , the presence of pseudo - attb sites ( 50%70.3% nt identity to attb sites ) has also been reported , but with an integration frequency 300 times lower than that with the corresponding attb site . however , the absence of this attb site from some actinomycete strains , such as saccharopolyspora erythraea , has also been reported .
we have previously reported the isolation from tunisian soil of two new actinomycete strains called streptomyces sp . us 24 and streptomyces sp . tn 58 , producing diverse interesting biological activities , and described the purification and structure elucidation of two and five active molecules from the streptomyces sp .
the active molecules produced by streptomyces sp . us 24 are the 3-indolethanol and a diketopiperazine ( dkp ) derivative , the cyclo ( l - phe , l - pro ) diketopiperazine .
dkp active molecules form a very important family because of their many potential uses as antibacterial , fungicidal , herbicidal , antiviral , immune - suppressor , and antitumor agents , and therefore they are very attractive for the production of novel hybrid active compounds by combinatorial biosynthesis . among the five active molecules characterised from the streptomyces sp .
tn 58 , two belong to the rhamnopyranoside family : the 1-o-(2-aminobenzoyl)--l - rhamnoside and the 4-hydroxybenzoyl -l - rhamnopyranoside .
these two active compounds possess inhibitory activity towards 3-hydroxysteroid dehydrogenase ( 3-hsd ) , a useful target for anti - inflammatory and antiphlogistic drugs .
it should be noted that until now , the streptomyces sp . tn 58 is the only described strain which produces these two molecules directly without any supplement addition into the culture media .
identification and expression of genes involved in biosynthetic pathways of these molecules constitute a real opportunity for the comprehension of their natural biosynthesis and subsequently the production of hybrid molecules .
the aim of this work was thus to succeed in carrying out intergeneric conjugation between e. coli et12567 and streptomyces sp . us
24 and tn 58 strains , to analyse the integration sites of the integrative vector pset152 into the chromosome of these two streptomyces strains , to study whether the site - specific integration of pset152 affects essential functions such as the production of active molecules in these two studied strains , and to verify the expression of a heterologous gene in streptomyces sp .
e.coli strain et12567 ( puz8002 ) [ 2 , 5 ] , used as the donor in intergeneric conjugation , is a methylation - defective strain ( dam-13 : : tn9 dcm-6 hsdm cmr ) .
e. coli strain dh5 ( f80 dlaczm15 (laczya - argf ) u169 enda1 reca1hsdr17 ( rk , mk ) deor thi-1 suse44 gyra96 rela1 ) and e. coli hb101 ( fhsds2 reca13 ara14 pro a2 lacy1 galk2 rps l20 xyl 5 mtl sup e44 ) were used as host strain , and e. coli atcc 8739 and micrococcus luteus lb 14110 were used as indicator micro - organisms for the antibacterial activity assays .
the two streptomyces strains us 24 and tn 58 were used as recipient for intergeneric conjugation .
cbs4 strain was the glucose isomerase deficient streptomyces violaceoniger in which the xyla gene of the streptomyces sk strain , under the control of the erme - up constitutive promoter , was integrated in its chromosome via the integrative vector pts55 .
pset152 is an integrative plasmid carrying c31 int and att functions and an apramycin resistance gene for selection in streptomyces and e. coli .
pgem - t easy vector is a ta cloning vector amp ( promega ) used for cloning of the pcr products .
ampicillin ( 50 g ml ) , chloramphenicol ( 25 g ml ) , nalidixic acid , and kanamycin ( 50 g ml ) were added to growth media when required and 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ( 40 g ml ) when appropriate .
transformation of e. coli dh5 or hb101 with pgem - t pset152 derivatives was carried out according to the manufacturer 's instructions ( promega ) .
24 and tn 58 strains were grown in tryptone soya broth ( tsb : 30 g tryptic soy broth plus 5 g yeast extract per 1000 ml distilled water ) medium for the preparation of genomic dna and in r2ye plates for the preparation of spore stocks .
tsb supplemented with 30 g of apramycin ml was used to grow exconjugants for the preparation of genomic dna and to maintain them .
as1 medium ( 1 g yeast extract , 5 g soluble starch , 0.2 g l - alanine , 0.2 g l - arginine , 0.5 g l - asparagine , 2.5 g nacl , 10 g na2so4 and 20 g agar , ph adjusted at 7.5 with koh ) was used for conjugation experiments . for bioactive molecules production , streptomyces sp . us 24 strain and the corresponding exconjugants were grown in tsb medium , supplemented at 1% ( w / v ) with starch and with a trace element solution : ( 7.5 ml l final ; 0.4
g l zncl2 ; 2 g l feso47h2o ; 0.065 g l h3bo3 and 0.135 g l mona2o42h2o ) .
streptomyces tn 58 strain and corresponding exconjugants were grown in tsb medium , supplemented with glycerol at 1% ( w / v ) and with potassium at 1 mmol l. for antibacterial activity determination , indicator microorganisms were grown overnight in lb medium at 30c for m. luteus lb14110 and at 37c for e. coli atcc 8739 , then diluted 1 : 100 in lb medium and incubated for 5 hours under constant agitation at 200 rpm at the appropriate temperature . for growth study of the two bacteria : streptomyces tn 58/xyla and cbs4 , spores at 10 ml of each strain were used to inoculate 1000 ml erlenmeyer flasks with four indents , containing 200 ml of minimum liquid medium ( [ nh4]2so4 2.5 g l , mgso4 0.3 g l , cacl2 0.2 g l , kh2po4 1 g l , yeast extract 1 g l and 1 ml of trace element solution ) in presence of xylose as solely carbon source at 10 g l. biomass of the two studied streptomyces strains was determined at different growth times by measurement by weighing to constant dry weight after drying at 105c .
intergeneric conjugation between e. coli and streptomyces us 24 and tn 58 strains was performed as described previously by flett et al . , with minor modifications .
e. coli et12567 ( puz8002/pset152 ) was grown to an absorbance of 0.40.6 at 600 nm .
the cells were pelleted by centrifugation , washed twice in an equal volume of lb , pelleted again , and finally resuspended in 1/10 volume of lb .
aliquots of the two studied streptomyces strain spores suspension stored at 20c were used as recipients .
spores ( eq10 ) were washed in 2 yeast extract tryptone medium , resuspended in 500 l of 2 yeast extract tryptone medium , and incubated at 50c for 10 minutes to induce germination .
donor cells ( 500 l approximately 10 cells ) were added to the treated spores , the mixture was pelleted by centrifugation , and finally the pellets were resuspended in the residual liquid , and the undiluted mixtures were plated .
the mating mixtures were spread on as1 plates containing 10 mm mgcl2 and incubated for 18 hours at 37c for the streptomyces us 24 and at 30c for the streptomyces tn 58 strain .
the plates were overlaid each with 1 ml of water containing 500 g of nalidixic acid and 1 mg of apramycin , incubated further for 5 days at the appropriate temperature , and the exconjugants were counted .
control experiments were performed as described above but without the addition of e. coli donor cells .
the viable count of the donor culture was determined by spreading the cells on lb agar plates supplemented with appropriate antibiotics .
small - scale plasmid preparations from e. coli were performed as described by .
digestion with restriction endonucleases , and separation of dna fragments by agarose gel electrophoresis , alkaline calf intestinal phosphatase , ligation of dna fragments , and transformation were done according to for e. coli and for streptomyces . for blot manipulations ,
about 1 g and 50 ng were loaded for the digested genomic dna and plasmids dna , respectively .
p - labeled probes were prepared using the random prime labelling system ( amersham ) .
pcr amplification of the attb sites of the two studied streptomyces strains was performed using the two primers attb1 and attb2 previously described by .
approximately 200 ng genomic template dna was used with 100 pmol of each primer per 50 l reaction volume . to improve the denaturation of the dna , 5% ( v / v ) dmso
amplifications were performed in a gene amp pcr system 2700 ( applied biosystems ) using 1u pfu dna polymerase ( stratagene ) and the recommended buffer system according to the following amplification profile : 94c ( 5 minutes ) followed by 45 cycles of denaturation at 94c ( 30 seconds ) , annealing at 60c ( 1 minute ) and extension at 72c ( 1 minute ) .
the pcr reaction mix was analysed by agarose gel electrophoresis , and the products having the expected sizes were purified then cloned into pgem - t easy vector .
reactions were performed with a thermo sequenase cycle sequencing kit ( amersham ) and specific primers .
for the extraction of the active molecules from the streptomyces sp . us 24 and tn 58
strains and their corresponding exconjugants ( four exconjugants randomly chosen for each strain ) , spores at 10 ml were used to inoculate 500 ml erlenmeyer flasks with four indents containing 100 ml of culture medium .
after incubation for 24 hours in an orbital incubator with shaking at 250 rpm at the appropriate temperature , the preculture was used to inoculate ( 5% v / v ) a total volume of 1000 ml culture medium having the same composition of the preculture .
after three days incubation at 37c or 30c for streptomyces us 24 and tn 58 strains , respectively , and their corresponding exconjugants , in an orbital incubator with shaking at 250 rpm , the culture broths were filtered to separate mycelium and supernatant .
each supernatant was extracted twice with an equal volume of ethyl acetate and then evaporated on a rotavapor ( laborata 4000 ) , and obtained crude extract was dissolved in 1 ml ethyl acetate and used for antibacterial activities as follows : a paper disk was impregnated with 80 l of the corresponding sample and then laid on the surface of an agar plates containing 3 ml of top agar inseeded by 40 l of a 5-hour old culture of m. luteus lb 14110 or e. coli atcc 8739 . after 2 hours at 4c , plates containing m. luteus were incubated at 30c and those inoculated with e. coli at 37c , all for overnight .
plates were examined for evidence of antibacterial activities represented by a zone of inhibition of growth of the corresponding indicator micro - organisms around the paper disk .
thin layer chromatography ( tlc ) was performed on silica gel plates sio2 ( merck ) .
sk placed under the control of the erme - up constitutive promoter from pmm6 was cloned into the streptomyces integrative vector pset152 linearised by bamhi , leading to the pss6 vector construct .
this plasmid was firstly obtained within e. coli hb101 strain and then transferred to e. coli et12567/puz8002 for the construction of the streptomyces tn 58/xyla strains by conjugal transfer .
preparation of the cell - free lysate of the streptomyces sk , tn 58 , tn 58/pset152 , tn 58/pss6 , and cbs4 ( used as positive control ) strains was achieved as follows .
the cells were grown in tsb medium for 48 hours and were harvested by centrifugation at 8000 rpm for 10 minutes , and the pellets were suspended in te - buffer with 10 mm mgcl2 and 1 mm cocl2 .
after incubation for an hour on ice in the presence of 5 mgml lysozyme , 100 gml pmsf , and 1 gml pepstatin a , cells were disrupted by sonication at 4c for 6 minutes
( pulsations of 3 seconds , amplify 90 ) using a vibra cell sonicator ( fisher - bioblock scientific ) , and debris were removed by centrifugation at 20 000 rpm for 30 minutes .
glucose isomerase activity was determined using fructose as substrate by quantitatively measuring the glucose production .
the activity was assayed in a reaction mixture containing the enzyme ( 100 l of an appropriate diluted purified or crude extract ) with 10 mm mgcl2 , 1 mm cocl2 and 15% fructose , in a volume of 400 l . in standard condition
, assays were incubated for 30 minutes at 80c , and the reaction was stopped by cooling the tubes on ice .
the amount of glucose generated was determined by glucose - oxidase ( god - pap ) enzyme system , and a650 was measured after 40 minutes at room temperature .
one unit of glucose isomerase activity is defined as the amount of enzyme needed to produce 1 mol of product per minute under the assay conditions .
protein concentration was determined by bradford 's method using the bovine serum albumin as standard .
the plasmid pset152 was mobilized from e. coli et12567 ( puz8002 ) into streptomyces sp . us 24 and tn 58 strains .
exconjugants were obtained at a frequency of approximately 5 10 and 3 10 per recipient spore of streptomyces sp . us 24 and tn 58 strains , respectively , which showed a high frequency of exconjugants .
indeed , nikodinovic et al . , consider that the transformation efficiency ( 5 10 exconjugants recipient ) of the streptomyces nodosus by conjugal transfer of dna from e. coli was a high transformation frequency .
the integrative plasmid pset152 can not replicate in streptomyces ; so stable exconjugants should be obtained only if pset152 has been integrated into the chromosome of studied strain . in order to identify the copy number of pset152 in the recipient streptomyces sp .
chromosomal dnas were extracted , digested by bamhi and probed with the whole linearised vector pset152 in a blot analysis . since pset152 had a single bamhi site , dna from exconjugants
was expected to show hybridisation of two fragments to probe in the case of single copy integration . for streptomyces
us24 exconjugants , obtained hybridisation patterns were identical and showed the presence of three bands of about 5.7 , 6.2 , and 7.8 kb ( figure 1(a ) ) .
the same probe did not hybridise to any dna fragment of the wild type genomic dna .
this result implied that the plasmid was presumably integrated at the same locus in all corresponding exconjugants .
however , among the three obtained bands , the one of 5.7 kb corresponds exactly to the size of the linearized vector pset152 .
this fact can be explained via the tandem integration of two copies of the pset152 vector in the chromosome of streptomyces sp .
the presence of tandemly repeated copies of pset152 vector in the attb site can be explained by the property of pset152 to integrate at attr or attl sites via attp / attr or attp / attl recombination .
so , in certain cases , we can assist to the multiple independent recombination events , and pset152 can integrate at attl or attr sites via attp / attl or attp / attr .
, if this event occurs , this reaction must be specific to the attl and attr sites as tandem repeats of pset152 in the pseudo - attb sites were not generally observed .
alternatively , the generation of tandem repeats of pset152 in the attb may be a consequence of the rate of integration .
if the rate of recombination into the attb site is rapid , integration occurs early during the mating period , and when the hyphae containing integrated pset152 receives a further copy of pset152 , this last one may , generally , integrate by homologous recombination to generate a tandem repeat . concerning streptomyces tn 58 ,
two identical hybridisation signals were obtained for all eight analysed exconjugants corresponding to two dna fragments of about 6.8 and 8 kb ( figure 2(a ) ) .
this result implied that a single copy of the pset152 plasmid was integrated at the same locus in all the exconjugants in strain streptomyces sp . tn 58 .
we can suggest that the chromosome of this strain contains a single functional attb site for vectors that integrate site specifically using the bacteriophage c31 att / int system ( figure 2(b ) ) .
all together , our data strongly suggest the presence of an efficiently recognised attb site in the chromosome of the two studied streptomyces strains .
24 and tn 58 strains chromosomes implies the existence of one attb site recognised by the c31 integrase . taking advantage of the two pcr primers ( attb1 and attb2 ) described by combes et al .
, , we recovered a unique pcr product at the expected size of approximately 0.3 kb from the chromosomal dnas of the two studied strains .
these fragments were cloned into the pgem - t easy vector yielding pss4 and pss5 plasmids for us 24 and tn 58 strains , respectively .
the two inserts possess a size of 292 bp and 293 bp for us 24 and tn 58 strains , respectively , and a high nucleotide identity of 86% ( figure 3 ) .
these two amplified dna fragments encode a homolog of the streptomyces coelicolor a3(2 ) sco3798 orf with a nucleotide identity of 92% and 86% for streptomyces
this orf is a putative chromosome condensation protein , with sequence similarity to a mammalian - encoded protein , pirin ( an identified nuclear protein that interacts with bcl-3 and nuclear factor i ) , in which the attb site of c31 lies in various streptomyces species . for streptomyces species , all characterised c31 attb sites showed high nucleotide sequence similarity varying from 82.4% to 100%
however , the presence of pseudo - attb sites ( 50%70.3% nt identity to attb sites ) into which integration occurred at a frequency 300 times lower than into the corresponding attb site has also been reported in this genus of bacteria .
the multiple - sequence alignment of attb sites of streptomyces sp . us 24 and tn 58 with several other streptomyces species is shown in figure 4 .
this alignment indicates that these sequences are conserved , and some positions in the minimal site can tolerate nucleotide changes .
kuhstoss and rao reported , by comparing the s. ambofaciens attb site with attp , that the core sequence ( i.e. , the region at which the crossover occurs ) is 5ttg .
combes et al . , had shortened this sequence to two nucleotides 5tt which was present in the attb site of both studied strains streptomyces sp . us
it has been reported that integration of the integrative vectors into the c31 attb site can cause detrimental effects on antibiotic production in some strains . in fact , it should be considered that the decrease in antibiotics production for many streptomyces strains carrying integrated site specific plasmids such as s. fradiae producer of tylosin , s. kanamyceticus producer of kanamycin and others has been reported .
therefore , while integrative vectors could be used for the development of stable recombinant producers of antibiotics , their possible negative effect on the level of antibiotic production should be considered .
thus , in order to determine whether the site - specific integration of the plasmid pset152 affected relevant functions of streptomyces sp . us 24 and streptomyces sp . tn 58 strains , we have followed the growth of both strain and their corresponding exconjugants carrying plasmid pset152 .
all exconjugants grew well in the minimal medium , and there was no difference compared to the wild type strains .
the presence of pset152 had no effects on growth and sporulation of the two strains .
this stability was determined as the proportion of colonies that retained resistance to apramycin ( at 50 g ml ) after several passages of the exconjugants under nonselective conditions .
plasmid pset152 was inherited every time for the all studied exconjugants under nonselective conditions for both strains .
meanwhile , antimicrobial activity was determined by observing bacterial growth inhibition of the wild type strains ( us 24 and tn 58 ) and corresponding exconjugants .
us 24 and streptomyces sp . tn 58 and their corresponding exconjugants ( figure 5 and table 1 ) .
in addition , the analysis on tlc plates of the ten studied active crude extracts revealed the presence of the expected active molecules in each strain and its corresponding exconjugants . for the streptomyces
us 24 strain and its four studied exconjugants , two bands were easily detected on tlc plates under uv light at 254 nm after spraying with anisaldehyde / sulphuric acid .
the first band gives an orange colouration with a retention factor , rf = 0.46 ( chcl3/meoh 10% ) and the second one a violet colouration , rf = 0.37 ( ch2cl2/9% meoh ) .
according to our previously work , these two bands correspond to the 3-indolethanol and the cyclo ( l - phe , l - pro ) diketopiperazine active molecules , respectively . concerning the streptomyces sp . tn 58 strain and its studied exconjugants , the five active molecules ( m1 to m5 ) were present in all five crude extracts .
m1 belongs to the acetyltryptamine family and gives a colouration orange and violet after anisaldehyde / sulphuric acid and erhlich 's reagent visualisation .
m2 , rf = 0.57 ( ch2cl2/1% meoh ) , and m3 , rf = 0.47 , ( chcl3/5% meoh ) belong to the thiazole and brevinamide families , respectively .
m2 gives a chestnut colouration on spraying with anisaldehyde / sulphuric acid and m3 gives a pinkish colouration after erhlich 's reagent visualisation .
the two other active molecules ( m4 and m5 ) characterised from the streptomyces sp . tn 58 strain , and belonging to the rhamnopyranosides family , do not have antimicrobial activities , but they possess inhibitory activity towards 3-hydroxysteroid dehydrogenase ( 3-hsd ) . indeed , the rhamnosylated aromatic compounds were easily detected on tlc plates due to their striking yellow / greenish colouration after visualisation with anisaldehyde vapour .
for all five analysed crude extracts , the wild type one ( from the streptomyces sp . tn 58 strain ) and those from the four corresponding exconjugants , we obtained similar tlc migration profiles and two yellow / greenish bands having a retention factor ( chcl3/15% meoh ) of 0.22 and 0.33 corresponding to the two molecules belonging to the rhamnopyranoside family .
for the five active molecules of the streptomyces sp . tn 58 and its four studied exconjugants , tlc profiles , bands colouration , and retention factors of the different active compounds were in perfect concordance with the results of previously works .
these data clearly demonstrated that the morphological differentiation and active molecule production of all studied pset152 integrated exconjugants were similar to those of wild type streptomyces sp .
consequently , conjugal transfer using attp / b site integration can be concluded to be a suitable means of gene transfer and expression for both studied strains .
the establishment of a gene transfer system and the stable integration of cloned genes at the specific loci in the genome of streptomyces sp .
xylose isomerase ( d - xylose ketol isomerase ec 5.3.1.5 ) catalyses the reversible isomerisation of d - xylose into d - xylulose .
it is also referred to as glucose isomerase ( gi ) because of its ability to convert d - glucose to d - fructose .
this property is widely exploited industrially for the production of high - fructose syrup from starch .
sp sk , and we attempted to examine whether this gene could express and fold effectively in strain tn 58 .
it should be noted that the wild type streptomyces tn 58 strain can not assimilate the xylose as solely carbon source .
based on intergeneric conjugal transfer , transformation efficiency of strain tn 58 by plasmid pss6 was achieved at high frequency ( 10 exconjugants recipient ) .
the chromosomal structure of three tn 58/xyla strains , arbitrary chosen , was checked by southern blot hybridisation using the 800 bp psti dna fragment internal to pset152 as probe , which contain the orit region . as expected , a unique and identical large bamhi band was obtained for the three tn 58/xyla strains chromosomal dna , whereas wild type strain tn 58 chromosomal dna does not hybridise to the probe ( data not shown ) .
one of the three recombinant strains studied was subcloned five times on a solid media in the absence of apramycin followed by cultivation for several generations in liquid media in the absence of selection pressure .
the plating of this culture on solid media , on both with and without selective pressures , showed that 100% of the colonies were apramycin resistant and able to grow in the presence of xylose as solely carbon source .
determination of the mycelial intracellular glucose isomerase activity of streptomyces tn 58 , streptomyces tn 58/pset152 , streptomyces
sk , cbs4 , and tn 58/xyla strains shows that glucose isomerase activity of the tn 58/xyla recombinant strain is of about eighteenfold higher than that of the sk strain and comparable to that of cbs4 strain .
no activity was detected for the wild type tn 58 strain and the recombinant strain tn 58/pset152 ( table 2 ) .
study of the growth of the two strains , streptomyces tn 58/xyla and cbs4 , in minimum liquid media containing xylose as solely carbon source showed that the two strains grew well , and for each time , the resulting biomass was quite similar .
it should be noted that glucose isomerase activity is correlated with biomass production , and optimum activity has been obtained after 48 hours of incubation .
according to these results , we can deduce that site - specific recombination directed by pset152 makes it possible to stably insert heterologous dna in strain tn 58 .
this should be very important for a number of cases such as genes expression , genetic complementation , and biosynthetic pathway manipulations of the active molecules of this strain especially those belonging to the rhamnopyranoside family .
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streptomyces sp . us 24 and streptomyces sp . tn 58 , two strains producing interesting bioactive molecules , were successfully transformed using e. coli et12567 ( puz8002 ) , as a conjugal donor , carrying the integrative plasmid pset152 . for the streptomyces
sp .
us 24 strain , two copies of this plasmid were tandemly integrated in the chromosome , whereas for streptomyces sp . tn 58 , the integration was in single copy at the attb site .
plasmid pset152 was inherited every time for all analysed streptomyces sp .
us 24 and streptomyces sp . tn 58 exconjugants under nonselective conditions .
the growth , morphological differentiation , and active molecules production of all studied pset152 integrated exconjugants were identical to those of wild type strains .
consequently , conjugal transfer using pset152 integration system is a suitable means of genes transfer and expression for both studied strains . to validate the above gene transfer system , the glucose isomerase gene ( xyla ) from streptomyces sp .
sk was expressed in strain streptomyces sp . tn 58 .
obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively .
glucose isomerase activity of the constructed tn 58 recombinant strain is of about eighteenfold higher than that of the streptomyces sp .
sk strain .
such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high - fructose syrup from starch .
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this worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus leptospira .
more than 1.7 million cases of severe leptospirosis are reported each year , with a case mortality rate of about 10% .
untreated leptospirosis can eventually lead to hepatorenal failure , pulmonary hemorrhage syndrome , and even death depending on bacterial virulence and the host immune response [ 3 , 4 ] .
the clinical symptoms are similar to a variety of other infectious diseases which are often prominent in the same geographic regions , including scrub typhus , dengue , and malaria .
although the infection can be cured with proper antibiotic therapy at the onset of the disease , the lack of rapid diagnostic tests often presents a barrier to early diagnosis . microscopic agglutination test ( mat ) is considered the gold standard of serologic diagnosis .
this assay requires ample time as it works by detecting the antibody titer increase in serum samples obtained weeks apart .
while this technique provides an efficient retrospective diagnosis , it does not provide early diagnosis .
other diagnostic tests include dark - field microscopy , enzyme - linked immunosorbent assay , and western blot and are known to have low sensitivity .
polymerase chain reaction ( pcr ) and quantitative real time pcr ( qpcr ) can be used for detecting leptospirosis in clinical samples within the first week of illness with low clinical sensitivity . only about 50% of culture and/or mat confirmed cases
both pcr and qpcr are costly and often not readily available in many laboratories , especially those where leptospirosis is endemic .
originally described by notomi et al . , loop - mediated isothermal amplification ( lamp ) offers an alternative dna amplification method .
lamp uses bst dna polymerase for strand displacement dna synthesis along with primers that create cauliflower - like structures with multiple loops .
visualization of amplified dna products on gel electrophoresis is the most accurate method which can differentiate true positives from false positives due to nonspecific amplification .
however the procedures involved in gel electrophoresis are not practical for resource - limited areas .
the reaction products can be seen by several alternative methods which are easy to perform , such as turbidity derived from magnesium pyrophosphate formation , using a fluorescent dye such as sybr green to be visualized under uv light or by metal indicators such as hydroxy naphthol blue ( hnb ) or calcein , which can be seen by the naked eye [ 11 , 13 ] .
we developed a loop - mediated isothermal amplification assay using a combination of primer sets targeting the lipl32 and lipl41 genes .
lipl32 encodes a major conserved subsurface protein and is expressed in all pathogenic leptospira .
detection , examined the specificity and limit of detection of the method , and implemented the method to mimic blood samples .
our results demonstrate the assay 's potential use in leptospira endemic areas where resource setting is limited .
oligonucleotide primers used for lamp were based on the lipl32 genes of l. interrogans serovar copenhageni strain fiocruz l1 - 130 ( atcc , va ) .
six sets of primers targeting lipl32 were designed by primer explorer v4 ( http://primerexplorer.jp/e/ ) .
the primer set used for lipl41 was the same as the one previously described by lin et al . .
all primers were synthesized by eurofins mwg operon ( huntsville , al ) and the final choice of primers is described in table 1 .
the lipl32 and lipl41 genes from l. interrogans serovar copenhageni strain fiocruz l1 - 130 ( atcc , va ) were cloned into a pet28a vector ( novagen , ca ) .
the closed circular plasmids ( pet28a - lipl32 ( pl32 ) and pet28a - lipl41 ( pl41 ) ) were purified using qiagen plasmid mini kit ( qiagen , stockach , germany ) following the manufacturer 's instructions .
the pure pl32 and pl41 were quantified using a nanodrop 2000 microsample spectrophotometer ( thermo scientific , wilmington , de ) and used as a positive control to determine the detection limit in the lamp assay .
the plasmid pl32 was also used as the template for the selection of the best primer set .
genomic dna used in lamp and qpcr was extracted from l. interrogans copenhageni strain fiocruz l1 - 130 by qiamp dna mini kit ( qiagen , stockach , germany ) following the manufacturer 's instructions .
genomic dna template from 25 leptospira - suspected cultures was also extracted in the same way and used as template in both lamp and pcr as described below . the genomic dna from other bacteria ( o. tsutsugamushi , r. typhi , r. conorii , r. rickettsii , c. burnetii , and b. bacilliformis ) , whose symptoms are commonly mistaken for leptospirosis , were used for cross - reactivity study .
normal human plasma ( seracare , milford , ma ) spiked with l. interrogans genomic dna were prepared .
a total of 200 l spiked plasma samples were used for dna extraction using qiamp dna mini kit and the extracted dna was eluted with 20 l elution buffer .
briefly , a 25 l reaction mixture contained 1.6 mm of each fip primer and bip primer , 0.8 mm of each lf primer and lb primer , 0.2 mm of each f3 primer and b3 primer , 20 mm tris - hcl ( ph 8.8 ) , 10 mm kcl , 8 mm mgso4 , 10 mm ( nh4)2so4 , 0.1% triton x-100 , 0.8 m betaine ( sigma - aldrich , st .
louis , mo ) , 1.4 mm dntp mixture ( new england biolabs , beverly , ma ) , 8 u bst dna polymerase ( new england biolabs , beverly , ma ) , and 5 l of dna template . the optimal reaction temperature of 63c was experimentally determined by varying the temperature from 58 to 63c .
each reaction was terminated by adding 5 l of 10x bluejuice ( invitrogen , carlsbad , ca ) for gel detection .
the reaction products were examined by electrophoresis on a 2% agarose gel stained with a 1 : 10,000 dilution of gelred ( phenix research products , asheville , nc ) .
other detection methods involved inclusion of dyes before amplification , such as addition of hnb into the reaction to enable direct visual detection or inclusion of sybr green to detect the reaction products by a uv light .
quantitative real time pcr was performed to compare and to confirm the sensitivity of the lamp assay .
serial dilutions of pl32 plasmid were used to obtain the standard curves to determine the copy number of the purified l. interrogans copenhageni strain fiocruz l1 - 130 genomic dna or to determine the limit of detection after the extraction of dna from spiked samples .
abi 7500 fast real time pcr system ( applied biosystems , foster city , ca ) was used to perform the qpcr and analyze the amplification data .
f3 and b3 primers of primer set l32 - 5 were used to determine the copy number of the genomic dna of l. interrogans present .
each reaction mixture contained 0.5 m of forward primer f3 , 0.5 m of reverse primer b3 , 1x rt2 sybr green qpcr mastermix ( sa - biosciences , frederick , md ) , and 5 l of dna template .
an initial 10-minute activation step at 95c was followed by 40 cycles of 95c for 15 seconds , 60c for 1 min , and a melting curve determination cycle .
25 leptospira - suspected human specimens were collected and the organisms were cultured from blood using emjh media in siriraj clinical research center at mahidol university in bangkok , thailand .
to identify the species of leptospira in each specimen , species - specific primers targeting the ompl1 gene sequence were used in a pcr assay previously described by reitstetter .
among the six primer sets designed by the lamp primer designing software , primer sets l32 - 3 and l32 - 5 performed the best at 63c .
both primer sets were able to detect 25 copies of pl32 and primer set l32 - 1 detected 50 copies of pl32 ( see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/147173 ) .
primer sets l32 - 2 , l32 - 4 , and l32 - 6 did not work at all ( data not shown ) .
primer set l41 was able to detect 50 copies of pl41 ( figure s2 ) very similar to the previous report at approximately 100 copies .
both primer sets l32 - 3 and l32 - 5 were able to detect 25 copies of genomic dna ( figure s3 ) .
the sensitivity of the assay was improved to 12 copies of genomic dna by combining primer set l32 - 5 targeting lipl32 and the primer set targeting lipl41(figure s4 ) .
therefore , the combination of primer sets l32 - 5 and l41 was used for the rest of the experiments .
dna sequence analysis of the 270 bp region of l. interrogans serovar copenhageni strain fiocruz l1 - 130 where the l32 - 5 lamp primer set was located showed greater than 99% sequence identity to pathogenic species l. interrogans , l. kirschneri , l. borgpetersenii , and l. noguchii .
the same region has 96% and 92% sequence identities to pathogenic species l. weilii and l. santarosai , respectively .
the specificity of the lamp assay was evaluated by using the genomic dna from other bacteria whose symptoms are commonly mistaken for leptospirosis .
lamp reactions containing different strains of o. tsutsugamushi ( karp , kato , gilliam , and ta763 ) , r. typhi , r. conorii , r. rickettsia , c. burnetii , and b. bacilliformis were tested .
about 10 copies of each genomic dna was used as template and all tested negative with the combination of primer sets l32 - 5 and l41 . to mimic a clinical situation , we tested lamp using normal human plasma spiked with l. interrogans genomic dna .
the lamp assays had a detection limit of 33 copies per reaction based on standard curves obtained from diluted plasmids ( table 2 ) .
out of the 25 samples analyzed , only 18 were pcr positive targeting ompl1 using species - specific primer sets .
the leptospira species identified included nine l. interrogans intergroup a , five l. interrogans intergroup b , two l. borgpetersenii , and two l. weilii .
further sequencing of the 16s rrna gene confirmed that these 7 pcr negative samples were not leptospira spp .
thus a total of 18 pcr confirmed leptospira - positive samples and 7 pcr / sequence confirmed leptospira - negative samples were used to evaluate the performance of the lamp assay blindly .
addition of hnb to the reaction produced a visual color change from purple to blue ( as the mg ions in solution were chelated by pyrophosphate ions ) .
the reaction results can be visualized by the naked eye as seen in figure 1(b ) .
the inclusion of sybr green in the reaction mixture to detect the reaction products by uv light was also tested ( figure 1(c ) ) .
both alternative methods allow the examination of the results without opening the tube and require no additional process .
because the clinical presentation of leptospirosis is very similar to malaria , scrub typhus , and dengue , it is not possible to reliably predict the cause of infection based on the clinical signs and symptoms .
although pcr and qpcr have advantages with respect to quantification , control of contamination , and sensitivity , both methods require specialized equipment . lamp on the other hand is a low technology diagnostic tool for resource - limited setting .
previously , lin et al . reported a lamp method targeting the lipl41 gene of l. interrogans . in their study , the lamp products were examined by electrophoresis on a stained agarose gel and the detection limit was approximately 100 copies per reaction . later on ,
several lamp assays targeting the 16s ribosomal rna gene were also developed [ 2325 ] .
the results of lamp reaction were determined on the basis of detecting a white precipitate with the naked eye after centrifugation .
they demonstrated a lower limit of detection of 10 genome equivalents ( 20 copies ) per reaction versus 100 copies per reaction by earlier method .
however , their amplification step takes as long as two hours . with primers specific for pathogenic leptospira ,
koizumi et al . were able to detect 10 genome equivalents per reaction by uv fluorescence .
they also described that using heat - denatured dna improved the assay sensitivity to 2 genome equivalents per reaction .
the same effect was observed when we developed a lamp assay for detection of o. tsutsugamushi .
when dna was denatured , the copies of exposed single stranded dna increased which allowed more primers to anneal to the dna to start a lamp reaction . we anticipate that by heating the template we will further improve our assay 's sensitivity similarly .
were able to detect 10 and 100 copies per reaction , respectively , for serovars tarassovi and icterohaemorrhagiae by eye with 90 min incubation time .
the lamp assay that we have developed is as sensitive as the best method mentioned above without heat - denaturing the dna .
the incubation time is shorter than several of the previous methods and most importantly there is no need for centrifugation or uv fluorescence for determination of results .
all we need is a heat source to provide a constant temperature at 63c for 60 min and the results can be read by eye without other instruments .
diagnostic testing , in particular early detection , is critical for leptospirosis , as most infected individuals have nonspecific symptoms that are easily confused with dengue and malaria , which require different treatments .
lamp is an attractive alternative to pcr - based methods for early detection since a thermocycler is not required .
a heating block or water bath to maintain a constant temperature around 60c is all that is required for the lamp assay , making it particularly suited to resource - limited settings .
our study also presents a variety of methods that not only can detect the lamp products without opening the reaction tubes to avoid a very common contamination problem but also have a shorter assay time .
the advantage of using these detection methods , by naked eye or by a small fluorescence reader , will greatly enhance our ability to quickly diagnose an individual for a leptospira infection .
this assay has the potential to be used as a rapid , robust , and easy - to - perform assay in the endemic regions . in the future
, we would like to prepare the lyophilized lamp reagents to avoid the requirement of cold chain .
using a stable lyophilized reaction mixture will be a perfect assay to be used in resource - limited settings where leptospirosis is endemic .
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leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of leptospira .
we have developed a loop - mediated isothermal amplification ( lamp ) assay to detect the dna of leptospira spp .
six sets of primers targeting the gene of the subsurface protein , lipl32 , were evaluated for their detection sensitivity .
the best primer set detected less than 25 copies of lipl32 per reaction of both plasmid dna template and purified leptospiral genomic dna . by combining primers targeting lipl32 with the previously published primer set targeting lipl41 ,
the sensitivity of the assay was improved to 12 copies of l. interrogans .
the specificity of the lamp assay was evaluated by using the genomic dna from other clinically encountered bacterial species such as different strains of orientia tsutsugamushi , rickettsia typhi , rickettsia conorii , rickettsia rickettsii , coxiella burnetii , and bartonella bacilliformis .
these genomic dna samples were all negative in our lamp assay .
the sensitivity of the lamp assay was very similar to that of quantitative real time pcr .
several detection methods for the amplified product of lamp assay were performed to demonstrate the simplicity of the assay . in summary ,
our results have suggested that this assay is rapid , robust , and easy to perform and has the potential to be used in endemic locations .
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solitary necrotic nodule ( snn ) of the liver is a rare benign lesion first reported in 1983 by shepherd and lee , who described four lesions with a completely necrotic core and a hyalinized fibrotic capsule containing elastic fibers .
they also described central reticulin fibers within the nodule , suggesting the origin as sclerosing hemangioma .
we here describe a case of snn , whose central reticulin fibers within the nodule suggest the origin as hepatocellular carcinoma ( hcc ) or other hepatocyte - origin tumors , treated with laparoscopic anatomical iv segmentectomy of the liver .
a 76-year - old japanese female , with no prior medical history and no symptom , visited our hospital with the heterogeneous hypoechoic lesion in the liver segment vi incidentally pointed out in abdominal ultrasonography ( us ) . there were no abnormal findings in her routine laboratory data , including liver function tests , serology profile for hepatitis b or c , and tumor markers including ca 19 - 9 , alpha - fetoprotein , and cea . a previous history of alcohol abuse was not documented .
computed tomography ( ct ) with contrast demonstrated a 1.1 cm sized low - density lesion with mild ring enhancement on the rim in the arterial phase , located in the liver segment vi ( figure 1 ) . without any definite diagnosis from clinical imaging ,
the specimen showed necrotic tissue with trabecular pattern alignment of reticulin fibers in silver stain .
the immunohistochemistry examination showed no positive staining with ck7 , ck19 , ck20 , and hepatocyte in the tissue . since the possibility of hepatocellular carcinoma with necrotic change could not be ruled out , she underwent laparoscopic anatomical iv segmentectomy of the liver . on the cut section of the surgical specimen ,
a 1.2 1.2 cm sized , homogeneous , whitish - yellow - colored nodule with central small cystic area was observed in the liver segment iv .
the nodule was well demarcated from surrounding normal liver tissue with the fibrous capsulation ( figure 2 ) .
histologically , the liver nodule was necrotic tissue without viable cells and signs of inflammation , which had fibrous capsule and central cystic change ( figure 3 ) .
in silver stain , the nodule showed trabecular pattern alignment of ghost cells and reticulin fibers orthogonal to the capsule ( figure 4 ) , which was similar to the hepatocyte - originated neoplasm .
also , the findings of chronic hepatitis were observed in the background liver ( figure 5 ) .
there were no signs of bacterial or fungal organism and calcification around or inside the lesion .
although the nodule was located next to the peripheral glissonian pedicle of segment iv , there were no abnormal vessels around or inside the lesion .
her hospital stay was uneventful and she is well without any signs of recurrence and liver diseases for two and half years after surgery .
snn is a rare hepatic lesion , pathologically characterized by central amorphous necrotic core , sometimes accompanied with central cystic change , and enclosed by a hyalinized fibrotic capsule [ 14 ] . in the majority of cases ,
most snns are single small lesions and found most commonly under the superficial capsule in the right lobe [ 24 ] .
these characteristics are comparable to the present case . although snn is reported to occur in adult males predominantly ( 68.6% of cases ) , the present case was in an adult female .
snn appears as heterogeneous hypoechoic nodule with unclear margins on us and shows hypodensity lesion with peripheral enhancement when enhanced on ct scan .
differential diagnosis is hard for snn from intrahepatic cholangiocarcinoma and necrotic metastasis by us and ct scan [ 6 , 7 ] . also , percutaneous liver needle biopsy may not be useful for distinguishing snn from necrotic malignant tumor . in the present case
, we also performed surgery under the suspicion of malignant tumor with necrosis , such as hcc .
several pathogenetic hypotheses of snn are suggested : evolution of hemangioma , lesion of traumatic etiology , and sequelae of previous infection such as parasite [ 1 , 2 , 9 , 10 ] .
there are previous reports described this entity as a burnt - out phase of a variety of lesions and most of them lack specific etiology [ 11 , 12 ] . however , the alignment of ghost cells and reticulin fibers in the present case is different from previously reported snn , which origins were suggested as hemangioma or metastasis .
trabecular pattern alignment of ghost cells and reticulin fibers orthogonal to the capsule in the nodule and the findings of chronic hepatitis observed in the background liver may suggest that the nodule is a
burnt - out phase of hepatocyte - originated neoplasm , such as hcc , although there is no other supporting histological / radiological evidence or definite etiology for hepatitis . on the other hand
, there had been reports of spontaneous regression of hcc previously [ 13 , 14 ] .
the cause of the regression is suggested as tumor hypoxia or a systemic inflammatory response .
although there are no evident findings of ischemia ( hypoxia ) or inflammation in the present lesion , it is possible that an snn is occurred from spontaneous regression of hcc . furthermore , laparoscopic hepatectomy for this lesion with the difficulty of diagnosis should be useful , since it is usually located near the surface of the liver and easy to resect laparoscopically .
even anatomical resection of segment iv for the possibility of hcc in the present case had been performed safely .
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solitary necrotic nodule of the liver is a rare benign lesion with a completely necrotic core and a hyalinized fibrotic capsule containing elastic fibers .
the pathogenetic mechanism is still unclear .
we here describe a case of snn , whose central reticulin fibers within the nodule suggest the origin as hepatocellular carcinoma or other hepatocyte - origin tumors , treated with laparoscopic anatomical segmentectomy of the liver .
a 76-year - old japanese female , with no prior medical history and no symptom , visited our hospital with the heterogeneous hypoechoic lesion in the liver segment vi incidentally pointed out in abdominal ultrasonography . computed tomography with contrast demonstrated a 1.1 cm sized low - density lesion with mild ring enhancement on the rim in the arterial phase . since the possibility of malignant tumor with necrotic change could not be ruled out , she underwent laparoscopic anatomical segmentectomy of the liver . in the histological examination of the surgical specimen ,
the liver nodule was necrotic tissue without viable cells and signs of inflammation , which had fibrous capsule and central cystic change and showed trabecular pattern alignment of ghost cells and reticulin fibers orthogonal to the capsule .
also , the findings of chronic hepatitis were observed in the background liver .
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most urinary stones can be treated with extracorporeal shock wave lithotripsy ( eswl ) , but ureteroscopic removal of stones ( urs ) by use of intracorporeal lithotripsy devices has also been widely used as a valid , minimally invasive alternative to eswl . a gradual decrease in ureteroscope size and the development of effective lithotriptors
smaller ureteral stones can be extracted intact with endoscopic baskets or grasping devices after ureteral dilation , if necessary . however , larger ureteral stones require lithotripsy for the safe extraction of the stone fragments . to date , four techniques are available for intracorporeal lithotripsy : electrohydraulic lithotripsy ( ehl ) , laser lithotripsy , ultrasonic lithotripsy , and ballistic lithotripsy .
ehl was attempted in 1955 by yutkin and was the first technique developed for intracorporeal lithotripsy ( quoted from ) . in recent years , the development of a new lithotriptor using a holmium : yttrium aluminum garnet ( yag ) laser has increased the efficacy of urs for the treatment of ureteral stones .
the holmium laser is one of the safest , most effective , and most versatile intracorporeal lithotripters .
however , because of excellent flexibility and cost advantage , ehl has also been used for the treatment of ureteral stones .
the success rate of ehl is reported to be similar to that of laser lithotripsy .
it has been demonstrated that ehl successfully fragments 84 to 90% of stones without major complications . to the best of our knowledge , there have been few reports regarding the correlation between stone components and the outcomes of urs using ehl in patients with distal ureteral stones , although a few previous studies have reported the outcomes of ehl .
the present study was conducted to evaluate the influence of ureteral stone components on the outcomes of urs by ehl in patients with distal ureteral stones .
we retrospectively analyzed the medical records of 193 patients ( 131 males and 62 females ) who underwent urs by ehl for distal ureteral stones at our institution between march 2005 and april 2011 .
the present study included patients with solitary , unilateral , distal ureteral stones with a stone size of 0.5 to 2.0 cm that were completely removed by urs by use of ehl .
patients who had tight stone impaction with significant tissue edema , inflammatory polyps , or ureteral strictures were excluded from the study . in addition , severe obesity , urinary tract malformations , ureteral strictures , active urinary tract infections , uncorrected coagulation disorders , previous stone manipulations , and previous ureteral surgeries were considered as exclusion criteria .
the study protocol was approved by the institutional review board of hallym university kangnam sacred heart hospital .
stone size was defined as the largest diameter measured on abdominal computed tomography ( ct ) scans .
the distal ureter was defined as the segment between the lower border of the sacroiliac joint and the ureterovesical junction in accordance with previous studies .
urs procedures were performed by a single urologist in the lithotomy position under spinal anesthesia .
access to the ureter was gained by using a 7.5-fr rigid ureteroscope ( wolf , knittlingen , germany ) .
the ehl modality used was a calcutript ( karl stortz , tttlingen , germany ) electrohydraulic generator with a 3-fr lithotriptor probe .
all stones were fragmented under direct endoscopic vision , with simultaneous sterile saline irrigation using single pulses ( frequency a ) at level 2 intensity .
frasmented stones were extracted by using a 3-fr zero tip nitinol stone retrieval basket ( boston scientific , natick , ma , usa ) .
operating time was defined as the time interval from ureteroscope insertion to the complete removal of stones .
briefly , morphological examination of both the surface and sections of the stones was followed by sequential fourier transform infrared ( ft - ir ) spectroscopic analysis , with quantification of stone components .
ureteral stones were classified into 5 categories on the basis of the main component accounting for 50% or more of the stone content as follows : calcium oxalate monohydrate ( com ) , calcium oxalate dihydrate ( cod ) , carbonate apatite ( cap ) , uric acid ( ua ) , and struvite ( st ) .
all statistical analyses were performed by using one - way analysis of variance , followed by the bonferroni post hoc test for multiple comparisons and the pearson chi - squared test for categorical variables .
multivariate analysis was performed by means of logistic regression to assess predictive factors for successful fragmentation .
spss ver . 13.0 ( spss inc . , chicago , il , usa ) was used to perform all statistical analyses .
the mean operating time was 25.18.2 minutes ( range , 10 to 55 minutes ) .
the mean stone size as measured by abdominal ct was 1.150.44 cm ( median , 1 cm ; range , 0.5 to 2.0 cm ) .
minor complications were hematuria ( n=16 ) and postoperative ureteric colic ( n=10 ) , followed by fever ( n=3 ) , submucosal dissection ( n=3 ) , and minimal ureteric perforation ( n=2 ) .
double - j ureteric stents were placed as an auxiliary procedure in 14 patients ( 7.3% ) .
of the co stones , the proportions of com and cod stones were 74.4% and 25.6% , respectively .
the co stones were followed by ua ( 19.7% ) , cap ( 8.3% ) , and st ( 7.2% ) stones .
ureteral stones were classified into 5 categories on the basis of their main component ( table 3 ) .
there were no significant differences in sex or age between the individual groups ( p=0.56 and p=0.79 , respectively ) .
the mean stone size was not significantly different between the individual groups ( p=0.08 ) .
however , overall significant , between - group differences in the mean operating time were found ( p=0.038 ) .
the mean operating time was significantly longer in the ua group ( 28.68.3 minutes ) than in the com group ( 24.07.8 minutes , p=0.04 ) .
no significant differences were observed between the other groups in terms of the mean operating time .
overall complication rates and the rates of each complication were not significantly different between the groups .
cases with a mean operation time < 30 minutes and residual fragments <3 mm were considered successful fragmentation , as previously described .
univariate analysis of the factors showed that stone size and ua composition were associated with successful fragmentation by urs with ehl ( table 4 ) . in the multivariate analyses ,
the stone size was negatively associated with the odds ratio ( or ) for successful fragmentation .
ua as a stone component ( or , 0.42 ; 95% confidence interval , 0.20 to 0.89 , p=0.023 ) was also significantly important as a negative predictive factor for successful fragmentation after adjustment for stone size ( table 5 ) .
the purpose of this study was to evaluate the influence of ureteral stone components on the outcomes of urs by ehl in patients with distal ureteral stones .
ureteroscopic fragmentation of ureteral stones by use of intracorporeal lithotripsy is a popular , effective , minimally invasive technique and is considered to be the treatment of choice for lower ureteral stones .
previous studies have reported success rates ranging from 72 to 100% , which is similar to the success rate in our study for patients who were treated by urs with ehl ( 98.5% ) .
ehl has been widely used for endoscopic treatment of urinary stones since its introduction by yutkin in 1955 ( quoted from ) .
green and lytton reported their experience with ehl of ureteral stones with the use of a rigid ureteroscope and a 5-fr ehl probe .
the initially developed ehl probe had a long diameter ( 9-fr ) and thus had a narrow margin of safety owing to its relatively large size .
later advances in technology have allowed for the development of 1.6- to 5-fr probes , which are safer and are able to pass through short - diameter , flexible ureteroscopes .
fragmentation capability does not differ among probes , but larger probes appear to be more durable .
our surgical outcomes are in agreement with those of previous studies , regardless of probe size . the efficacy of ehl in a stone is determined by the fragility of the stone and the pressure generated by the lithotripsy device .
we used a single pulse ( frequency a ) and a level 2 intensity in all cases .
the collected stones were assessed by sequential ft - ir spectroscopic analysis and were categorized according to the main component .
ft - ir spectroscopy is currently the most widely used method for investigating the composition and structure of urinary stones [ 18 - 20 ] . in the present study ,
most stones showed mixed components , and the most common component was co , including com and cod , which accounted for 64.8% of all ureteral stones .
the proportions of stone components are consistent with those of previous studies . in our analysis ,
stone components influenced the mean operating time , which was significantly longer in the ua group than in the co group .
some studies have suggested that fragmentation failure may be due to a variety of stone components .
analyzed the fragmentation rate of 189 stones treated by ehl in relation to stone components . in that study
, they reported that 7 cases were ehl - resistant and that fragmentation failure may depend on the proportions of co and apatite and the organic matrix of the stones .
raney performed electrohydraulic cystolithotripsy on 37 patients and suggested that urate , phosphate , and carbonate stones are easy to crush , whereas oxalate and ua are more resistant to fragmentation .
other studies have demonstrated that fragmentation failure also depends on the surface characteristics of urinary stones .
the more lamellated and harder a stone , the more difficult it is to break .
stone surface characteristics may also play a role in fragmentation efficiency because rough stones have been reported to fragment more easily than smooth ones .
stones with smooth and round surfaces are difficult to fragment because it is hard to keep a probe in contact with their surface .
although we did not analyze surgical outcomes according to stone surface characteristics , our results may be explained by the surface characteristics and properties of ua stones .
ua stones usually form pebble - like objects with a smooth surface and are the hardest among human urinary stones .
a cross - section of the ua stone usually shows concentric layers of microcrystalline material that form typical laminations with radial striations converging to the core area .
we found that the mean operating time increased with increasing percentage of ua in ureteral stones .
ua stones exist most often in a pure state , and thus they are rarely accompanied by phosphate or calcium oxalate . in our study , ua stones were more resistant to ehl fragmentation than were stones containing the other components .
previous studies have reported that the major disadvantage of ehl is its propensity to damage the ureteral mucosa and its association with ureteral perforation and the risk of perforation , especially in impacted stones associated with significant mucosal edema .
concerning postoperative complications , submucosal dissection occurred in 3 patients ( 1.6% ) and minimal ureter perforation occurred in 2 patients ( 1.0% ) , but no major complications were observed .
the reason for this lack of major complications and the lower prevalence of minor complications is that high - risk groups were excluded .
instead , we measured the mean operating time from ureteroscope insertion to the complete removal of ureteral stones . considering ureteroscope manipulation time , the efficacy of ehl fragmentation may have been underestimated
. however , most of the operating time as defined by our method was taken up by fragmentation ; thus , the difference may be minimal .
to the best of our knowledge , there have been few reports on the influence of stone components on the outcomes of urs using ehl in patients with urinary stones . despite the limitations of our study , our study has the strength of evaluating operating time in relation to stone components .
many recent studies have demonstrated that holmium : yag laser lithotripsy is superior to ehl in terms of stone fragmentation and postoperative complications .
however , ehl is the cheapest intracorporeal lithotripsy method because it requires only an inexpensive generator and probes .
ehl can fragment urinary stones , regardless of stone type , and can be a good alternative to laser lithotripsy .
the results of this study suggest that successful fragmentation by urs with ehl may be associated with the proportion of the ua component .
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purposewe evaluated the influence of urinary stone components on the outcomes of ureteroscopic removal of stones ( urs ) by electrohydraulic lithotripsy ( ehl ) in patients with distal ureteral stones.materials and methodspatients with a single distal ureteral stone with a stone size of 0.5 to 2.0 cm that was completely removed by use of ehl were included in the study .
operating time was defined as the time interval between ureteroscope insertion and complete removal of ureteral stones .
ureteral stones were classified into 5 categories on the basis of their main component ( that accounting for 50% or more of the stone content ) as follows : calcium oxalate monohydrate ( com ) , calcium oxalate dihydrate , carbonate apatite ( cap ) , uric acid ( ua ) , and struvite ( st).resultsa total of 193 patients ( 131 males and 62 females ) underwent ehl . the mean operating time was 25.18.2 minutes and the mean stone size was 1.150.44 cm .
calcium oxalate stones accounted for 64.8% of all ureteral stones , followed by ua ( 19.7% ) , cap ( 8.3% ) , and st ( 7.2% ) stones .
the mean operating time was significantly longer in the ua group ( 28.68.3 minutes ) than in the com group ( 24.07.8 minutes , p=0.04 ) .
in multivariate analyses , the stone size was negatively associated with the odds ratio ( or ) for successful fragmentation .
ua as a main component ( or , 0.42 ; 95% confidence interval , 0.20 to 0.89 ; p=0.023 ) was also found to be significantly important as a negative predictive factor of successful fragmentation after adjustment for stone size.conclusionsthe results of the present study suggest that successful fragmentation by urs with ehl could be associated with the proportion of the ua component .
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this condition of abnormal visceral rotation was known in animals since the days of aristotle .
it is associated with a number of other conditions such as kartagener 's ( bronchiectasis , sinusitis , situs inversus ) and cardiac anomalies .
there is no current evidence showing increased incidence of cholelithiasis in patients with situs inversus totalis .
after mouret performed first laparoscopic cholecystectomy in 1987 , it has become a gold standard in treatment of cholelithiasis .
the first known laparoscopic cholecystectomy in situs inversus has been reported by campos and sipes in 1991 . due to the very nature of reversed anatomy and possibility of associated anomalies
a 47-year - old female presented with pain since 15 days in left hypochondrium and left iliac fossa region .
ultrasonogrphy of abdomen revealed cholelithiasis with probe tenderness at left mcburney 's point with situs inversus .
she was posted for laparoscopic cholecystectomy and appendicectomy . or arrangement - mirror image of routine laparoscopic cholecystectomy .
anaesthetist was at the head end , monitor was placed on the patient 's left side and surgeon with camera assistant was on the right side of the patient .
a 10 mm camera port was placed at supraumbilical region , epigastric 10 mm port to the surgeon 's left and right hand as and when necessary . a 5 mm mid clavicular port to the surgeon 's right hand occasionally to be hold by assistant .
a 5 mm port was placed in the anterior axillary line at the level of umbilicus for gall bladder retraction .
x - ray chest showing dextro cardia ct scan showing left sided liver operating instruments , hook with diathermy was passed from epigastric port and managed with right / left hand of surgeon while traction at infundibulum was exerted by the assistant .
dissection at fossa was done by surgeon 's right hand at mid clavicular port and gall bladder manipulated by surgeon 's left hand through epigastric port .
intra operative picture showing gall bladder on left of ligamentum teres dissection of cystic duct for simultaneous appendicectomy , same ports were used even though the positions were cumbersome .
anterior axillary port was used for surgeon 's right hand , while for left hand ; mid - clavicular port was used .
appendix with cecum was visualised in the left iliac fossa region somewhat high up in position .
this entity is considered to have a genetic predisposition that is autosomal recessive with the defect being localised on the long arm of chromosome 14 .
there is no evidence that situs inversus predisposes to cholelithiasis , but it may be a cause of diagnostic confusion .
delay in the diagnosis was due to the left upper abdominal pain and unknown situs inversus .
internet search has revealed 36 reported cases of cholecystectomy in situs inversus including our case [ table 1 ] . of these , 11 are males and 25 are females .
the age group is from 20 to 80 years . of the reported cases , 26 are situs inversus totalis , while in 4 cases ; the condition has not been specified .
the procedure becomes difficult because of the fact that the anatomy is mirror image of that of routinely seen and most surgeons are right handed .
this needs alteration in technique of cholecystectomy . when chances of about 25% biliary anatomical variations are considered , the chances of iatrogenic injuries increases .
publications on situs inversus and laparoscopic cholecystectomy the dissection is difficult , as commonly used right hand has to be used for retraction and left for dissection . if this anatomical variation makes the picture confusing , then early conversion should be thought . in our patient ,
a number of papers have suggested different port positions , but we used mirror image of standard laparoscopic cholecystectomy ports .
the pubmed search has revealed the use of laparoscopy for various procedures in cases of situs inversus .
the difficulties faced by all the surgeons are mainly because of the mirror image anatomy and change in the use of the dominant hand while dissecting . in conclusion ,
it needs modification in technique , proper planning and with cautious dissection ; the procedure can be safely completed .
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situs nversus totalis is a rare condition which presents difficulty in clinical diagnosis as well as laparoscopic surgery for cholelithiasis .
the mirror image anatomy makes the laparoscopic intervention difficult even for an experienced surgeon .
presenting here is a case report and review of literature .
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corynebacterium species are non - spore - forming aerobic gram - positive bacilli that are spread widely in the environment and constitute part of the normal flora on skin and mucosa in humans . in a hospital setting , non - diphtheriae corynebacterium are commonly considered contaminants when found in blood cultures because of their low virulence .
recently , there have been increasingly frequent reports of infection by non - diphtheriae corynebacterium in immunocompromised patients who were hospitalized or in immunocompetent patients who had implanted medical devices .
corynebacterium striatum is a commonly - isolated corynebacteria in the clinical microbiology laboratory , although confirmed infections from sterile sites are relatively rare .
c. striatum can cause pulmonary abscess , meningitis , septic arthritis , vertebral osteomyelitis , catheter - related blood stream infection and native- and prosthetic - valve endocarditis .
the aid of molecular diagnostic methods , especially the 16s rrna and rpob gene sequencing , has greatly improved the ability to detect c. striatum in clinical specimen . here , we report a case of native - valve endocarditis caused by c. striatum , which was accurately identified by 16s ribosomal rna sequencing in a non - immunocompromised patient .
a 55-year - old man with no significant medical history was transferred to our hospital with a 14-day history of fever and lethargy . over the previous 5 weeks
two weeks prior to transfer , a high fever suddenly developed and persisted despite administration of broad - spectrum antibiotics including ceftriaxone , piperacillin - tazobactam , or meropenem in combination with moxifloxacin .
there was a healing abrasion on his hand after the car accident , but there were no stigmata of endocarditis .
on admission to our hospital , his blood pressure was 150/100 mmhg , pulse rate was regular at 100 beats per minute , and temperature was 38.0c .
laboratory data were as follows : hemoglobin , 11.2 g / dl ; platelets , 251,000/mm ; c - reactive protein , 79.7 mg / dl ; procalcitonin 0.18 ng / ml ; and erythrocyte sedimentation rate , 52 mm / h .
electrocardiogram showed normal sinus rhythm . computed tomography ( ct ) of the chest showed pulmonary emphysema , and abdominal ct was normal .
the patient initially received 2 g ceftazidime intravenously every 8 hours in combination with oral metronidazole ( 500 mg every 8 hours ) . on the third day of admission , 2 separate sets of blood cultures were positive for gram - positive cocci .
after the second set of blood cultures , intravenous vancomycin ( 1 g every 12 h ) was added empirically . on the sixth day , kocuria kristinae was identified in both blood cultures using the vitek 2 system ( biomrieux , marcy - letoile , france ) in the absence of definitive laboratory guidelines for determining the antibiotic susceptibility . while the initial blood culture was regarded as contaminated
, when the same pathogen was isolated from the consecutive blood culture , we determined that k. kristinae might be the true pathogen . at this time , initial empiric ceftazidime and metronidazole were discontinued and intravenous vancomycin was maintained . because k. kristinae is not a common infective endocarditis pathogen
, we also performed 16s rrna sequencing analysis to confirm the identification of the pathogen in the blood isolates .
the universal eubacterial primers fu1 ( 5-ttggagagtttgatcctggctc-3 ) and ru2 ( ggactaccagggtatctaa-3 ) were used . the 16s rrna gene sequence ( 766 bp )
the sequence was 99.00% identical to that of c. striatum was identified ( genbank accession number jf342700.1 ) .
transthoracic echocardiography showed 2 mobile oscillating masses at the tip of the mitral valve leaflet .
transesophageal echocardiography revealed two hypermobile echogenic masses on the anterior and posterior mitral valve leaflets , 10 mm and 8 mm , respectively ( fig .
1 ) . there was no involvement of either the subvalvular structure or paravalvular structure .
large , hypermobile vegetations were attached to the middle scallop of the anterior ( arrowhead ) and posterior mitral valve leaflets ( arrow ) , 10 mm and 8 mm , respectively .
vegetation on the posterior mitral valve leaflet showed a 7 mm lineal mobile structure , which indicates a high embolic risk .
thus , the patient was diagnosed with c. striatum infective endocarditis not k. kristinae infective endocarditis .
interestingly , k. kristinae , which was found in the blood cultures , was also cultured from mitral valve vegetation despite administration of vancomycin for 14 days .
the patient completed 6 weeks of intravenous vancomycin for prosthetic valve endocarditis . at a follow - up over one year
non - diphtheriae corynebacterium are commonly isolated from clinical specimens but are typically considered contaminants .
in addition to c. diphtheriae , corynebacterium are considered organisms that are normal inhabitants of human skin and respiratory tract . also , with the use of molecular diagnostic methods , the taxonomy of corynebacterium species has changed and has been reclassified from earlier defined taxa in recent years .
there is an increased frequency of reported non - diphtheriae corynebacterium infections , particularly as a cause of nosocomial infection in hospitalized and immunocompromised patients , though these pathogens are widely distributed in the environment and mucous membranes of humans .
common nosocomial pathogens include corynebacterium amycolatum , corynebacterium jeikeium , corynebacterium urealyticum , and c. striatum . c. striatum is widely distributed in the environment , especially in hospital settings associated with nosocomial infection .
it colonizes on the skin and mucous membranes of normal hosts and hospitalized patients , and disruption of its integument may lead to bacteremia and subsequent septic complications in either immunocompromised patients or patients with medical devices .
in addition , while c. striatum is frequently isolated in polymicrobial infections , its degree of pathogenicity is unclear , and differentiation of colonization from pathogen - causing infection has been difficult .
this organism can cause pneumonia , empyema , meningitis , septic arthritis , vertebral osteomyelitis , and endocarditis . c. striatum is an uncommon cause of infective endocarditis .
an english language literature search in medline revealed 22 previously reported cases of endocarditis due to c. striatum ( table 1 ) .
more than half of cases are hospital - acquired infection , and 11 cases ( 50% ) are associated with medical devices , including 4 cases of prosthetic valve , 3 cases of pacemaker lead , 3 cases of vascular access for hemodialysis , and 1 case of ventriculo - atrial shunt .
valve replacements are performed in one quarter of the cases and overall mortality approaches 27% . in our case
, the patient had recently experienced a long - term hospital stay and had a scar from an abrasion received during a traffic accident , but had not required a central venous catheter .
tte , transthoracic echocardiography ; tee , transesophageal echocardiography ; avr , aortic valve replacement ; yr , years ; m , months ; dm , diabetes mellitus ; chf , congestive heart failure ; esrd , end - stage renal disease ; anca , anti - neutrophil cytoplasmic antibody ; d , days ; ihd , ischemic heart disease ; mvr , mitral valve replacement ; hd , hemodialysis ; arf , acute renal failure ; af , atrial fibrillation ; cva , cerebrovascular accident ; crf , chronic renal failure . one interesting finding is that c. striatum can be misidentified as k. kristinae using automated systems ( biomrieux vitek 2 gp card ) .
though api coryne system is a useful tool for identifying corynebacterium species in the clinical laboratory , this may not incorporate all of the tests necessary for the identification of every corynebacterium species . in recent years
, the introduction of molecular methods , especially 16s rrna gene and rpob gene sequencing , has improved the ability to identification of corynebacterium species . in our case
, the causative organism was misidentified as k. kristinae by the commercial identification kit but was confirmed as c. striatum by 16s rrna sequence analysis .
there is a previous similar case report of misidentification of c. striatum as k. kristinae using the commercial identification kit . in literature review , there are only 3 cases of infective endocarditis caused by c. striatum identified by 16s rrna sequencing .
clinically , when organisms such as c. striatum or k. kristinae are identified from subsequent blood cultures in unusual clinical scenarios , we suggest a genotypic assay , such as 16s rrna , to confirm species identity . additionally , 16s rrna sequencing of resected endocardial specimen is useful tool for verifying the causative agent .
bosshard et al . suggested that the sensitivity , specificity , and positive and negative predictive values of 16s rrna sequencing were 94% , 100% , 100% , and 90% , for cases of native valve endocarditis . in our case , unfortunately , 16s rrna sequencing on vegetation acquired in the operative field was not performed .
however , because organisms detected from blood and vegetation were both misidentified as k. kristinae using the commercial identification kit , the microorganisms in the vegetation might be c. striatum . despite the excellent specificity of 16s rrna sequencing
, clinicians have to be aware of the interpretation of a positive result within clinical context ; even several months after completion of successful therapy for endocarditis , 16s rrna sequencing results may still be positive .
however , c. striatum may be susceptible to vancomycin but resistant to penicillin , ciprofloxacin , erythromycin , rifampin , and tetracycline ; it has variable susceptibility to other -lactams and aminoglycosides .
recently , the emergence of multidrug - resistant strains acting as nosocomial pathogens was reported in long - term hospitalized patients with underlying disease , and the most effective antibiotic was vancomycin .
there are no definitive laboratory guidelines for determining the antibiotic susceptibility of coryneform bacteria , and in our case , susceptibility testing was not available .
we treated the patient with vancomycin , which was reported as the most active antibiotic against corynebacteria in the literature , and clinical response was good .
defervescence was achieved after 3 days of vancomycin treatment . in a literature review of c. striatum endocarditis
, there were more cases treated with medical therapy alone than with valve replacement ( table 1 ) . in our case , we performed mitral valve replacement therapy due to high embolic risk .
interestingly , resected vegetation was culture - positive despite 14 days of vancomycin treatment , so we administrated antibiotics for another 4 weeks after valve resection .
this bacterial persistence might be associated with multidrug resistance or a high tolerance to antibiotic - induced killing in c. striatum . according to yoo et al .
, we need awareness of the emergence of multidrug - resistant c. striatum in korea ; however , further investigation is required . because corynebacterium species are usually considered to be contaminants and are not routinely identified to the species level , c. striatum infection rates are probably underestimated . in unusual clinical scenarios , blood cultures
positive for corynebacterium species or k. kristinae , as determined by a commercial automatic culture system , should not be overlooked .
the possibility of c. striatum infective endocarditis should be considered even in a patient without structural heart disease or prosthetic valves , and the genotypic assay , such as 16s rrna sequence analysis , may be very useful .
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corynebacterium species are non - fermentous gram - positive bacilli that are normal flora of human skin and mucous membranes and are commonly isolated in clinical specimens .
non - diphtheriae corynebacterium are regarded as contaminants when found in blood culture .
currently , corynebacterium striatum is considered one of the emerging nosocomial agents implicated in endocarditis and serious infections .
we report a case of native - valve infective endocarditis caused by c. striatum , which was misidentified by automated identification system but identified accurately by 16s ribosomal rna sequencing , in a 55-year - old male patient .
the patient had two mobile vegetations on his mitral valve , both of which had high embolic risk . through surgical valve replacement and an antibiotic regimen ,
the patient recovered completely .
in unusual clinical scenarios , c. striatum should not be simply dismissed as a contaminant when isolated from clinical specimens .
the possibility of c. striatum infection should be considered even in an immunocompetent patient , and we suggest a genotypic assay , such as 16s rrna sequencing , to confirm species identity .
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acute kidney injury early after transplantation may arise from the donor 's condition , prolonged cold ischemia time , and early posttransplant injuries like rejection and drug toxicity .
immediate clinical correlates of this injury may be delayed graft function or a less - than - optimal glomerular filtration rate .
however , acute kidney injury carries adverse long - term consequences as patients with acute tubular injury early posttransplantation have an inferior long - term allograft function .
therefore , markers , which facilitate early diagnosis of acute kidney injury or even provide prognostic information , would be helpful in the posttransplant care of these patients .
traditionally , serum parameters as creatinine and urine output are used to monitor kidney function in transplanted patients . however
, a rise in serum creatinine already implies a significant amount of kidney damage , which limits its ability to detect impaired graft function at early stages .
recently , novel biomarkers such as kim-1 , il-18 , and neutrophil gelatinase - associated lipocalin ( ngal ) have been proven useful in detecting nontransplant acute kidney damage .
ngal , a 25-kda protein , is a member of the lipocalin family , which is expressed in many human tissues , including the kidney . in children and adults with cardiopulmonary surgery ,
ngal was specifically and early elevated in those patients that developed acute renal failure a few days after surgery .
reported that urinary ngal could be an early predictor of dgf after kidney transplantation . in a previous study by mishra et al . with immunochemical staining of renal allografts , a correlation was established between an increased expression of ngal and prolonged cold ischemia time , elevated serum creatinine levels , and dialysis requirement . in another study ,
urinary ngal was more accurate in predicting dialysis within one week of transplantation than serum creatinine .
acute kidney injury primarily affects renal tubules and ngal is produced in injured tubular epithelial cells .
therefore , we addressed the question whether urinary ngal correlates with histologically confirmed acute tubular injury using protocol biopsies taken within the first six months after transplantation .
the study population consisted of 140 adult kidney transplant recipients randomly chosen from our protocol biopsy archive ( n = 35 patients who never had any signs of ati and n = 105 patients with at least one finding of ati during the protocol biopsy program ) .
controls included 9 healthy individuals ( 2-kidney controls ) , 13 individuals after kidney donation ( 1-kidney control ) , and 5 icu patients with acute kidney failure .
renal protocol biopsies are regularly performed 6 weeks and 3 and 6 months after kidney transplantation in the transplant center of the medical school of hannover since 2001 .
all patients are eligible for the protocol biopsies unless certain medical conditions like bleeding disorders and other comorbidities advice against a biopsy .
in addition to these regular protocol biopsies , biopsies for cause are performed in case of unclear allograft impairment .
demographic and clinical data of patients participating in this program were collected prior to and at the time of transplantation .
the demographics for patients with and without ati are depicted in table 1 . for patients without ati n
= 23 samples were taken at 6 weeks , n = 17 at 3 months , and n = 16 at 6 months after transplantation . for patients with signs of ati
n = 44 samples were taken at 6 weeks , n = 3 samples at 3 months , and n = 55 samples at 6 months .
after transplantation , clinical data and routine laboratory results were collected corresponding to the time points of the three protocol biopsies .
data and sample collections were performed with an informed consent of the patients and with the approval of the ethics board of the medical school hannover .
urinary ngal was measured in the spot urine using the quantikine human lipocalin-2/ngal immunoassay ( cat.no .
urinary ngal excretion was normalized to urinary creatinine excretion to correct for differences in ngal due to urine dilution .
urinary ngal excretion is presented as the amount of urinary ngal in ng per g of urine creatinine .
comparisons of categorical data between groups were performed with fisher 's exact test and the chi - square test for two or more samples .
numerical data were compared with the kruskal - wallis test and the mann - whitney test .
linear regression analyses were performed with log transformed ngal values as the dependent variable and the demographical , clinical , and laboratory factors that had been significantly different in univariate analyses .
variables from the univariate analyses were separately examined for the samples at 6 weeks and 6 months using the backward selection .
note that for better visualisation the logarithm to base 10 was used in all figures with ngal results , because these values were highly variable and not normally distributed .
first we compared the ungal excretion levels of renal allograft recipients without ati to different control groups .
there was no significant difference in ungal excretion in transplanted patients versus healthy individuals with 1 kidney ( i.e. , kidney donors ) and healthy individuals with 2 kidneys ( figure 1 ) .
in nontransplanted patients with acute renal failure , the ngal / creatinine ratio was significantly higher 2.0490e + 006 ng / g ( 382498 to 1.0470e + 007 ) compared to healthy individuals with one or two kidneys and to transplanted patients without signs of ati in the biopsies up to 6 months after transplantation ( p < 0.001 ) .
subsequent analyses represent a cross - sectional study of samples where not all patients provided urine samples at every time point , showing no time - associated differences in samples taken at 6 weeks , 3 months , and 6 months after transplantation ( figure 2(a ) ) .
figure 2(b ) depicts the dynamic change of ungal excretion at 6 weeks , 3 months and 6 months after transplantation in individual patients without ati .
next , we tested the sensitivity of urinary ngal as a biomarker to indicate ati in transplanted patients . at six weeks , patients with ati had urinary ngal values that were two - fold higher than the values from patients without ati in the biopsies , yet there was a considerable overlap of values between the two groups . at 6 months , no differences between patients with and without ati were apparent ( figure 3(a ) ) .
multiple findings of ati did not correlate with higher ungal levels ( figure 4(a ) ) .
moreover , the extent of ati , whether diffuse or focal , did not cause a difference of the ngal ratios ( figure 4(b ) ) . in an explorative analysis , we correlated ngal levels to allograft damage ( e.g. , acute tubular injury , delayed graft function , donor age ) and to additional clinical , laboratory , and demographic variables ( for a complete list see supplementary materials available online at doi:10.1155/2012/563404 ) .
female recipient gender was associated with significant higher ngal ratios at 6 weeks and 6 months after transplantation . regarding donor gender , a numerical trend towards higher
a lower gfr at the time of sampling was weakly associated with higher ngal ratios at 6 months .
furthermore , we analyzed whether acute impairment of the allograft function at the time of biopsy had any relation to ngal levels .
serum creatinine levels at the day of biopsy showed changes relative to the baseline values before the biopsy from 29 to 129% in patients without acute tubular injury and from 40 to 60% in patients with acute tubular injury . however , a correlation of the change in creatinine with ungal excretion could not be established for these patients ( r 0.1 ; data not shown ) .
isometric vacuolization and prevalence of chronic graft changes ( cgrade>0 ) according to the current banff classification at 6 weeks after transplantation were associated with significantly higher ungal levels . however , after 6 months , ungal ratios were lower in patients with isometric vacuolization .
in addition , patients with urinary tract infection at the time of sampling had significantly higher ungal ratios at 6 weeks as well as 6 months ( table 2 ) .
next , linear regression analyses were performed with log transformed ngal values to determine the contribution of the identified variables of the explorative univariate analysis to the observed ngal levels .
significant variables from the univariate analyses were separately examined for the samples at 6 weeks and 6 months using the backward selection . for the 6-week samples ( table 3(a ) ) , presence of urinary tract infection and isometric vacuolization of tubular epithelial cells in the concomitant biopsy were associated with higher ngal levels ( r = 0.296 ; unadjusted r square of model fit ) .
recipient gender was not a significant factor in the regression model at 6 weeks ( r = 0.138 ; p = 0.131 ) . in the 6-month samples ( table 3(b ) ) , urinary tract infection , female recipient gender , and absence of isometric vacuolization were associated with higher ngal levels ( r = 0.357 ) .
since the univariate analysis indicated a relation between donor and recipient gender and ngal levels , the possible gender combinations are additionally presented ( table 4 ) .
significant differences were observed between the male donor / male recipient group compared to all other possible combinations , except the female / male combination at six weeks ( p = 0.20 ) .
acute tubular injury ( ati ) is a frequent finding in biopsies of renal allograft recipients and is linked to an inferior long - term allograft function . in this study , we tested whether urinary ngal ( ungal ) may be applicable as a sensitive and noninvasive biomarker to monitor ati in renal allograft recipients .
ngal can be produced locally in the kidney by injured tubular cells and can be secreted by activated neutrophils / macrophages or inflamed vasculature . in a recent experimental study ,
this group generated an ngal reporter mouse , which showed a close correlation between tubular stress and ungal .
thus , ungal levels could reflect kidney damage in real time , as discussed by mori and nakao . in animal models ,
ngal has shown to be the most upregulated gene and overexpressed protein with a high predictive value for acute kidney injury [ 1113 ] .
we established the baseline ungal excretion in our transplanted patient cohort without findings of ati .
ungal levels in this group were comparable to healthy controls and remained stable 6 weeks , 3 months , and 6 months after transplantation .
as proper controls for patients with a renal allograft , we collected sample material from healthy individuals with one kidney ( donors with normal kidney function ) , in addition to healthy controls with 2 kidneys .
our observation of a tendency towards a higher ngal excretion in healthy individuals with 2 kidneys versus 1 kidney may point to a confounding effect by the kidney mass .
this finding should be confirmed with a larger cohort . in patients with ati ,
median ungal ratios were significantly higher at 6 weeks after transplantation than in patients without signs of ati .
while these findings may indicate a correlation of ungal excretion to ati in patients early after transplantation , we could not demonstrate increased ungal levels in patients with ati at 6 months after transplantation . on the contrary ,
median ungal levels in patients with ati were even lower at 6 months compared to the controls without ati .
further analyses showed no differences of ngal excretion depending on the frequency of ati or the distinction between diffuse versus focal ati .
ngal levels were weakly inversely correlated to the egfr ; however , no link could be established between ungal levels and acute rises in serum creatinine at the time of ati diagnosis .
tubulointerstitial lesions in these patients were characterized by tubular atrophy , interstitial fibrosis , and inflammatory cell infiltration .
acute tubular injury in our transplanted patients differed from these features as it was defined by epithelial swelling with lucency of the cytoplasm , loss of brush border , and/or luminal dilatation with flattening of the epithelium and cytoplasmatic vacuolization . furthermore , even though a 2-fold increase in ngal was present in our patients at 6 weeks , the substantial overlap with values from patients without ati precludes the use of ngal as a sensitive marker for kidney injury in transplant patients .
hollmen et al . found that high urinary ngal levels until day 14 after transplantation in patients with early graft function ( egf ) were associated with worse kidney function at 3 weeks , but not at 3 months or 1 year after kidney transplantation .
most studies validating ngal as a marker for allograft function emphasize its predictive value first and foremost in early urine or serum samples , up to 2 weeks after transplantation [ 4 , 5 ] .
ngal showed to be an early marker for aki and subsequent dialysis or dgf in transplanted patients by means of immuneohistochemical staining , urinary excretion levels , and serum levels [ 5 , 6 , 15 , 16 ] . in our patient population with ati findings , a considerable proportion ( 68.8% )
had delayed graft function and need for posttransplantation , yet this was not reflected by a relevantly elevated urinary ngal value at 6 weeks posttransplantation .
further , in patients with stable graft function , subclinical tubulitis was associated with higher ngal levels . in our regression analyses , ungal ratios correlated significantly with isometric vacuolization 6 weeks after transplantation , which could be related to immunosuppressant toxicity and damage , whereas isometrical vacuolization at 6 months was associated with lower ngal levels .
this raises the question whether ungal ratios in transplanted patients really reflect the ongoing tubular damage or whether ngal may be more of an indicator for regenerative capacities of the kidney .
plasma ngal measurements can be influenced by numerous clinical variables , such as hypertension , systemic infections , age , and a higher validity in children .
in our linear regression analyses , we confirmed that in patients with concomitant urinary tract infection at 6 weeks and at 6 months posttransplantation ungal ratios were significantly higher .
this could be related to the leukocytes present in the urine as shown by decavele et al . who demonstrated a correlation of one urinary leukocyte to an increase of 12 pg of ungal .
one other important but unexpected finding of our study was that female patients with grafts from female kidney donors as well as female graft recipients with male or female donors had higher ungal ratios compared to male recipients with kidneys from male donors .
further parameters such as coronary heart disease and blood transfusion before transplantation could be correlated to higher ngal levels after 6 months but not at 6 weeks after transplantation .
these variables showed no significant association to ungal levels in subsequent multivariate analyses but may indicate other potential confounders in transplanted patients .
the limitations to our study are that we present a single - centre study and that urine samples were stored at 80c for more than a year and some protein degradation may have occurred during the freeze - thaw cycle thus , this study should be confirmed with immediate ungal measurements in fresh urine samples .
in addition we do not have information about the ungal levels in our patients prior to transplantation or in the donors and some patients did not provide urine samples at all time points when protocol biopsies were taken .
thus , further studies are necessary to evaluate the diagnostic value of ungal measurements in patients after kidney transplantation . in conclusion , even though ngal may be useful in classifying patients with established aki or glomerulonephritis in native kidneys or may be a predictor for delayed graft function and recovery early after kidney transplantation [ 1416 , 23 ] , it is not a sensitive tool for monitoring for ati at later times after transplantation .
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urinary neutrophil gelatinase - associated lipocalin ( ungal ) is known to predict the prolonged delayed graft function after kidney transplantation .
we examined the relation of ungal with histological findings of acute tubular injury ( ati ) .
analyses were made in biopsies taken at 6 weeks , 3 months , and 6 months after kidney transplantation .
ungal was measured in the spot urines , normalized to urinary creatinine excretion , and correlated to biopsy findings and clinical , laboratory , and demographic variables .
controls included healthy individuals , individuals after kidney donation and icu patients with acute kidney failure .
renal transplant recipients without ati did not display elevated ungal levels compared to the healthy controls .
transplant patients with ati had a higher ungal excretion at 6 weeks than patients without ati ( 27,435 versus 13,605 ng / g ; p = 0.031 ) .
this increase in ungal was minor compared to icu patients with acute renal failure ( 2.05 106 ng / g ) .
patients with repeated findings of ati or severe ati did not have higher urinary ngal levels compared to those with only one ati finding or moderate ati .
female recipient gender and urinary tract infection were identified as potential confounders .
ungal has a relation with histological signs of acute tubular injury .
the usability of this biomarker in renal allograft recipients is limited because of the low sensitivity .
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it has long been considered as resulting from environmental factors such as ultraviolet light exposure .
every year , more than 200,000 people were diagnosed with melanoma and it results in more than 50,000 deaths worldwide .
the incidence of melanoma has been increasing at a high speed in recent years . until now
, surgical resection and chemotherapy are still the best curative options for the treatment of melanoma .
however , the postoperative 5-year survival rate is only 510% due to the high rate of metastasis in patients who have had resection .
this study aimed to identify novel biomarkers and to elucidate the molecular mechanisms underlying melanoma metastasis . in recent years , extensive studies have indicated that micrornas ( mirnas ) are widely involved in the prognosis , metastasis , and overall patient survival of melanoma .
mirna is a kind of small noncoding rna that regulates gene expression at post - transcriptional level .
the major function of mirna is to regulate the gene expression by binding to the 3- untranslated region of target mrnas . until now , more than 2000
these mirnas participate in many key cellular processes including proliferation , apoptosis , and metastasis .
de - regulation of mirna expression has been identified in many cancer types , including melanoma .
their functions as etiologic factors in melanoma have been widely revealed . however , these studies are very limited , and the results were inconclusive .
the underlying pathogenesis of melanoma , especially for their metastasis , remains far from understood . to derive a more precise estimation of the association between these mirnas and melanoma ,
a meta - analysis is needed to help us to better understand the possible risk factor for melanoma metastasis . in this study
, we collected several mirna expression profiling datasets for melanoma , and then a meta - analysis was performed to identify the differentially expressed mirnas between metastatic and nonmetastatic samples .
functional annotation of the mirna target genes was further performed to explore the putative links between these mirnas and melanoma metastasis .
we demonstrated that signaling pathways involved in many fundamental biological processes are linked to the metastasis of melanoma , such as focal adhesion pathway and mitogen - activated protein kinase ( mapk ) signaling .
our results provide clues for the mechanism of melanoma metastasis and could be used for mirna - based drug development for preventing this disease .
we first searched the pubmed and gene expression omnibus database to collect mirna expression profiling datasets about melanoma .
key words including melanoma , mirna , microarray , and gene expression profiling were used for data searching . only the original experimental works published before june 2016 for analyzing the metastasis of melanoma were retained ,
finally , a total of seven expression profiles which included 82 metastatic and 87 nonmetastatic samples were collected [ table 1 ] .
characteristics of datasets included in this analysis geo : gene expression omnibus . for each dataset ,
the missing values in the original expression values were imputed by using the r package of impute , and then the expression values were logarithmically transformed ( base 2 ) and quantile normalized for each dataset .
the average expression value was used if there were multiple probes for a given mirna in each sample . to make the expression profiles from different comparable studies , the z - score transformation approach was used to calculate the expression intensities for each gene expression profile to remove the inconsistency and heterogeneity .
z - score was defined as the difference of raw intensity data for each gene and the average gene intensity within a single experiment divided by the standard deviation of all of the measured intensities .
the predicted targets of mirnas were obtained from targetscan , miranda , and pita databases .
the predicted targets obtained from at least two programs were regarded as the reliable target genes . based on the interactions of mirnas and target genes , the mirna function network was built .
the gene ontology ( go ) enrichment analysis was performed to investigate the main function of target genes by using the web - based software gorilla .
adjusted p values were computed for the go terms by multiple test adjustment of benjamini - hochberg ( bh ) .
in addition , for identifying the biological pathways associated with the metastasis of melanoma , we also performed the kyoto encyclopedia of genes and genomes ( kegg ) enrichment analysis of target genes . to get the overview of the global shifts of gene expression in metastasis samples ,
the limma algorithm was used to identify the differentially expressed mirnas between metastasis and control samples .
the bh method was used for multiple test correction . false discovery rate ( fdr ) < 0.01 was selected as the criteria for calculating significant differences .
we first searched the pubmed and gene expression omnibus database to collect mirna expression profiling datasets about melanoma .
key words including melanoma , mirna , microarray , and gene expression profiling were used for data searching . only the original experimental works published before june 2016 for analyzing the metastasis of melanoma were retained ,
finally , a total of seven expression profiles which included 82 metastatic and 87 nonmetastatic samples were collected [ table 1 ] .
for each dataset , the missing values in the original expression values were imputed by using the r package of impute , and then the expression values were logarithmically transformed ( base 2 ) and quantile normalized for each dataset .
the average expression value was used if there were multiple probes for a given mirna in each sample . to make the expression profiles from different comparable studies , the z - score transformation approach was used to calculate the expression intensities for each gene expression profile to remove the inconsistency and heterogeneity .
z - score was defined as the difference of raw intensity data for each gene and the average gene intensity within a single experiment divided by the standard deviation of all of the measured intensities .
the predicted targets of mirnas were obtained from targetscan , miranda , and pita databases .
the predicted targets obtained from at least two programs were regarded as the reliable target genes . based on the interactions of mirnas and target genes , the mirna function network was built .
the gene ontology ( go ) enrichment analysis was performed to investigate the main function of target genes by using the web - based software gorilla .
adjusted p values were computed for the go terms by multiple test adjustment of benjamini - hochberg ( bh ) .
in addition , for identifying the biological pathways associated with the metastasis of melanoma , we also performed the kyoto encyclopedia of genes and genomes ( kegg ) enrichment analysis of target genes .
to get the overview of the global shifts of gene expression in metastasis samples , the limma algorithm was used to identify the differentially expressed mirnas between metastasis and control samples .
the bh method was used for multiple test correction . false discovery rate ( fdr ) < 0.01 was selected as the criteria for calculating significant differences .
in recent years , many studies have indicated that mirnas are involved in the development of melanoma .
high throughput methods such as microarray have been widely used to get mirna expression profiling for many cancer types including the melanoma . however , due to the different platforms used for these researches , only few of the identified mirnas can be mutually identified . to overcome this problem
, we collected seven expression profiles for metastatic samples of melanoma to perform the meta - analysis [ figure 1 ] .
the number of the samples for these studies ranged from 12 to 37 , with the total number of 169 samples for all the studies .
then , the z - score transformation method was used to make the global normalization .
we used the limma package to identify the differentially expressed mirnas between metastatic and control samples . with the threshold of fdr < 0.05 ,
a total of 63 mirnas were identified as differentially expressed . among these genes , 35 were upregulated and 28 were downregulated .
the top ten upregulated and downregulated mirnas are listed in table 2 , which include the hsa - mir-205 , hsa - mir-203 , hsa - mir-508 , hsa - mir-514 - 1 , hsa - mir-509 - 1 , and hsa - mir-506 .
mirna with the most high upregulation was hsa - mir-508 , with a 15-fold higher expression in metastatic samples .
we also performed an unsupervised hierarchical clustering analysis of these differentially expressed mirnas and revealed distinct expression patterns in metastatic and control samples [ figure 2 ] , suggesting the potential of expression profiles of these top differentially expressed genes to distinguish sample status for melanoma .
the top ten upregulated and downregulated micrornas full lists of the differentially expressed mirnas en = 10 ( n>0 ) .
hierarchical clustering of the micrornas whose expressions were significantly altered in metastatic samples . to get a more comprehensive understanding of the biological roles of the differentially expressed mirnas in melanoma metastasis ,
the targets of these differentially expressed mirnas were predicted by using targetscan , pita , and miranda .
a total of 816 protein - coding genes were predicted to be regulated by the mirnas identified above .
then , we constructed the mirna regulatory by using the differentially expressed mirnas and the predicted targets .
this network consisted of 880 nodes and 1576 edges [ figure 3 ] . within this network ,
9 mirnas were associated with only one kind of downstream gene , while 18 mirnas were associated with more than 30 target genes .
the top - ranked mirna included hsa - mir-548b , hsa - mir-181b-2 , hsa - mir-141 , hsa - mir-766 , and hsa - mir-203 .
for mirna targets , ksr2 , suds3 , sh3tc2 , mvb12b , and lonrf2 were all critical genes that demonstrated to have the highest connectivity in the mirna regulatory network , suggesting that these genes could be of functional importance for the metastatic processes .
red nodes represent the upregulated micrornas and green nodes represent the downregulated micrornas , whereas the blue nodes represent microrna target genes . to get a more comprehensive understanding of the biological roles of these differentially expressed mirnas in melanoma
, we also performed the go categories enrichment analysis for the mirna target genes by using the gorilla online software . go category provides a descriptive framework of functional annotation for gene set analysis . with
the fdr < 0.05 , we found go terms for biological processes significantly enriched in nuclear division , cell division , mitotic cell cycle , etc .
go terms mainly included hydrolase activity , transmembrane transporter activity , and protein kinase binding [ supplementary figure 1 ] .
in addition , we also performed the kegg pathway enrichment analysis to further evaluate the biological significance for the differentially expressed genes .
the top - enriched biological pathways mainly included pathways in cancer , mapk signaling pathway , cell junction , focal adhesion , and cell adhesion [ table 3 ] .
enriched gene ontology terms ( biological process ) of predicted targets of differentially expressed micrornas in metastatic samples
. enriched gene ontology terms ( molecular functions ) of differentially expressed micrornas in metastatic samples .
fdr : false discovery rate ; kegg : kyoto encyclopedia of genes and genomes ; mapk : mitogen - activated protein kinase .
in recent years , many studies have indicated that mirnas are involved in the development of melanoma .
high throughput methods such as microarray have been widely used to get mirna expression profiling for many cancer types including the melanoma . however , due to the different platforms used for these researches , only few of the identified mirnas can be mutually identified . to overcome this problem
, we collected seven expression profiles for metastatic samples of melanoma to perform the meta - analysis [ figure 1 ] .
the number of the samples for these studies ranged from 12 to 37 , with the total number of 169 samples for all the studies .
then , the z - score transformation method was used to make the global normalization .
we used the limma package to identify the differentially expressed mirnas between metastatic and control samples . with the threshold of fdr < 0.05 ,
a total of 63 mirnas were identified as differentially expressed . among these genes , 35 were upregulated and 28 were downregulated .
the top ten upregulated and downregulated mirnas are listed in table 2 , which include the hsa - mir-205 , hsa - mir-203 , hsa - mir-508 , hsa - mir-514 - 1 , hsa - mir-509 - 1 , and hsa - mir-506 .
mirna with the most high upregulation was hsa - mir-508 , with a 15-fold higher expression in metastatic samples .
we also performed an unsupervised hierarchical clustering analysis of these differentially expressed mirnas and revealed distinct expression patterns in metastatic and control samples [ figure 2 ] , suggesting the potential of expression profiles of these top differentially expressed genes to distinguish sample status for melanoma .
the top ten upregulated and downregulated micrornas full lists of the differentially expressed mirnas en = 10 ( n>0 ) .
to get a more comprehensive understanding of the biological roles of the differentially expressed mirnas in melanoma metastasis , the targets of these differentially expressed mirnas were predicted by using targetscan , pita , and miranda .
a total of 816 protein - coding genes were predicted to be regulated by the mirnas identified above .
then , we constructed the mirna regulatory by using the differentially expressed mirnas and the predicted targets .
this network consisted of 880 nodes and 1576 edges [ figure 3 ] . within this network ,
9 mirnas were associated with only one kind of downstream gene , while 18 mirnas were associated with more than 30 target genes .
the top - ranked mirna included hsa - mir-548b , hsa - mir-181b-2 , hsa - mir-141 , hsa - mir-766 , and hsa - mir-203 . for mirna targets , ksr2 , suds3 , sh3tc2 , mvb12b , and lonrf2
were all critical genes that demonstrated to have the highest connectivity in the mirna regulatory network , suggesting that these genes could be of functional importance for the metastatic processes .
red nodes represent the upregulated micrornas and green nodes represent the downregulated micrornas , whereas the blue nodes represent microrna target genes .
to get a more comprehensive understanding of the biological roles of these differentially expressed mirnas in melanoma , we also performed the go categories enrichment analysis for the mirna target genes by using the gorilla online software . go category provides a descriptive framework of functional annotation for gene set analysis . with
the fdr < 0.05 , we found go terms for biological processes significantly enriched in nuclear division , cell division , mitotic cell cycle , etc .
go terms mainly included hydrolase activity , transmembrane transporter activity , and protein kinase binding [ supplementary figure 1 ] .
in addition , we also performed the kegg pathway enrichment analysis to further evaluate the biological significance for the differentially expressed genes .
the top - enriched biological pathways mainly included pathways in cancer , mapk signaling pathway , cell junction , focal adhesion , and cell adhesion [ table 3 ] .
enriched gene ontology terms ( biological process ) of predicted targets of differentially expressed micrornas in metastatic samples .
enriched gene ontology terms ( molecular functions ) of differentially expressed micrornas in metastatic samples .
fdr : false discovery rate ; kegg : kyoto encyclopedia of genes and genomes ; mapk : mitogen - activated protein kinase .
, the cause of melanoma metastasis is still far from understood . a better understanding of the pathogenesis of metastasis could be of paramount importance for drug development and treatment of this disease . in recent years , gene expression profiling
analysis has been shown to be a useful tool to investigate the pathophysiology of complex genetic tracts , such as cancer .
a large number of mirnas involved in many critical cellular pathways of cancer progression have also been identified to have an aberrant expression in melanoma . until now
, studies focusing on the mirna expression profiling analysis on the metastasis of melanoma are of comparatively limited value . in this study , we collected those mirna expression profiling datasets of melanoma and performed a systemic meta - analysis to retrieve metastasis - associated mirnas .
a total of 63 differentially expressed mirnas could successfully classify the different types of samples .
many of these mirnas identified are known to be involved in a variety of cancer types .
specifically , mir-23 has been identified as upregulated in many cancer types that represent putative oncogene , whereas mir-625 could function as a tumor suppressor that cause downregulation in esophageal squamous cell carcinoma and colorectal cancers .
in addition , many predicted targets of the differentially expressed mirna have also been suggested to have an association with cancer development .
for instance , ksr2 was essential to tumor cell energy homeostasis and was critical to the integration of mitogenic and metabolic signaling pathways .
the followed functional implication analysis indicated several physiological impact pathways for the metastasis of these diseases .
moreover , we utilized go and kegg pathway enrichment analyses to further interpret their biological functions .
this systematic meta - analysis of gene profiling took a step in investigating the mechanisms underlying the metastasis of melanoma .
these findings have dramatically expanded our knowledge of pathophysiology of this disease . in conclusion , by collecting the mirna expression datasets from different platforms , multiple biological markers were identified for the metastasis of melanoma .
this work was important to characterize the specific roles of those genes involved in the pathogenesis of melanoma .
functional analysis of these genes may provide additional insights into the complex process of these diseases .
in addition , this analysis may help improve the diagnosis and treatment of this disease .
supplementary information is linked to the online version of the paper on the chinese medical journal website .
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background : melanoma is a type of cancer that develops from the pigment - containing cells . until now
, its pathological mechanisms remain largely unknown .
the aim of this study was to identify metastasis - related microrna ( mirnas ) and gain an understanding of the biological functions in the metastasis of melanoma.methods:we searched the pubmed and gene expression omnibus database to collect mirna expression profiling datasets about melanoma , with key words of melanoma , mirna , microarray , and gene expression profiling .
only the original experimental works published before june 2016 for analyzing the metastasis of melanoma were retained , other nonhuman studies , reviews , and meta - analyses were removed .
we performed a meta - analysis to explore the differentially expressed mirna between metastatic and nonmetastatic samples .
moreover , we predicted target genes of the mirnas to study their biological roles for these mirnas.results:we identified a total of 63 significantly differentially expressed mirnas by meta - analysis of the melanoma expression profiling data . the regulatory network constructed by using these mirnas and the predicted targets identified several key genes involved in the metastasis of melanoma .
functional annotation of these genes indicated that they are mainly enriched in some biological pathways such as mitogen - activated protein kinase signaling pathway , cell junction , and focal adhesion.conclusions:by collecting the mirna expression datasets from different platforms , multiple biological markers were identified for the metastasis of melanoma .
this study provided novel insights into the molecular mechanisms underlying this disease , thereby aiding the diagnosis and treatment of the disease .
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duane retraction syndrome ( drs ) was recognized by stilling in 1887 and turk in 1896 .
as a result , drs is also referred to in the literature as stilling - turk - duane syndrome.1 drs is characterized by the following main features : 1 ) complete or partial absence of abduction of the affected eye ; 2 ) partial , or rarely complete , deficiency of adduction of the affected eye ; and 3 ) retraction of the affected eye into the orbit on adduction .
more recently , these characteristics have been named as congenital cranial dysinnervation disorders ( ccdds ) in the literature .
electromyography ( emg ) studies of patients with drs have shown that there is gross abnormality of the innervational pattern in this condition .
using emg , huber2 classified drs into 3 types :
type i : abduction restriction , normal or restricted adduction with associated retraction of the globe , and widening of the palpebral fissure on attempted abduction.type ii : restricted adduction and abduction and retraction of the globe on adduction , frequently associated with exotropia.type iii : severe restriction of both abduction and adduction , with either minimal esotropia orexotropia or near orthophoria and retraction on adduction and widening of the palpebral fissure on abduction .
type i : abduction restriction , normal or restricted adduction with associated retraction of the globe , and widening of the palpebral fissure on attempted abduction .
type ii : restricted adduction and abduction and retraction of the globe on adduction , frequently associated with exotropia .
type iii : severe restriction of both abduction and adduction , with either minimal esotropia orexotropia or near orthophoria and retraction on adduction and widening of the palpebral fissure on abduction .
the aims of treatment for drs are to achieve eye alignment in the primary position , to eliminate abnormal head posture ( ahp ) , and to prevent amblyopia .
some patients do not require surgical correction because they have neither great deviation nor face turn . in this study
all charts of the patients who were seen at our hospital s strabismus department between 2000 and 2015 and who were given a diagnosis of drs were reviewed .
this study was in compliance with the principles of the declaration of helsinki and was conducted in accordance with beyoglu gz training and research hospital institutional review board regulations .
patients with drs who underwent medical treatment with prismatic glasses and who had a minimum follow - up of 3 months were included in the study .
the following information was recorded : age , sex , family history of strabismus , systemic disease , refractive status , presence or absence of amblyopia , ahp , and history of strabismus surgery .
ocular alignment was graded as follows : orthotropia , with no movements noted on cover uncover test ; small - angle eso- or exodeviation , with phoria or tropia within 10 pd ( prism dioptri ) and significant eso- or exotropia , with deviation > 10 pd .
ocular misalignment data were obtained using the prism cover test and krimsky test . this management has been suggested for patients who do not want botulinum toxin a or surgery , in addition to having a small deviation and ahp .
ductions were graded on a scale from 0 ( full duction ) to 4 ( indicating an inability to move the eye past the midline ) .
head - turn measurements were recorded and evaluated at distance and at near fixation with best optical corrections .
an estimation of ahp was arrived at by an orthoptic method ( the nose being the focus ) , photographed , and the patient s distance- and near - reading positions observed .
noses were used to evaluate the ahp , and patients were separated into 2 groups with ahp , namely , those with < 10 and > 10 face turn .
palpebral fissures were investigated for retraction in adduction in all patients . at the same time , the upshoot or downshoot of the affected eye was assessed in adduction .
stereoacuity for near vision was recorded using the titmus fly stereo test for all patients .
all patients were treated with appropriate prismatic prescriptions and evaluated at 1 month and 3 months .
data were analyzed using minitab statistical software , release 20 ( minitab inc , state college , pa , usa ) .
a total of 638 records of strabismus patients diagnosed with drs were available for review .
the study group consisted of 7 ( 58.3% ) girls and 5 ( 41.7% ) boys .
the left eye was involved in 7 patients ( 66.7% ) and for 1 patient , the condition was bilateral ( 8.3% ) .
ahp was evaluated ; 8 ( 66.7% ) patients had < 10 , 4 ( 33.3% ) patients had > 10 face turn .
the mean follow - up period was 12.217.7 months ( range : 348 months ) .
esotropia was measured in 2 ( 16.7% ) patients at near and distance fixation upon admission .
the mean esodeviations were 12 pd and 10 pd at near and distance fixation , respectively .
all our patients had ahp ( face turn ) . at the same time , our patient s palpebral fissures were normal and equal on both sides in the primary position .
upshoot or downshoot was not observed . at the patients 3-month clinical examinations , ocular misalignment in the primary position
this study reports the results of medical management using prismatic glasses to treat patients with drs .
the prevalence of drs is about 1/1,000.1 drs prevalence was 1.2% in the strabismus patients included in our study .
it occurs sporadically in 90% of cases and is inherited in a small percentage as an autosomal dominant disorder.36 bilateral cases represent 15%20% of all cases of drs .
this study found 8.3% of drs patients with bilateral involvement.4,68 drs patients should receive individualized treatment according to the clinical manifestations observed .
the aims of management in drs include the elimination or improvement of abnormal head position ( face turn ) , the treatment of significant or slight ocular misalignment , the reduction of severe globe retraction , and the improvement of upshoots and downshoots .
refractive error must be corrected in patients with drs as the first step in medical treatment .
the prevalence of amblyopia in drs patients ranges from 3% to 48% in the literature.4,9,10 amblyopia was found in only 1 patient ( 8.3% ) in our study and we advised this patient occlusion therapy .
omalley et al11 and mohan et al12 reported amblyopia in 14% of their study group .
corrective lenses or contact lenses are used to correct refractive errors , and standard occlusion therapy is used to treat amblyopia.13 the correction of hypermetropic refractive error in children may reduce the angle of strabismus and drs symptoms .
kekunnaya et al14 demonstrated that , in 1 case , face turn and esotropia in the primary position were eliminated with corrective lenses alone .
park et al15 found in their study that orthotropia occurred in the majority ( 59% ) , followed by exotropia in 23.1% , and esotropia in 17.9% .
similarly , isenberg and urist s study reported that 28% were esotropic , 29% were exotropic , and 43% were orthotropic .
orthotropia was the most common primary position , and esotropia was found in only 2 ( 16.7% ) patients in our study .
our patients with esotropia had 12 pd deviation in near vision and 10 pd in distance vision .
strabismus can be called comitant or incomitant strabismus based on whether deviation is equal in all positions of gaze or varies with the direction of gaze.16 concomitant strabismus includes conditions such as palsy of the 4th and 6th cranial nerves , drs , brown syndrome , inferior oblique overaction , as well as a and v patterns .
the goal of the orthoptic treatment is the strongest possible fusion in primary gaze , as well as maximization of both monocular and binocular accommodative skills , including amplitude and flexibility .
a major finding of this study was that prismatic lenses may improve ahp and ocular misalignment , especially in cases with small deviation and ahp .
our study showed that prismatic glasses improved ahp completely in 41.6% of cases and reduced it to an acceptable level in 58.3% of the selected drs patients .
at the same time , we obtained improvement in ocular misalignment in the primary position .
a surgical approach was required for a drs patient with prominent ocular misalignment , marked ahp , cosmetically unacceptable upshoots and downshoot , and severe globe retraction on adduction .
however , there is a possibility of treatment with prismatic glasses in selected patients if deviation is within 10 pd and ahp is 10. the total drs patient sample size is the main strength of this study .
however , the study group size is limited because only drs patients with face turn < 10 , deviation 12 pd , and followed for at least 3 months in our clinic , were included in the study .
further study with wider case series and longer follow - up should be undertaken to support the results of this study .
to our knowledge , this study is the first study with the aim of presenting the treatment results of prismatic eyeglasses in patients with drs .
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purposeto report the results of using prismatic glasses for duane retraction syndrome ( drs).methodsdata were obtained from the records of patients who were evaluated during the year 2000 in the strabismus unit of the beyoglu eye training and research hospital .
the average follow - up was 12.217.7 months . in all cases ,
2 main variables were evaluated : horizontal deviation in the primary position and face turn .
prismatic glasses were provided to patients according to the degree of shift in the primary position.resultsthe mean age of patients was 11.2 years .
an analysis was performed on the data collected from 12 cases ; 7 patients were females ( 58.3% ) and 5 were males ( 41.7% ) , with 11 ( 91.7% ) cases being of type i drs .
all 12 patients had abnormal head posture ( face turn ) and an angle of mean deviation equaling 10 pd ( prism dioptri).conclusiontreatment was individualized on a case - by - case basis .
prismatic glasses are useful for eliminating abnormal head posture and ocular misalignment in selected cases .
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periodontal disease , or periodontitis , is one of the most underdiagnosed human diseases ; it affects about 80% of the adult population.1 the mean percentage of the maximum score of the community periodontal index in adults between the ages of 35 and 44 years indicates that only around 10% of individuals do not seem to have any kind of gingival inflammation.2 in the early stages of the disease , there is an accumulation of bacterial biofilm in the supragingival region that stimulates inflammation of the gingival tissue .
when the inflammation is restricted to soft tissue , it is called gingivitis.3,4 the presence of bleeding , edema , and increased crevicular fluid resulting from inflammation mediated by bacteria or endotoxins , and the immunological humoral response of the host with gingival hyperemia are common signs in both gingivitis and periodontitis.5 edema of the gingival tissue and bleeding in periodontitis can lead to bone resorption ; the final stage of this process is the loss of teeth due to total elimination of support tissue.6 bleeding is unpleasant while brushing the teeth , and often discourages patients from performing oral hygiene .
studies have shown that aminaphtone interferes in bleeding.79 recently , studies have shown that this drug helps to control bleeding in patients on oral warfarin therapy and in cases of ocher dermatitis.7,10,11 gingival bleeding has proven to be a viable model in the therapeutic assessment of aminaphtone .
the objective of the current study was to evaluate the efficacy of aminaphtone to control gum bleeding .
fifteen male and 15 female children aged between 10 and 18 years with a mean age of 13.4 years and with gingival bleeding were enrolled in this randomized , double - blind , placebo - controlled , phase iv clinical trial .
the inclusion criteria were that the participants were students at the professora iria barbieri vita state school in mirassol , brazil and had gingivitis and periodontitis with gingival bleeding .
patients with gingivitis were selected and invited to participate in the study after parents gave their informed consent .
thirty identical boxes containing blister packs of identical pills of either aminaphtone or placebo were produced and coded with unique numbers by the manufacturer ( baldacci laboratory , brazil ) and donated for this trial .
the significance of each coded number was not revealed until after the practical part of the trial had been completed .
a research assistant administered the medication or placebo to patients , with fifteen patients receiving aminaphtone ( capilarema 75 mg ) and the other 15 receiving placebo twice daily for 5 days .
all participants were examined by a single dentist in her office located inside the school , where she is employed as a dentist .
the clinical histories of the patients were taken , and they were submitted to an intraoral examination to calculate the bleeding or gingival index .
the gingival index uses a probe to identify the presence or absence of bleeding on the four faces of the teeth ( mesial , distal , vestibular , and lingual or palatal ) . for this , the tip of a periodontal probe ( hu - friedy 1511bbr pc ) was inserted into the periodontal pocket or next to the root extension .
it was inserted parallel to the long axis of all teeth with smooth but firm force , and run around the tooth between its surface and the free gingival margin or the inner wall of the periodontal pocket .
a check was made up to 10 seconds after delicately opening the gingival sulcus.12 the plaque index was obtained using the visible plaque index , as described by ainamo and bay;13 visible plaque was evaluated on teeth after staining both arches with a specific dye .
records of the dental examinations were noted on special sheets together with the patient s identification and a questionnaire on relevant data .
on probing , bleeding of at least 50% of teeth was adopted to characterize periodontal disease .
intraoral clinical evaluations were made before starting medication / placebo and then at 3 and 5 days after administration .
the sample size was calculated based on a pilot study ( godoy and godoy , unpublished data , 2012 ) on the reduction of light bleeding ( nosebleeds ) . according to this study ( -error = 0.05 , power = 0.80 )
on anticipating a dropout rate of 10% , the total sample size was increased to 15 participants .
data were analyzed using the statsdirect statistical software , ( statsdirect ltd , altrincham , uk ) .
calculations of the mean and standard deviation were made using parametric tests ( student s t - test ) for continuous variables with gaussian ( normal ) distribution .
the study was approved by the ethics committee of the medical school in so jose do rio preto ( 008/2012 ) and the brazilian clinical trial register ( req 1284 ) .
fifteen male and 15 female children aged between 10 and 18 years with a mean age of 13.4 years and with gingival bleeding were enrolled in this randomized , double - blind , placebo - controlled , phase iv clinical trial .
the inclusion criteria were that the participants were students at the professora iria barbieri vita state school in mirassol , brazil and had gingivitis and periodontitis with gingival bleeding .
patients with gingivitis were selected and invited to participate in the study after parents gave their informed consent .
thirty identical boxes containing blister packs of identical pills of either aminaphtone or placebo were produced and coded with unique numbers by the manufacturer ( baldacci laboratory , brazil ) and donated for this trial .
the significance of each coded number was not revealed until after the practical part of the trial had been completed .
a research assistant administered the medication or placebo to patients , with fifteen patients receiving aminaphtone ( capilarema 75 mg ) and the other 15 receiving placebo twice daily for 5 days .
all participants were examined by a single dentist in her office located inside the school , where she is employed as a dentist .
the clinical histories of the patients were taken , and they were submitted to an intraoral examination to calculate the bleeding or gingival index .
the gingival index uses a probe to identify the presence or absence of bleeding on the four faces of the teeth ( mesial , distal , vestibular , and lingual or palatal ) . for this
, the tip of a periodontal probe ( hu - friedy 1511bbr pc ) was inserted into the periodontal pocket or next to the root extension .
it was inserted parallel to the long axis of all teeth with smooth but firm force , and run around the tooth between its surface and the free gingival margin or the inner wall of the periodontal pocket .
a check was made up to 10 seconds after delicately opening the gingival sulcus.12 the plaque index was obtained using the visible plaque index , as described by ainamo and bay;13 visible plaque was evaluated on teeth after staining both arches with a specific dye .
records of the dental examinations were noted on special sheets together with the patient s identification and a questionnaire on relevant data .
on probing , bleeding of at least 50% of teeth was adopted to characterize periodontal disease .
intraoral clinical evaluations were made before starting medication / placebo and then at 3 and 5 days after administration .
the sample size was calculated based on a pilot study ( godoy and godoy , unpublished data , 2012 ) on the reduction of light bleeding ( nosebleeds ) . according to this study ( -error = 0.05 , power = 0.80 )
on anticipating a dropout rate of 10% , the total sample size was increased to 15 participants .
data were analyzed using the statsdirect statistical software , ( statsdirect ltd , altrincham , uk ) .
calculations of the mean and standard deviation were made using parametric tests ( student s t - test ) for continuous variables with gaussian ( normal ) distribution .
the study was approved by the ethics committee of the medical school in so jose do rio preto ( 008/2012 ) and the brazilian clinical trial register ( req 1284 ) .
after the final evaluation of bleeding of all patients , the pharmaceutical company revealed which children received aminaphtone and which received a placebo .
table 1 shows that bleeding was reduced after treatment in both groups ( paired t - test , p<0.0001 for both groups ) .
figures 1 and 2 show the variations in bleeding , comparing before and after treatment , for the placebo and aminaphtone groups , respectively ( paired t - test , p<0.0001 ) . on comparing the number of bleeding points before and after treatment between the aminaphtone and placebo groups , we found significantly higher reduction with the medication ( p<0.0001 ) .
the mean reduction in the number of points for the placebo group was 6.5 , and for the aminaphtone group it was 14.4 .
the present study evaluated the effect of aminaphtone on the reduction of gingival bleeding in young patients with gingivitis . both groups in this study
there are no publications in the literature on the issue of using aminaphtone or any other drug that may affect capillary fragility .
the hypothesis for this bleeding is that capillary fragility occurs during inflammatory processes , and so drugs that reduce this fragility may improve control of bleeding .
there are few studies evaluating the use of aminaphtone in processes that involve bleeding.8,9 recently however , a series of case reports were published that suggest that this drug reduces capillary fragility .
these studies show the control of nosebleeds and report on improvement in cases of ocher dermatitis.7,10,11 bleeding gums are normally controlled with oral hygiene .
the explanation for the reduction in bleeding in the placebo group was better oral hygiene as a result of participating in this study .
therefore , the improved hygiene that was associated with treatment using aminaphtone may have had a synergistic effect in reducing gingival bleeding .
another aspect that catches the attention is that the control in bleeding was 44% of the patients in the aminaphtone group but zero in the placebo group .
the objective of this study was to evaluate the clinical control of bleeding ; gingivitis has proven to be an attractive research model to study capillary fragility .
another aspect is the reduction of edema in patients who take aminaph - tone , with a faster improvement in gum color than for those who took the placebo , thereby suggesting control of capillary permeability , which is also expected with the use of aminaphtone.14 bleeding is unpleasant when using dental floss , and the reduction of edema and gingivitis improves adherence to treatment .
aminaphtone reduces gum bleeding in gingivitis and may have a supportive role in the control of bleeding , but the main approach to treatment must be to brush the teeth well and use dental floss .
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aimthe objective of the current study was to evaluate the efficacy of aminaphtone to control gum bleeding.patients and methodsfifteen male and 15 female children , aged between 10 and 18 years with a mean age of 13.4 years and with gingival bleeding , were enrolled in this randomized , double - blind , placebo - controlled , phase iv clinical trial .
the inclusion criterion was gingivitis with gingival bleeding .
participants were prescribed either aminaphtone or placebo .
thirty identical boxes containing blister packs of identical pills of either aminaphtone or placebo were produced and coded with unique numbers by the manufacturer ( baldacci laboratory , brazil ) and donated for this trial .
a research assistant administered aminaphtone ( capilarema 75 mg ) to fifteen patients or placebo to fifteen patients twice daily for 5 days .
intraoral clinical evaluations of bleeding were made before starting medication / placebo and then at 3 and 5 days after administration.resultson comparing the number of bleeding points before and after treatment between the aminaphtone and placebo groups , we found significantly higher reductions with the medication ( p<0.0001).conclusionaminaphtone reduces gum bleeding in gingivitis , and may have a supportive role in the control of bleeding .
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patients with schizophrenia exhibit a wide range of cognitive dysfunctions,1 including deficits in working memory , verbal and spatial memory , motor and processing speed , attention , and executive function.2,3 cognitive impairment determines the level of patients daily function and social - occupational outcome.4,5 therefore , a cognitive test battery that can be efficiently and feasibly used to assess cognitive deficits in patients with schizophrenia is essential for outcome measurement in both clinical settings and research environments.6,7 the ideal test battery should contain a broad range of cognitive dimensions , satisfy reliability and validity criteria , and be repeatable to assess changes over long - term follow - up periods.8 numerous neuropsychological testing batteries have been developed to determine the magnitude of cognitive impairment in patients with schizophrenia.9,10 for example , the well - known measurement and treatment research to improve cognition in schizophrenia is used in clinical trials for assessing cognitive function in patients with schizophrenia.11,12 however , most of the present cognitive assessment tools are time - consuming , costly , or inconvenient to use in practice .
the brief assessment of cognition in schizophrenia ( bacs ) is a brief test battery which measures impairment of verbal memory , working memory , motor speed , verbal fluency , attention and processing speed , and executive function.13 these domains of cognitive deficits have been demonstrated to reliably reflect the functionality of patients with schizophrenia.14,15 the bacs is easily performed in clinical settings and can be administered by medical professionals in 40 minutes .
the original version of bacs was in english and has undergone a validation study.16 the bacs has been translated into over 30 languages , and the validities of the japanese,17 french,18 italian,19 spanish,20 german,21 persian,22 and brazilian portuguese23 versions have been proven .
the normative data of the chinese version of the bacs have been established using a representative community sample in taiwan ( unpublished data ) , and notable differences in verbal tests and executive function were found when compared to the norms developed in the us.13 however , the bacs has not been validated in a chinese - speaking population . since chinese
is one of the most commonly spoken languages in the world , adaptation and validation of the bacs could advance the knowledge about cognition in schizophrenia , facilitate the comparison of patients with schizophrenia in various countries , and help to determine the cross - cultural validity of the bacs .
therefore , the current study aimed to validate the bacs by comparing 66 patients with schizophrenia to an age- and sex - matched sample of healthy controls .
this cross - sectional study was conducted in chang gung memorial hospital , and was approved by the institutional review board of chang gung hospital in taiwan .
eligible patients with schizophrenia at an outpatient department in the kaohsiung chang gung memorial hospital were selected for this study if they met the following inclusion criteria : 1 ) were aged 1865 years ; 2 ) were diagnosed with schizophrenia as defined by the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv - tr)24 after assessment using the chinese version of the mini international neuropsychiatric interview ( mini ) ( which was translated from english by the taiwan society of psychiatry25 and known to have good inter - rater reliability);26 3 ) had received a stable dose of an antipsychotic drug and had relatively stable psychotic symptoms ; 4 ) had no history of major physical illnesses that would influence cognitive performance , and 5 ) could speak and read chinese to perform the test . in total , 66 patients with schizophrenia were recruited in this study ( mean age : 41.2 years , 57.6% male ) .
information regarding the antipsychotic usage by patients and daily dose were obtained through patients medical records .
the dose of antipsychotic medication in use was calculated according to the defined daily dose ( ddd ) recommended by the who collaborating centre for drug statistics methodology.27 the healthy individuals were recruited from the staff of kaohsiung chang gung memorial hospital and keelung chang gung memorial hospital , and from the community volunteers in kaohsiung city and keelung city , taiwan .
the recruitment criteria were as follows : 1 ) absence of a history of major psychiatric disorders ( eg , psychotic disorders , bipolar disorders , major depressive disorders , organic mental disorders , and substance user disorders ) confirmed using dsm - iv criteria and the mini ; 2 ) absence of any known systemic or neurological diseases that would influence cognitive performance ; 3 ) han chinese ethnicity ; and 4 ) capacity to speak and read chinese to perform the test .
we recruited 382 healthy individuals to establish the norms of the bacs ( unpublished data ) .
finally , 66 healthy individuals who were age- and sex - matched to our patients with schizophrenia were selected as the control group .
a total of 38 patients with schizophrenia were willing to receive bacs retesting , and the rest 28 patients either dropped out or refused to receive the retest .
the bacs test battery is used for neuropsychological assessment of patients with various psychotic disorders,26 and the subtests of the bacs are listed in the following section .
list learning test ( verbal memory ) : patients are presented with 15 phrases and then asked to recall as many as possible . this procedure is repeated five times .
eight alternative forms are designed to minimize the practice effect . in the current study ,
the first form was chosen for testing all participants , and the second form was chosen for retest purposes . digit sequencing task ( working memory ) : patients are presented with clusters of numbers of increasing length and are asked to repeat the numbers in order , from lowest to highest .
token motor task ( motor speed ) : patients are given 100 plastic tokens and asked to place them into a container as quickly as possible for 60 seconds .
verbal fluency : performance is evaluated based on the total number of words / phrases adequately responded to in the category instances test and controlled oral word association test . in the category instances test ( semantic fluency ) , patients are given 60 seconds to list as many words as possible relating to the category of animals .
the outcome measure is the total number of unique words listed for animals . in the controlled oral word association test ( letter fluency )
, patients are given 60 seconds to list as many phrases as possible that begin with the mandarin word
symbol coding test ( attention and processing speed ) : as quickly as possible , patients are asked to write numerals 19 that matched the symbols on a response sheet for 90 seconds .
the tower of london test ( executive function ) : patients are asked to look at two pictures simultaneously .
each picture shows three differently colored balls arranged on three pegs , with the balls in a unique arrangement in each picture .
the patients are asked to calculate the smallest number of times that the balls in one picture would have to be moved to arrive at an arrangement of balls as seen in another picture .
the first form was used for testing all participants , and the second form was used for retesting .
finally , a composite score was calculated by summing scores on each measure for each patient and healthy control .
the z - score compared the sum of each patient with that of the corresponding control.13 a z - score of 0 for each scale indicates average functioning with reference to the normal population of the same age range and sex , and every 1 point represents one standard deviation ( sd ) .
the upsa - b is modified from the ucsd performance - based skills assessment ( upsa ) which is designed for assessing daily functioning in patients with severe mental illness.28 the upsa - b consists of two subscales : a financial portion and communication domain . in the financial portion , the participant would be asked to count specific amounts of real currency , make change , and request a bank check to pay a bill .
the communication domain requires the participant to correctly call directory assistance to obtain a telephone number and then reschedule an appointment in a hospital .
the raw scores of the ubsa - b were standardized to a 100-point scale.29 the chinese version of the upsa - b was developed and has been validated in patients with mental illnesses among chinese populations.30 the upsa - b served as the validity criterion for the bacs in this study .
data were analyzed using the statistical software package spss , version 21.0 ( spss inc . ,
the data are presented as either mean ( sd ) or frequency ( % ) .
categorical variables were compared between patients and controls using either the chi - square test or fisher s exact test , depending on case numbers , and an independent t - test was used to compare continuous variables between groups . in a two - tailed test , p<0.05 was considered statistically significant .
the test retest reliability of all subtests of the bacs was measured with intraclass correlations ( icc ) .
the paired samples t - test was used to examine whether there were significant practice effects . the principal components analysis ( pca ) with varimax rotation was applied for determining the factor structure of the bacs subtests .
pearson s correlation was performed to analyze the relationships between each bacs subscale and the correlation between bacs composite scores and upsa - b scale scores , separately in patients and controls .
scaled z - scores for a subset of variables and a composite z - score were calculated based on the normative data of the bacs established in our previous work ( unpublished data ) .
the receiver operating characteristic ( roc ) analyses and area under curve ( auc ) were further applied to examine the validity of using the z - score of each bacs subtest to differentiate patients with schizophrenia from healthy controls .
sensitivity and specificity of the best cutoff point of the bacs composite score were calculated . a kruskal
wallis test was performed to examine the potential difference in bacs composite scores among schizophrenia patients with different antipsychotic use .
pearson s correlation test was performed to analyze the correlation between ddd of antipsychotics and bacs composite scores .
this cross - sectional study was conducted in chang gung memorial hospital , and was approved by the institutional review board of chang gung hospital in taiwan .
eligible patients with schizophrenia at an outpatient department in the kaohsiung chang gung memorial hospital were selected for this study if they met the following inclusion criteria : 1 ) were aged 1865 years ; 2 ) were diagnosed with schizophrenia as defined by the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv - tr)24 after assessment using the chinese version of the mini international neuropsychiatric interview ( mini ) ( which was translated from english by the taiwan society of psychiatry25 and known to have good inter - rater reliability);26 3 ) had received a stable dose of an antipsychotic drug and had relatively stable psychotic symptoms ; 4 ) had no history of major physical illnesses that would influence cognitive performance , and 5 ) could speak and read chinese to perform the test . in total , 66 patients with schizophrenia were recruited in this study ( mean age : 41.2 years , 57.6% male ) .
information regarding the antipsychotic usage by patients and daily dose were obtained through patients medical records .
the dose of antipsychotic medication in use was calculated according to the defined daily dose ( ddd ) recommended by the who collaborating centre for drug statistics methodology.27 the healthy individuals were recruited from the staff of kaohsiung chang gung memorial hospital and keelung chang gung memorial hospital , and from the community volunteers in kaohsiung city and keelung city , taiwan .
the recruitment criteria were as follows : 1 ) absence of a history of major psychiatric disorders ( eg , psychotic disorders , bipolar disorders , major depressive disorders , organic mental disorders , and substance user disorders ) confirmed using dsm - iv criteria and the mini ; 2 ) absence of any known systemic or neurological diseases that would influence cognitive performance ; 3 ) han chinese ethnicity ; and 4 ) capacity to speak and read chinese to perform the test .
we recruited 382 healthy individuals to establish the norms of the bacs ( unpublished data ) .
finally , 66 healthy individuals who were age- and sex - matched to our patients with schizophrenia were selected as the control group .
the cognitive functions of all participants were assessed using the bacs . a total of 38 patients with schizophrenia were willing to receive bacs retesting , and the rest 28 patients either dropped out or refused to receive the retest .
the bacs test battery is used for neuropsychological assessment of patients with various psychotic disorders,26 and the subtests of the bacs are listed in the following section .
list learning test ( verbal memory ) : patients are presented with 15 phrases and then asked to recall as many as possible . this procedure is repeated five times .
eight alternative forms are designed to minimize the practice effect . in the current study ,
the first form was chosen for testing all participants , and the second form was chosen for retest purposes . digit sequencing task ( working memory ) : patients are presented with clusters of numbers of increasing length and are asked to repeat the numbers in order , from lowest to highest .
token motor task ( motor speed ) : patients are given 100 plastic tokens and asked to place them into a container as quickly as possible for 60 seconds . the outcome measure is the total number of tokens placed in the container . verbal fluency : performance is evaluated based on the total number of words / phrases adequately responded to in the category instances test and controlled oral word association test . in the category instances test ( semantic fluency ) , patients are given 60 seconds to list as many words as possible relating to the category of animals . the outcome measure is the total number of unique words listed for animals . in the controlled oral word association test ( letter fluency ) , patients are given 60 seconds to list as many phrases as possible that begin with the mandarin word
symbol coding test ( attention and processing speed ) : as quickly as possible , patients are asked to write numerals 19 that matched the symbols on a response sheet for 90 seconds .
the tower of london test ( executive function ) : patients are asked to look at two pictures simultaneously .
each picture shows three differently colored balls arranged on three pegs , with the balls in a unique arrangement in each picture .
the patients are asked to calculate the smallest number of times that the balls in one picture would have to be moved to arrive at an arrangement of balls as seen in another picture .
the first form was used for testing all participants , and the second form was used for retesting .
finally , a composite score was calculated by summing scores on each measure for each patient and healthy control .
the z - score compared the sum of each patient with that of the corresponding control.13 a z - score of 0 for each scale indicates average functioning with reference to the normal population of the same age range and sex , and every 1 point represents one standard deviation ( sd ) .
the upsa - b is modified from the ucsd performance - based skills assessment ( upsa ) which is designed for assessing daily functioning in patients with severe mental illness.28 the upsa - b consists of two subscales : a financial portion and communication domain . in the financial portion , the participant would be asked to count specific amounts of real currency , make change , and request a bank check to pay a bill .
the communication domain requires the participant to correctly call directory assistance to obtain a telephone number and then reschedule an appointment in a hospital .
the raw scores of the ubsa - b were standardized to a 100-point scale.29 the chinese version of the upsa - b was developed and has been validated in patients with mental illnesses among chinese populations.30 the upsa - b served as the validity criterion for the bacs in this study .
the cognitive functions of all participants were assessed using the bacs . a total of 38 patients with schizophrenia were willing to receive bacs retesting , and the rest 28 patients either dropped out or refused to receive the retest .
the bacs test battery is used for neuropsychological assessment of patients with various psychotic disorders,26 and the subtests of the bacs are listed in the following section .
list learning test ( verbal memory ) : patients are presented with 15 phrases and then asked to recall as many as possible . this procedure is repeated five times .
eight alternative forms are designed to minimize the practice effect . in the current study ,
the first form was chosen for testing all participants , and the second form was chosen for retest purposes . digit sequencing task ( working memory ) : patients are presented with clusters of numbers of increasing length and are asked to repeat the numbers in order , from lowest to highest .
token motor task ( motor speed ) : patients are given 100 plastic tokens and asked to place them into a container as quickly as possible for 60 seconds . the outcome measure is the total number of tokens placed in the container . verbal fluency : performance is evaluated based on the total number of words / phrases adequately responded to in the category instances test and controlled oral word association test . in the category instances test ( semantic fluency ) , patients are given 60 seconds to list as many words as possible relating to the category of animals . the outcome measure is the total number of unique words listed for animals . in the controlled oral word association test ( letter fluency ) , patients are given 60 seconds to list as many phrases as possible that begin with the mandarin word
symbol coding test ( attention and processing speed ) : as quickly as possible , patients are asked to write numerals 19 that matched the symbols on a response sheet for 90 seconds .
the tower of london test ( executive function ) : patients are asked to look at two pictures simultaneously .
each picture shows three differently colored balls arranged on three pegs , with the balls in a unique arrangement in each picture .
the patients are asked to calculate the smallest number of times that the balls in one picture would have to be moved to arrive at an arrangement of balls as seen in another picture .
the first form was used for testing all participants , and the second form was used for retesting .
finally , a composite score was calculated by summing scores on each measure for each patient and healthy control .
the z - score compared the sum of each patient with that of the corresponding control.13 a z - score of 0 for each scale indicates average functioning with reference to the normal population of the same age range and sex , and every 1 point represents one standard deviation ( sd ) .
the upsa - b is modified from the ucsd performance - based skills assessment ( upsa ) which is designed for assessing daily functioning in patients with severe mental illness.28 the upsa - b consists of two subscales : a financial portion and communication domain . in the financial portion , the participant would be asked to count specific amounts of real currency , make change , and request a bank check to pay a bill .
the communication domain requires the participant to correctly call directory assistance to obtain a telephone number and then reschedule an appointment in a hospital .
the raw scores of the ubsa - b were standardized to a 100-point scale.29 the chinese version of the upsa - b was developed and has been validated in patients with mental illnesses among chinese populations.30 the upsa - b served as the validity criterion for the bacs in this study .
data were analyzed using the statistical software package spss , version 21.0 ( spss inc . ,
the data are presented as either mean ( sd ) or frequency ( % ) .
categorical variables were compared between patients and controls using either the chi - square test or fisher s exact test , depending on case numbers , and an independent t - test was used to compare continuous variables between groups . in a two - tailed test , p<0.05 was considered statistically significant .
the test retest reliability of all subtests of the bacs was measured with intraclass correlations ( icc ) .
the paired samples t - test was used to examine whether there were significant practice effects . the principal components analysis ( pca ) with varimax rotation was applied for determining the factor structure of the bacs subtests .
pearson s correlation was performed to analyze the relationships between each bacs subscale and the correlation between bacs composite scores and upsa - b scale scores , separately in patients and controls .
scaled z - scores for a subset of variables and a composite z - score were calculated based on the normative data of the bacs established in our previous work ( unpublished data ) .
the receiver operating characteristic ( roc ) analyses and area under curve ( auc ) were further applied to examine the validity of using the z - score of each bacs subtest to differentiate patients with schizophrenia from healthy controls .
sensitivity and specificity of the best cutoff point of the bacs composite score were calculated . a kruskal
wallis test was performed to examine the potential difference in bacs composite scores among schizophrenia patients with different antipsychotic use .
pearson s correlation test was performed to analyze the correlation between ddd of antipsychotics and bacs composite scores .
a total of 66 patients with schizophrenia ( schizophrenia group ) and 66 age- and sex - matched healthy control subjects ( control group ) were recruited in this study .
. a higher proportion of the control group were married ( p<0.001 ) and employed ( p<0.001 ) .
compared with the control group , patients in the schizophrenia group were impaired in all dimensions of cognitive function assessed by the bacs and upsa - b ( p<0.001 ) .
the test retest reliability and potential practice effects of the bacs are summarized in table 2 . in the 38 patients with schizophrenia who were tested and retested , the iccs of the bacs subtests ranged from 0.59 ( verbal fluency ) to 0.86 ( attention and processing speed ) , and all were significant ( p<0.001 ) .
the paired samples t - test revealed that none of the bacs subtests showed statistically significant practice effects .
pca demonstrated that the first and second components accounted for 60.9% ( eigenvalue : 3.66 ) and 14.9% ( eigenvalue : 0.90 ) of the variance , respectively .
this suggests that a one - factor solution is the best fit for our dataset .
table 3 shows the correlation matrix of each bacs subtest of patients and controls . in patients with schizophrenia ,
the bacs composite score was positively correlated with all bacs subtest scores ( p<0.001 ) , and all bacs subtest scores were positively correlated with each other ( p<0.05 ) , with the exception of the correlation between working memory and motor speed .
the composite score was significantly correlated with all subtest scores in controls ( p<0.01 ) and with upsa - b scores ( figure 1 ) in patients ( r=0.70 , p<0.001 ) and controls ( r=0.44 , p<0.001 ) . the roc analysis
( figure 2 ) showed that all bacs subtest scores could significantly differentiate patients with schizophrenia from healthy controls ( p<0.001 ) .
the bacs composite score had the best discriminant validity ( auc : 0.94 , p<0.001 ) , followed by the scores for motor speed ( auc : 0.91 , p<0.001 ) , attention and processing speed ( auc : 0.90 , p<0.001 ) , working memory ( auc : 0.88 , p<0.001 ) , verbal memory ( auc : 0.84 , p<0.001 ) , verbal fluency ( auc : 0.84 , p<0.001 ) , and executive function ( auc : 0.77 , p<0.001 ) , respectively ( table 4 ) .
the roc output indicated that a composite z - score of 0.71 had the best discriminant validity , with a sensitivity of 0.91 and a specificity of 0.89 . among patients with schizophrenia , no significant differences in bacs composite scores were observed between different antipsychotic users ( p=0.066 ) .
the bacs composite scores were not significantly correlated to the ddd of antipsychotics ( r=0.07 , p=0.561 ) .
the practice effects in all bacs domains estimated by paired samples t - test were nonsignificant .
consistent with previous findings regarding the validation of bacs in other language versions,16,17,21,23 we suggest that the chinese version of the bacs is a reliable assessment tool for understanding the cognitive function of patients with schizophrenia .
keefe et al16 suggested that forms of the list learning test and tower of london test need to be changed to retest patients and assess changes in their cognitive abilities independent of practice effects ( such as memorization resulting from repeated test taking ) .
therefore , all patients were given an alternative form of the list learning test and tower of london test at the time of retesting in our study .
notably , the category instances test ( verbal fluency ) differs between the english and the chinese versions of the bacs .
there are two alternative forms of the category instances test ( naming supermarket items or tools ) in the english bacs and only one form ( naming animals ) in the chinese bacs .
therefore , no alternative form of the verbal fluency test could be used in the current study .
the pca performed in our study ( similar to previous studies of the spanish,20 persian,22 and portuguese versions of the bacs)23 revealed that one factor accounted for 60.9% of the total variance .
nevertheless , a significantly better model fit was also associated with a three - factor solution in the original english bacs16 and japanese bacs,17 and a two - factor solution in the french bacs.18 the components extracted by pca might be different between studies because of discrepancies in sample characteristics , sample size , language or culture , and clinical features.31,32 nevertheless , all aforementioned studies consistently indicated that a single factor could explain a large amount of the variance ( general cognitive function ) . taken together , these findings indicate that the bacs can be used to assess specific cognitive domains , as well as general cognitive function in schizophrenia . in agreement with
the previous validation studies,16,2022 our study showed strong correlation between bacs composite scores and all bacs subscales , in both patients and controls and between bacs composite scores and upsa - b scores .
previous researchers also found strong correlation between the bacs composite score and standard neurocognitive assessments other than the bacs.1618,20,22,23,33 these findings indicate that the bacs has satisfactory intercorrelations and concurrent validity .
however , the magnitude of our correlation coefficients between bacs subtests ( table 3 ) , as well as between bacs and upsa - b scores ( figure 1 ) , was lower in healthy controls than in patients with schizophrenia .
these results may be explained by the global nature of cognitive decline in patients but not healthy controls , giving rise to strong correlation among specific domains of cognitive function .
in addition , the upsa - b score is a measure of basic real - world functioning in patients with mental illnesses,29 and an obvious ceiling effect may be present in healthy individuals without cognitive impairments .
the roc analysis ( figure 2 ) showed the high sensitivity and specificity of the bacs composite score to distinguish patients from controls ( auc = 0.94 ) .
this finding is in line with german bacs21 and persian bacs findings.22 this result suggests that the chinese bacs has good discriminant validity for distinguishing patients from controls based on cognitive function . among the bacs subtests , motor speed and attention and processing speed also individually had a high discriminant validity ( auc > 0.9 ) , while executive function , measured by the tower of london test , had a relatively low discriminant validity ( auc = 0.77 ) .
likewise , keefe et al16 previously found that discriminant validity between patients and controls was smaller for executive function than for other bacs subtests .
this implies that the tower of london test is less sensitive to cognitive deficit detection in schizophrenia .
furthermore , cognitive function has been associated with clinical symptoms or prescription of psychotropic drugs in patients with schizophrenia.3436 the large deficits in motor speed and attention of schizophrenia patients may possibly be due to disease course or antipsychotic drug therapy .
this study has several limitations : first , the sample size in this study was small . further study with a larger sample size is warranted to examine the reliability and validity of the bacs across different clinical groups ( eg , paranoid type vs disorganized type ; first - episode psychosis vs chronic schizophrenia ) .
second , only patients with schizophrenia , but not healthy control subjects , were given bacs retests .
in addition , the second form of the list learning test was constantly used for bacs retesting .
therefore , we could not determine iccs and practice effects in healthy individuals , nor compare test retest reliability across different versions of the list learning test .
third , this study used only one cognitive test ( upsa - b ) as the validity criterion for the bacs .
a standard battery with less ceiling effect is needed to certify the concurrent validity of the bacs in future study .
finally , chinese contains a group of related language varieties , and the participants in this study were tested in mandarin ( the most widely used dialect in taiwan and people s republic of china ) .
it remains uncertain whether the findings in this study could be generalized to population who speak other dialects .
in summary , the chinese version of the bacs provides satisfactory psychometric properties , including high test
retest reliability , high internal consistency , acceptable concurrent validity , and good overall and domain - specific discriminant validity .
we suggest that the chinese bacs is a reliable and practical tool for assessing cognitive function in patients with schizophrenia .
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objectivea test battery that measures cognitive function impairment in patients with schizophrenia , the brief assessment of cognition in schizophrenia ( bacs ) , has been translated into various languages and validated .
this study aimed to test the reliability and validity of the chinese version of the bacs in a chinese - speaking population.methodsall participants in this study ( 66 patients with schizophrenia [ mean age : 41.2 years , 57.6% male ] and 66 age- and sex - matched healthy controls ) were from taiwan and assessed using the bacs and the university of california , san diego ( ucsd ) performance - based skills assessment , brief version ( upsa - b ) .
thirty - eight of the 66 patients with schizophrenia received a reassessment using the bacs.resultsthe bacs had good test
retest reliability , and all bacs subtests had statistically insignificant practice effects .
principal components analysis demonstrated that a one - factor solution best fits our dataset ( 60.9% of the variance ) . in both patients and controls , the bacs composite scores were positively correlated with all bacs subscales ( p<0.001 ) and upsa - b scales ( p<0.001 ) .
furthermore , all bacs subtests ( verbal memory , working memory , motor speed , verbal fluency , attention and processing speed , and executive function ) significantly differentiated patients with schizophrenia from healthy controls ( p<0.001 ) , and the bacs composite score had the best discriminative validity ( p<0.001).conclusionthe chinese version of the bacs exhibits satisfactory psychometric properties , including high test
retest reliability , high internal consistency , acceptable concurrent validity , and good discriminant validity .
we suggest that the bacs is a reliable and practical tool for assessing cognitive function in patients with schizophrenia .
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polymyalgia rheumatica ( pmr ) is the
most common inflammatory rheumatic disease in the elderly .
there is
considerable uncertainty related to diagnosis and outcomes in patients
presenting with the polymyalgic syndrome .
therefore the european league against
rheumatism ( eular ) and the american college
of rheumatology
( acr ) are supporting a study that is being conducted to establish
classification criteria for pmr .
ultrasound ( us ) of shoulders and hips was
selected among several other candidate criteria that had been recruited by a
delphi survey from a work group of
27 international physicians with interest in pmr ( rheumatologists ,
nonrheumatologists , statisticians , and methodologists ) who met to pursue a
consensus - based process for the development of classification criteria in pmr
in cambridge , uk , in 2005 .
participants agreed that currently , there was no role for
routine use of magnetic resonance imaging or positron emission tomography in
the evaluation of suspected pmr .
however musculoskeletal us was thought to have
utility as a diagnostic criterion for pmr due to widespread availability ,
feasibility , and results of preliminary studies in this condition [ 27 ] .
us depicts characteristic pathologic
findings of shoulder joints and the hip region that may aid in distinguishing
pmr from other diseases that may mimic it .
typical findings on us include
glenohumoral joint synovitis , subdeltoid bursitis , and biceps tendon
tenosynovitis of the shoulders [ 26 ] .
these findings occur in most pmr
patients , but they are often mild in contrast to elderly onset rheumatoid
arthritis ( eora ) . in the hips ,
us often reveals
hip joint synovitis and trochanteric bursitis in patients with pmr [ 2 , 5 , 7 ] . before considering us as a potential
tool for the classification , there was agreement regarding the need to
standardise the examination and assess the inter - observer agreement for
distinguishing lesions typical of pmr from other lesions like extensive
effusion or bursitis , rotator cuff tears , rotator cuff calcifications , and
osteoarthritis . to pursue this aim , members of
centres participating in the pmr classification criteria study met to
standardise the us examination in pmr patients for each centre and work towards achieving a high
degree of inter - reader agreement in the us
examination for the pmr
classification criteria study .
the study included both , patients with pmr and
other related diseases with shoulder pain such as rheumatoid arthritis ( ra ) .
while we present a validation study , our study does not address the question of
defining us characteristics of pmr .
fifteen rheumatologists and one
radiologist of varying us experience from the participating centres ( listed in
the acknowledgement ) met in oranjewoud ( the netherlands ) to evaluate the
possibility of including us in the upcoming pmr classification criteria study .
the meeting took place just following the 12th eular basic ultrasound course
and before the annual eular congress in june 2006 .
each group consisted of four assessors : one supervisor ,
one performing the us
scans , one measuring distances between structures , and one documenting the
results . the ultrasound examiners were with the exception of the supervisor
alumni
of the basic eular sonography course .
none were career specialists in the field of musculoskeletal
ultrasonography .
prior to the patient examination , each
supervisor gave a review of the us
examination including specifics of the standard scans to be performed for
operator training .
the group as a
whole decided if they regarded the us
findings as normal or abnormal .
to ensure standardised documentation , each participant was given a report sheet
that listed possible pathologic findings recorded as
no , indicating
the presence or absence of each particular finding . all us
assessments
were performed independently by each group without contact with other groups
and without knowledge of the patient 's disease or joint status .
each group was
given a maximum of 15 minutes ( shoulder joint ) and 10 minutes ( hip joint ) ,
respectively , for us examination per joint region , rotating on a preset plan from one us
station to
the next .
each healthy control and each patient was assigned to every us
station in
rotation .
two joints ( one shoulder and one hip ) were
examined in 4 healthy controls and in 4 patients with ra
or pmr .
four rounds ( normal shoulder , normal hip , pathologic shoulder , and
pathologic hip ) for 4 groups were necessary to ensure that an examination was
performed by each group , control / patient and joint region . patients and healthy
individuals were recruited from the medisch centrum leeuwarden by gawb . all
patients gave their consent to participation in the study .
examinations were performed with several us machines
which are in use in practice and simulate the conditions of the pmr
classification study .
stations 1 and 2 were
equipped with a mylab 70 ( esaote , genoa ,
italy ) .
shoulder and hip joints were examined with a linear array probe ( la 186 mhz ) ,
respectively .
station 3 was equipped with a logic e ( general electrics , milwaukee , usa ) ,
and station 4 with a voluson i ( general electrics , milwaukee , usa ) .
shoulder and hip joints were examined with a linear array probe ( 12l - rs , 135 mhz ) , respectively .
two equipment specialists each from esaote and general
electrics were present to help in case of problems with regard to machine
adjustments during the examinations .
scanner settings were uniform for all
measurements : frequency setting , b - mode gain , and 100% ; one focus point position in
the region of measurement .
an introduction to the us
device was given to the
observers prior to us examinations .
standard scans according to the eular
guidelines for musculoskeletal us were applied . for the shoulder joint ,
examination of the
biceps tendon , rotator cuff ( subscapularis , supraspinatus , and infraspinatus
tendons ) , glenohumeral joint , acromioclavicular joint , humeral bone surface
subacromial - subdeltoid bursa , and axillary artery was required .
effusion ,
synovitis , tenosynovitis , bone erosions , osteophytes , and bursitidies were
evaluated .
evaluation of the hip joints included assessment for
effusion , synovitis , osteoarthritis , and trochanteric bursitis .
synovitis , effusion , tenosynovitis ,
and erosions were defined according to the omeract definitions for musculoskeletal
ultrasound
all analyses were calculated
with statistical product and
service solutions ( spss ) 15.0 ( chicago , ill , usa ) .
the overall agreement between the 4
groups of sonographers with regard to all normal and pathologic findings for
shoulders and hips was 87% , reflecting substantial agreement .
for the healthy controls , overall agreement for the shoulder was 88.8%
( substantial agreement ) .
for the patient group , overall agreement for the shoulder joint was
85.2% ( moderate agreement ) .
for the pathologic hip joint , overall agreement was
74.3% ( substantial agreement ) .
the overall agreements with regard to the different pathologies for the
shoulder and hip joints are displayed in tables 3 and 4 .
figures 1(a ) and 1(b ) show
typical joint pathologies in pmr patients , for example , subdeltoid bursitis and hip
joint synovitis .
it is important to rigorously
evaluate the utility of us in pmr since there are no recognised laboratory or
imaging diagnostic tests for the condition .
recent studies have shown the importance
of us in depicting characteristic pathologies that aid in distinguishing pmr
from other mimicking diseases . the most frequent us
soft tissue alterations in
patients with pmr
have been described for the shoulders ( subdeltoid bursitis ,
tenosynovitis of the biceps tendon , and glenohumeral synovitis ) [ 26 ] and for
the hips ( synovitis and trochanteric bursitis ) [ 2 , 5 , 7 ] . however , to date us has not yet been
included in any diagnostic or classification criteria for pmr .
therefore , a
study group has met to evaluate the possibility of including us in the upcoming
pmr classification criteria study .
our standardized protocol and study results
demonstrated that us examinations for both , shoulder and hip joints can be a
useful tool for assessment of pathology in a future study of pmr ; since it can
be performed by competent examiners across centres with reliable result .
some former studies have
investigated the prevalence for the detection of inflammatory changes of the
shoulder [ 26 ] as well as the hip [ 2 , 5 , 7 ] joints in pmr by us .
performed a case control study of shoulder us
in pmr patients including 57
consecutive patients with pmr and 114 controls with bilateral shoulder pain and
stiffness due to ra , psoriatic arthritis , spondyloarthritis , osteoarthritis ,
fibromyalgia , or connective tissue disease .
bursitis was detected in all patients , glenohumeral
synovitis in 88% , and biceps tendon tenosynovitis in 88% .
other authors have
reported that inflammatory changes are discrete in pmr but much more severe in
eora .
frediani et al . performed a case control study that describes the
us
findings in 50 consecutive patients with pmr , spondyloarthritis , and ra ,
respectively .
they detected biceps tenosynovitis in 44% of spondyloarthritis
patients and in 38% of patients with ra , glenohumoral joint synovitis in 16% of
patients with spondyloarthritis , and in 54% of patients with ra , and subdeltoid
bursitis in 34% of patients with spondyloarthritis and in 44% of patients with
ra .
since a high prevalence of synovitis is
seen in shoulder and hip joints of pmr patients , the current study focused on
whether overall agreement could be achieved between readers for typical joint
pathologies seen in pmr patients ( including subdeltoid bursitis , biceps
tenosynovitis , and glenohumoral synovitis for the shoulder joint ,
resp . ) .
we found substantial overall agreement for detecting these pathologies
in normal as well as pathologic shoulder and hip joint pathologies .
these
findings underline the fact that us is not as observer dependent as formerly
thought .
furthermore , our results demonstrated the feasibility of
performing a multicentre study using us for evaluation of patients with
shoulder and hip lesions typical of pmr .
us is an important imaging tool for
the visualisation of inflammation in shoulders and hips joints of pmr . in this
exercise substantial agreement
the results of this study confirm that us can be
evaluated as a diagnostic technique in studies of pmr including the pmr classification
criteria study .
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objective . a study supported by the eular and
the acr being conducted to establish classification criteria for polymyalgia rheumatica ( pmr ) will include ultrasound examination of the shoulders and hips .
ultrasound ( us ) depicts glenohumeral joint effusion , biceps tenosynovitis , subdeltoid bursitis , hip joint synovitis , and trochanteric bursitis in pmr .
these findings may aid in distinguishing pmr from other diseases .
the purpose of this study was to assess standards and us interreader agreement of participants in the pmr classification criteria study .
methods .
sixteen physicians in four groups examined shoulders and hips of 4 patients and 4 healthy adults with ultrasound .
overall agreement and interobserver agreement were calculated .
results . the overall agreement ( oa ) between groups was 87% .
the oa for healthy shoulders was 88.8% , for healthy hips 100% , for shoulders with pathology 85.2% , and 74.3% for hips with pathology , respectively .
conclusion .
there was a high degree of agreement found for the examination of healthy shoulders and pathologic hips .
agreement was moderate for pathologic shoulders and perfect for healthy hips . us of shoulder and hips performed by different examiners
is a reliable and feasible tool for assessment of pmr related disease pathology and can be incorporated into a classification criteria study .
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the third stage of labor is still associated with considerable maternal morbidity and mortality .
the major complication is postpartum hemorrhage ( pph ) , which affects about 5% of deliveries [ 1 , 2 ] .
therefore it is the leading cause of maternal morbidity and mortality worldwide . in western countries , such as the united kingdom ,
it is the fifth most common reason for maternal death after thromboembolism , preeclampsia / eclampsia , genital tract sepsis , and amniotic fluid embolism .
some ten years ago , an editorial titled the retained placenta new insights into an old problem was raising hopes that this problem is to be solved soon .
whereas in the event of pph due to atony of the uterus there exist numerous guidelines , recommendations , and flowcharts for its management ; in the treatment of retained placenta the general consensus is more difficult to establish .
retained placenta is an important cause of pph and has an incidence of 1 : 100 to 1 : 300 births [ 6 , 7 ] . with this paper
our aim was to attract the obstetricians ' attention to the potential risk of retained placenta in the low risk setting where it occurs without prior warning and to present a possible flowchart for the timing of treatment to reduce blood loss and therefore maternal morbidity .
in general it can be said that already the duration of the third stage of labour is contributing to the risk of pph as the risk of major bleeding is believed to increase with time elapsed after birth .
hence , active management of third stage of labour using prophylactic oxytocics is accepted as standard of care .
active management of the third stage of labour involves administration of intravenous oxytocin , early cord clamping , transabdominal manual massage of the uterus , and controlled traction of the umbilical cord .
should this appear insufficient , the next step is usually manual removal of the placenta ( mrop ) .
however , the timing of this manoeuver is difficult as the risk of pph from leaving the placenta in situ has to be weighed against the knowledge that manual removal can itself cause hemorrhage .
it should also be borne in mind that the placenta may be delivered spontaneously up to 30 minutes or more after delivery of the child , without major additional blood loss .
the management questions that thus need answering are when and how to detect increased blood loss ?
observation of routine practice shows that mrop is regularly deferred beyond the limits recommended . in the absence of immediate evidence of increased vaginal bleeding , management is often conservative and expectant , open to several different options , and paying little attention to the time elapsed since birth . in a study of over 12,000 births , combs and laros
similarly , magann et al . found that the risk of hemorrhage increased with time . in their study , the risk of pph was already significantly increased at 10 minutes and , using a receiving operator characteristic ( roc ) curve , they demonstrated that the optimal cut - off time for the prediction of pph was 18 minutes , with a sensitivity of 31% and a specificity of 90% .
however , delaying the manual removal will lead to the spontaneous delivery of many placentas . despite scant evidence
it is commonly advised that if the placenta has not been expelled 30 minutes after delivery despite active management , mrop should be carried out under anaesthesia .
clearly , in the published recommendations the choice of timing for manual removal depends on the facilities available and the local risks associated with both pph and mrop .
thus the 2007 intrapartum guidelines produced for the uk government agency the national institute for health and clinical excellence ( nice ) suggest 30 minutes , whereas the who manual for childbirth suggests 60 minutes .
accordingly , a survey in europe showed that time until manual removal of placenta in the absence of bleeding varies widely between different countries , from under 30 minutes ( spain and hungary ) to 60 minutes and more ( the netherlands ) .
there are different reasons for retained placenta and there is a wide variety in the nomenclature for disturbances in placental disruption .
we believe the following classification is sound : placenta adherens is caused by failed contraction of the retroplacental myometrium , incarcerated placenta is caused by a closed or closing cervix , and placenta accreta is caused by abnormal placental implantation .
a part of the placenta or the entire placenta is abnormally adherent to the uterine wall without underlying decidua basalis . in placenta
increta the placental villi invade into the myometrium , while percreta placenta is classified as placental villi penetrating through the uterine serosa or the adjacent organs , usually the bladder [ 14 , 15 ] . as there is a fair probability for detecting cases with percreta placenta before the onset of labour due to ultrasound and/or magnetic resonance imaging and therefore operative delivery
can be planned with all necessary precautions , it is almost impossible for the clinician to detect or even distinguish between placenta accreta and increta despite numerous attempts to do so with several imaging techniques .
weeks observed a considerable variation in the retained placenta rate between countries . in less developed countries
it is less common ( about 0.1% of all deliveries ) but has a high fatality rate . in more developed countries
it affects about 3% of all vaginal deliveries but is very rarely associated with maternal death .
it is suggested that interventions common in the most developed countries such as abortions , uterine intervention , labour induction , and use of oxytocin could be contributing to the increase in retained placenta rate with increasing development . commonly named risk factors for disturbances in placental disruption , such as placenta accreta , are history of retained placenta , previous caesarean section , maternal age over 35 years , preterm labour , induced labour , multiparity , previous uterine injury or surgery , uterine malformations , infection , and preeclampsia [ 1 , 3 , 68 , 1418 ] .
it is believed that placenta accreta is becoming more common due to the rising caesarean section rate and advancing maternal age , both independent risk factors for placenta accreta [ 2 , 17 ] .
history of caesarean section and placenta previa are often of special interest as risk factors for placenta accreta . in a prospective observational cohort study of over 30,000 women who had caesarean delivery without labour ,
placenta accreta was present in 0.24% of women undergoing their first up to 6.74% of women undergoing their sixth or more caesarean delivery .
in women with placenta previa the risk for placenta accreta was 3% , 11% , 40% , 61% , and 67% for first , second , third , fourth , fifth , and sixth or more caesarean deliveries . with every additional caesarean delivery the risk for emergency hysterectomy was rising as well .
hysterectomy was required in 0.65% for their first caesarean delivery and increased up to 8.99% for their sixth or more caesarean delivery . in another study
the incidence of placenta accreta in case of placenta previa was 5% . with a previous caesarean section ,
some studies showed promising results by injecting oxytocin into the umbilical cord , as it increased the rate of spontaneous expulsions of the placenta and fewer manual removals of the placenta , but two cochrane reviews , either investigating umbilical cord injection of saline or oxytocin in the routine management of the third stage of labour or for the reduction of mrop , were not able to detect a significant reduction in the need for mrop .
nevertheless , umbilical vein injection of oxytocin solution is an inexpensive and simple intervention that could be performed while placental delivery is awaited .
however , high - quality randomized trials show that the use of oxytocin has little or no effect .
the same review showed a statistically lower incidence in manual removal of placenta if prostaglandin solution was used .
eller et al . published a study including 57 cases with placenta accreta , where all women underwent hysterectomy . in 15 cases
an attempt was made to remove the placenta manually , but these entire women required immediate hysterectomy for uncontrollable bleeding .
the authors of this study concluded that , in case of suspected placenta accreta , scheduled caesarean hysterectomy without attempting placental removal is associated with a significantly reduced rate of early morbidity compared with cases in which placental removal is attempted .
diagnosis of placenta accreta is not based on universally valid standard criteria but rather a diagnosis based on the obstetricians ' impression and subjective judgement .
some authors use only clinical criteria for the diagnosis of placenta accreta , while others use histopathological criteria , which is not always possible for obvious reasons .
some authors distinguish between total and partial placenta accreta , a diagnosis even more difficult to make .
as well for the term placenta adherens there is no consensus regarding exact criteria for the definition .
this may also be contributing to the highly variable incidence of placenta accreta , with rates reported in literature between 1 : 93,000 and 1 : 110 . aside from patients with placenta previa and patients with a high risk of morbidly adherent placenta due to obstetrical history , the diagnosis of placenta accreta is usually made at the time of delivery . a prenatal screening for placenta accreta , especially for woman with risk factors , would be eligible .
a prenatal diagnosis would allow a more planned approach and minimize maternal blood loss . in literature greyscale ultrasonography , colour doppler imaging , and magnetic resonance imaging ( mri )
have been described as alleged successful approaches to diagnose placenta accreta antenatally [ 6 , 15 , 17 , 18 ] .
esakoff and colleagues stated that ultrasound examination is a good diagnostic test for accreta in women with placenta previa and found this in consistency with most other studies in the literature . a recent meta - analysis involving
3707 pregnancies showed a sensitivity of 90.72% and a specificity of 96.94% of ultrasound for the antenatal detection of invasive placentation .
there is a general consensus that sensitivity and specificity of ultrasound are superior to those of mri ( sensitivity 8085% , specificity 65100% ) , but often both imaging techniques are used in conjunction in women at risk .
however , prenatal diagnosis of placenta accreta in absence of further abnormalities of placentation remains a challenge .
there are also few biochemical markers named which are thought to have a diagnostic potential , such as elevated levels of maternal serum creatinine kinase , alpha fetoprotein , and -human chorionic gonadotropin .
others promisingly described cell - free fetal dna , placental mrna , and dna microarray as potential tools for the diagnosis of abnormalities of placental invasion [ 15 , 26 , 27 ] .
but so far there exists no diagnostic tool ready to use in daily routine for prenatal diagnosis of placenta accreta .
the sensitivity of theoretically possible test methods also depends on the degree and extent of the abnormal placental invasion . in our experience prenatal diagnosis
is almost impossible in the low risk population , where often the parturient is seen in the maternity hospital only for childbirth .
we only can assume that these patients most probably have not undergone prenatal ultrasound examination with the very question of morbidly adherent placenta .
nevertheless , the use of colour doppler sonography in the third stage of labour has been promisingly introduced by krapp et al .
they examined the third stage of labour using greyscale and colour doppler ultrasound . in cases with normal placental separation they found cessation of blood flow between placenta and myometrium immediately after birth .
suggestive of placenta accreta was persistent blood flow from the myometrium deep into the placenta demonstrated by colour doppler ultrasound .
according to the authors this method allows a quicker diagnosis of placenta accreta and maternal blood loss can be minimized by early manual removal . as an ultrasound machine
should be easily available in a well - equipped delivery unit it is advisable to use ultrasound in the third stage of labour complicated by retention of the placenta . with a gain of experience in judging
the separation of the placenta from the uterine muscle ultrasound imaging may develop into a useful tool in the management of pathologic third stage of labour .
audureau et al . were able to show that the implementation of a multifaceted intervention scheme for the prevention and management of postpartum hemorrhage can be successful . in such way
the median delay for second - line pharmacological treatment was significantly shortened from 80 min before introduction to 32.5 min afterwards .
comparable to a strict work flow as already developed and implemented in most large delivery units for emergency caesarean section ( target of decision delivery time < 20 min ) a similar standardized protocol for manual removal of placenta might be useful . in figure 1
we present a showcase flowchart for cases with retained placenta with special emphasis on the time frame .
we believe that already strict observation of time , use of ultrasound for evaluation of the grade of placental detachment , and early involvement of support staff ( i.e. , second midwife , anaesthesiologist ) might contribute to a reduction of maternal morbidity .
needless to say , the suggested time frame is only applicable in the absence of increased vaginal bleeding , and its efficacy has to be proven in a controlled trial . in case of an increased blood loss during third stage of labour
ideally standardized operating procedures are already implemented . in conclusion , retained placenta remains a problem of the third stage of labour , which in the low risk setting usually is occurring without prior warning . in daily routine adherence to a strict protocol of active management of third stage of labour may be helpful to minimize time interval between birth and delivery of placenta and therefore minimize postpartum complications .
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the third stage of labor is associated with considerable maternal morbidity and mortality .
the major complication is postpartum hemorrhage ( pph ) , which is the leading cause of maternal morbidity and mortality worldwide .
whereas in the event of
pph due to atony of the uterus there exist numerous treatment guidelines ; for the management of retained placenta the general consensus is more difficult to establish .
active management of the third stage of labour is generally accepted as standard of care as already its duration is contributing to the risk of pph . despite scant evidence
it is commonly advised that if the placenta has not been expelled 30 minutes after delivery , manual removal of the placenta should be carried out under anaesthesia .
pathologic adhesion of the placenta in the low risk situation usually is diagnosed at the time of delivery ; therefore a pre- or intrapartum screening opportunity for placenta accreta would be desirable . but diagnosis of abnormalities of placentation other than placenta previa remains a challenge .
nevertheless the use of ultrasound and doppler sonography might be helpful in the third stage of labor .
an improvement might be the implementation of standardized operating procedures for retained placenta which could contribute to a reduction of maternal morbidity .
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eukaryotic genomic dna is packaged with histone proteins to form chromatin [ 1 , 2 ] .
the most fundamental repeating unit of chromatin is the nucleosome , which consists of an octamer of histones ( 2 copies of each histone protein : h2a , h2b , h3 , and h4 ) and the genomic dna wrapped around the octamer [ 3 , 4 ] .
modification ( e.g. , acetylation , methylation , and phosphorylation ) of the nucleosomal core histones influences chromatin structure and biological functions [ 57 ] .
the modified nucleosome should be formed at the genomic position or in the genomic region . in this paper , i will focus on nucleosome positioning ( not histone modification ) , because nucleosome positioning is not only related to compacting the genomic dna but also to gene regulation [ 817 ] . due to the development of dna sequencing technology and genomic tiling array technology ,
genome - wide nucleosome mapping has been performed in a wide range of eukaryotic species , including the budding ascomycetous yeast , saccharomyces cerevisiae ; the nematode , caenorhabditis elegans ; the fruit fly , drosophila melanogaster ; humans , homo sapiens ; the malaria parasite , plasmodium falciparum ; the filamentous ascomycete , aspergillus fumigatus ; the fission ascomycetous yeast , schizosaccharomyces pombe ; the plant , arabidopsis thaliana ; several ascomycetous yeasts ; the mouse , mus musculus ; the basidiomycete , mixia osmundae ; the amoebozoa , dictyostelium discoideum .
genome - wide analyses of nucleosome positioning have revealed that dna sequence preference exists for nucleosome occupancy [ 29 , 38 , 39 ] .
the nucleosome occupancy reflects average nucleosome positioning levels on a given region of dna in a population of cells [ 4043 ] .
for example , the dinucleotide sequences aa and tt are depleted in nucleosome - forming regions in different organisms [ 29 , 39 , 44 ] , whereas the g + c content is highly correlated with nucleosome occupancy [ 45 , 46 ] .
in addition , it has been reported that nucleosomal dna cytosines are more highly methylated than nucleosome linker dna cytosines in humans and the plant arabidopsis .
these results suggest that dna sequence preference in nucleosome occupancy has been conserved during eukaryotic evolution .
genome - wide nucleosome positioning data suggest that nucleosome occupancy restricts the range of genomic g + c content .
bacteria and archaea , which lack nucleosomes , have a wide range of g + c content .
in contrast , the genomic g + c content distribution of eukarya is completely different from that of bacteria and archaea ( figure 1 ) .
this distribution difference may be related to the differences in the conservation level of histones and nucleoid - associated proteins ; although histone proteins are highly conserved between different organisms , nucleoid - associated proteins vary among bacteria and archaea [ 4750 ] .
nucleosome depletion in the vicinity of the transcription start site ( tss ) has been indicated [ 5153 ] . indeed , nucleosome - free regions are pervasive in the gene promoters of yeast [ 26 , 54 , 55 ] .
moreover , the nucleosome organization around tsss is very similar among different organisms [ 20 , 29 , 30 , 39 , 54 , 55 ] . the nucleosome position profile is sharper in the downstream region of the tss .
nucleosomes downstream from the nucleosome - free region are well positioned , with positioning decaying with increasing distance into the protein - coding region .
nucleosome positioning is more conserved in gene promoters than in gene bodies , suggesting that nucleosome positioning in the gene promoter plays an important role in gene transcription [ 19 , 27 , 52 , 56 , 57 ] . on the other hand ,
nucleosome positioning in vivo differs from that in vitro , indicating that systems other than dna sequence preference are involved in nucleosome positioning [ 40 , 41 , 58 ] .
recently , it was reported that the most conserved nucleosome position ( the + 1 nucleosome ) , which is the sharpest in the nucleosome position profile , is maintained by atp - dependent factors in s. cerevisiae [ 59 , 60 ] .
it remains uncertain whether nucleosome positioning in the gene promoter has been evolutionarily conserved as a major driving force in gene expression [ 15 , 27 , 36 ] or not [ 57 , 61 , 62 ] .
genome - wide nucleosome mapping analyses of the ascomycete s. cerevisiae revealed that nucleosome depletion is also found around translation end sites as well as tsss [ 63 , 64 ] . in the basidiomycete m. osmundae , dinucleosome but
these results suggest that the nucleosome linker dna length of m. osmundae around tsss and translation end sites is shorter than that of s. cerevisiae .
nucleosome depletion around transcription end sites is also found in drosophila and dictyostelium [ 21 , 30 ] .
the regions around both transcription start and end sites have dna sequences rich in adenine and thymine , which disfavor core histones [ 21 , 30 , 54 ] .
recently , some chromatin remodelers have been reported to locate around transcription start and end sites in s. cerevisiae .
genome - wide nucleosome mapping analyses have shown that the nucleosome occupancy level in exons is higher than that in introns [ 6872 ] .
dna sequence differences between exons and introns are correlated with nucleosomal dna preferences , as exon dna sequences have a higher g + c content than intron dna sequences . as described above , nucleosomal dna prefers ( g + c)-rich sequences .
although the nucleosome positioning system differs between the ascomycetous budding yeast s. cerevisiae and the ascomycetous fission yeast sch .
pombe , genome - wide comparative analyses of nucleosome positions have revealed that nucleosome positioning shares a common feature among different organisms .
nucleosomal dna has a higher g + c content and a higher level of cytosine methylation than nucleosome linker dna ( figure 2 ) .
in addition , nucleosome positioning is found more frequently in exonic than in intronic regions . transcription start sites and translation ( or transcription ) end sites
thus , not only the structures of core histone proteins but also the nucleosome positioning systems have been greatly conserved during eukaryotic evolution .
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nucleosome positioning is not only related to genomic dna compaction but also to other biological functions .
after the chromatin is digested by micrococcal nuclease , nucleosomal ( nucleosome - bound ) dna fragments can be sequenced and mapped on the genomic dna sequence .
due to the development of modern dna sequencing technology , genome - wide nucleosome mapping has been performed in a wide range of eukaryotic species .
comparative analyses of the nucleosome positions have revealed that the nucleosome is more frequently formed in exonic than intronic regions , and that most of transcription start and translation ( or transcription ) end sites are located in nucleosome linker dna regions , indicating that nucleosome positioning influences transcription initiation , transcription termination , and gene splicing .
in addition , nucleosomal dna contains guanine and cytosine ( g + c)-rich sequences and a high level of cytosine methylation .
thus , the nucleosome positioning system has been conserved during eukaryotic evolution .
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small cell osteosarcoma ( os ) of the bone is an unusual variant of os .
it may present a diagnostic challenge on cytological assessment owing to its rarity and its morphologic similarity to other small round cell tumors , including ewing 's sarcoma ( es ) , peripheral neuroectodermal tumor ( pnet ) , malignant lymphoma , metastatic neuroblastoma , rhabdomyosarcoma and mesenchymal chondrosarcoma .
distinguishing these tumors from each other is important clinically as optimal therapy differs according to the tumor type .
only rare accounts of the cytopathological findings of small cell os on fine needle aspiration ( fna ) exist in the literature .
this article highlights the features that help in diagnosing this distinctive microscopic variant of os on fna cytology .
a 10-year - old male child presented with complaints of pain and swelling in the lower third of the right thigh for 6 months . the swelling was diffuse and measured 5 cm 6 cm .
radiograph showed a tumor involving the metaphysis and diaphysis of the femur with soft tissue extension and periosteal reaction .
magnetic resonance imaging ( mri ) revealed both intraosseous and extraosseous extension ; however , there was no intra - articular involvement .
fna was performed using a 22 gauge needle attached to a 10 ml syringe on cameco handle after careful anatomic localization of the tumor with the help of the radiograph and mri films .
smears were air dried and stained with may - grnwald - giemsa ( mgg ) and also wet fixed and stained with hematoxylin and eosin ( h and e ) .
the cytological smears were moderately cellular and showed clusters and singly scattered tumor cells [ figure 1 ] .
the cells were small and pleomorphic and had a high n / c ratio with round hyperchromatic nuclei and inconspicuous nucleoli and the cytoplasm showed vacuolations that were periodic acid - schiff ( pas ) positive [ figure 1 , inset ] .
many atypical mitotic figures , a few tumor giant cells and osteoclastic giant cells were noted . on careful search ,
a focal area showing presence of pink / metachromatic osteoid - like material was identified .
based on these findings , a diagnosis of malignant small round cell tumor closest to small cell os was made and biopsy was advised .
inset shows periodic acid - schiff ( pas ) positivity in the tumor cells ( pas , 400 ) the fna diagnosis was subsequently confirmed on trucut biopsy , which showed small round malignant cells directly laying down pink lacy osteoid .
the patient received neoadjuvant chemotherapy for 3 months , which was followed by resection of the right femur .
the chemotherapy was continued but the patient succumbed to neutropenia - related complications 6 months after the surgery .
small cell os is a distinctive microscopic variant of os , originally described as a neoplasm having microscopic features of both os and es .
this is a rare form of os with an incidence of 1.3% of all oss .
the clinical features including age , sex and skeletal distribution are similar to those of conventional os .
the radiological features are not consistently typical for small cell os because there is very little production of mineralized matrix . however , if the mineralization is identified within the tumor and in areas of soft tissue extension , it favors os .
the widespread use of pre - operative fna of bone lesions necessitates recognition of the various bone tumors as this may drastically alter the management .
small cell os poses unique diagnostic difficulties as it may be difficult to distinguish from other small cell malignancies .
primary small round cell tumors of the bone include small cell os , es / pnet , lymphoma and mesenchymal chondrosarcoma .
metastatic neuroblastoma and rhabdomyosarcoma are the other differential diagnoses in the younger age group . on cytology , small cell os is composed of small- to intermediate - sized cells
. the cells are mostly dyscohesive oval to round having a high n / c ratio .
sheets of monotonous small round cells with minimal cytoplasm are seen in either of the tumors .
the amount of osteoid produced however is often scanty , making the diagnosis difficult , as was noted in the current case .
in addition , it may be difficult to differentiate osteoid from hyalinized collagen that can be seen in es / pnet .
osteoid is seen as clumps of amorphous or finely fibrillar material that is faintly eosinophilic in h and e and bright red or pink in mgg .
in contrast with collagen , osteoid has a more wispy and lacy quality with ill - defined borders and lacks naked fibroblastic nuclei . in the absence of osteoid ,
the presence of homer wright rosettes and pseudorosettes supports the diagnosis of es / pnet .
although cytoplasmic glycogen is a feature of es , its positivity does not rule out a diagnosis of small cell os . in a histopathologic series of os ,
10/21 cases of small cell os were positive for glycogen , which was highlighted by the pas stain .
es / pnet is usually strongly positive for cd99 ; however , small cell os can also be immunoreactive , adding to the difficulty in diagnosis .
a negative result however supports the diagnosis of small cell os over es / pnet .
molecular testing can be very useful as the t ( 11;22 ) ( q24;q12 ) is diagnostic for es / pnet . however , this technique is not routinely available in most centers of resource - poor countries .
mesenchymal chondrosarcoma occurs in the older age group and is characterized by two cell populations : a small round cell component and lacunar cells associated with chondroid matrix .
the chondroid foci stain for s100 protein and the primitive mesenchymal cells are positive for cd99 . the cells of lymphoma as compared with small cell os generally have larger nuclei with vesicular chromatin and prominent nucleoli and show presence of lymphoglandular bodies .
in addition , neuroblastomas are immunoreactive for synaptophysin and chromogranin but are virtually never immunoreactive for cd99 . in rhabdomyosarcoma ,
eccentric , triangular , strap - shaped rhabdomyoblasts with dense eosinophilic cytoplasm may be recognized .
the diagnosis rests primarily on the identification of osteoid production by the malignant cells , and this should be carefully searched for . in the absence of osteoid , cellular pleomorphism favors a diagnosis of small cell os .
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small cell osteosarcoma ( os ) is a rare histological variant of os that poses unique diagnostic difficulties .
we present a case of a 10-year - old child who underwent fine needle aspiration cytology ( fnac ) from a mass in the right thigh .
the cytological findings were those of a malignant small round cell tumor , closest to small cell os .
the fnac findings were confirmed on histopathology .
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canonical transient receptor potential 4 ( trpc4 ) channels are calcium - permeable , nonselective cation channels that are widely distributed in mammalian cells .
trpc4 channels are activated by gq/11-phospholipase c ( plc ) pathway or directly activated by gi / o proteins .
it is also known as dexras1 ( dexamethasone - induced ras - related protein 1 ) or ags1 ( activators of g - protein signaling 1 ) .
our focus here will be on the roles of small g proteins rasd1 in the activation of trpc4 channels .
also , the roles of glucocorticoids and leptin via trpc4 in insulin secretin will be discussed .
the trpc subfamily consists of 7 proteins designated as trpc1 to 7 , which can be further divided into 4 subgroups based on their sequence homology and functional similarities : ( 1 ) trpc1 , ( 2 ) trpc4 and trpc5 , ( 3 ) trpc3 , trpc6 and trpc7 , and ( 4 ) trpc2 .
molecules downstream of plc , such as ip3 and dag , and pip2 hydrolysis have been suggested as activators and as an activation mechanism for trpc channels , respectively .
trpc4 channel consist of 6 transmembrane domains , intracellularly located amino and carboxyl termini and a putative pore - forming region . within the trpc family ,
trpc4 and trpc5 represent a structurally distinct subgroup that is characterized by its ability to form homo- and heteromultimeric channels with each other as well as with trpc1 . trpc4 and trpc4 are the most abundantly expressed and functionally characterized .
trpc4 channel lacks a domain of 84 amino acids in the c - terminal region containing a putative binding site for calmodulin ( cam ) binding and inositol 1 , 4 , 5-triphosphate ( ip3 ) receptors .
the deleted regions of trpc4 is also responsible for the channel activity by pi(4 , 5)p2 .
trpc4 has the 4 short amino acid sequence motifs ( m1-m4 ) that show typical features of trp .
m1 motif is discovered upstream of tm1 and is conserved in this form throughout the trpc subfamily .
this channel contains a coiled - coil domain , 4 ankyrin - like repeats , and predicted multimerization domain in the n - terminus as potential protein - protein interaction motifs .
trpc4 is expressed in diverse organs and cell types including the dendrites and soma of various types of neurons , smooth muscles , the cardiovascular system , including endothelial and cardiac cells , skeletal muscle cells , the myometrium , the kidney , and immune cells .
trpc4 channels differ extremely in their permeability and pharmacological modulation , other biophysical properties , and mode of activation depending on the cellular environment .
activation of trpc4 channels by agonists induced ca entry directly or indirectly via depolarization and activation of voltage - gated ca channels .
trpc4 channel was reported that this channel , as well as phospholipase c 1 and 2 interact with the first pdz domain of nherf , regulatory factor of the na / h exchanger .
the c - terminal pdz ( dlg , zo-1 , psd-95 ) motif of trpc4 has been implicated in the control of channels surface expression and localization .
the pdz deletion motif ( t - r - l ) of trpc4 dramatically reduced the plasma membrane levels of the channel , which led the authors to suggest that the interaction between htrpc4 and nherf is required for the retention and stabilization of htrpc4 channels in the cell membrane .
caveolin-1 ( cav-1 ) is associated with dynamic protein complex consisting of trpc4 , trpc1 and ip3rs .
the loss of cav-1 blunted the localization of trpc4 and agonist - stimulated complex formation .
trpc4 channel has been proposed to be activated by gq / phospholipase c signaling , release of intracellular castores , or vesicular translocation to the membrane .
gq - plc signaling mediated activation of plc increased ip3 that binds to the ip3 receptor located on the endoplasmic reticulum and released intracellular ca . a conformational change of trpc channel bound to ip3 receptors , which bind to the c - terminal end of the trpc channels via the ip3/calmodulin receptor binding domain . among trp channels , trpc4 channels are unique in that they activated by trivalent cations gd and la independently of gpcr .
mutation and modeling demonstrated that the k715 and r716 regions are important for the interaction between gi2 and trpc4 channel .
thus , we suggest that the regions 700720 containing the k715 and r716 regions are important for the interaction between gi2 and trpc4 channel . g subunits are not obligatory for the action of trpc4 channels .
recently , ml204 was identified as a blocker of both recombinant and endogenous trpc4 channel that lacks activity on most voltage - gated channels and other trps .
selectivity for block of trpc4 channels was examined in fluorescent and electrophysiological experiments against closely related trpc channels and more distantly related trpv , trpa and trpm channels , and against non - trp ion channels .
ml204 afforded good selectivity ( fold19- ) against trpc6 channels and more modest selectivity against trpc3 and trpc5 ( fold9- ) channels .
little or no block of trpv , trpa , trpm or voltage - gated ion channels was observed .
ml204 decreased the increase in intracellular ca mediated by trpc4 after activation of -opioid receptors .
recently , receptor - operated or / and store - operated mechanisms contribute to the activation of trpc4 channels is especially important for the activation mechanism of epidermal growth factor ( egf ) on trpc4 .
trpc4 mediated ca signaling pathway evoked by egf was identified specifically in sub - confluent , proliferating clusters of human microvascular endothelial cells .
direct mechanism is that 2 tyrosine residues of the c - terminus in human trpc4 , tyr-959 and tyr-972 were phosphorylated following egf receptor ( egfr ) stimulation of cos-7 cells .
this phosphorylation was mediated by src family tyrosine kinases ( stks ) , with fyn appearing to be the dominant kinase .
pka activation in turn has a negative feedback effect on egf - induced stimulation of the mapk cascade at the level of raf-1 in the erk limb of this superfamily .
similar study regarding leptin and glucose regulation of trpc4 was recently published . in this study , leptin is shown to activate trpc4 channel and ca influx through trpc4 activates ampk that triggers translocation of atp - dependent k channel ( katp ) to the plasma membrane to hyperpolarize the resting membrane potential in pancreatic -cell .
leptin activates trpc4 in pomc , and kisseptin - secreting hypothalamic neuron . through pomc ,
however , the exact mechanism how leptin activate trpc4 at the molecular level , via tyrosine phosphorylation of trpc4 like egf or jak2-pi3k - pip3-plcgamma1 pathway , needs more experiments .
the physiological role of these channels was recently established , demonstrating that trpc4 and trpc6 are the molecular candidates for non - selective cation channels activated by the muscarinic receptor stimulation ( micat ) in visceral smooth muscle cells .
studies in knock - out showed that trpc4 plays and important role in vascular physiology .
trpc4 mice have markedly reduced store- and receptor - induced endothelial ca entry and impaired endothelium - dependent vasorelaxation .
knockout of trpc4 strongly reduced acetylcholine - activated non - selective cation currents in visceral smooth muscle cells that are involved in the regulation of gastric motility .
airway tracheal and bronchial smooth muscles express trpc4 and trpc5 that are recognized as possible candidates in the pathogenesis of asthma and chronic obstructive pulmonary disease ( copd ) .
recently , single - nucleotide polymorphisms ( snps ) in the trpc4 gene are associated with human diseases .
for example , the trpc4 mutation of snp in the trpc4 gene is associated with a reduced risk of myocardial infarction .
this mutation leads to an increase in receptor - operated cation currents and ca entry .
this mechanism underlying protection is an improved endothelial function , but this needs more investigation . in other study , photoparoxysmal responses ( ppr ) characterized by abnormal visual sensitivity of the brain to photic stimulation which is generally related to idiopathic generalized epilepsies ( ige ) are related to various snps in the trpc4 gene .
this relation was not significant after corresponding corrections , this trend toward relation of trpc4 variants and ppr / ige is of especial interest and deserves further investigation since it could be shown that trpc4 is involved in numerous functions related with epilepsy .
rasd1 was first discovered as a dexamethasone inducible monomeric ras protein in att-20 mouse corticotroph cells in the year 1998 and is expressed at high concentrations in brain and at lower concentrations in heart , liver , kidney , skeletal muscle , pancreas , and placenta .
the upregulation of rasd1 expression by glucocorticoids was due to glucocorticoid response element ( gre ) in the 3'- flanking region of the human rasd1 gene .
rasd1 has all of the conserved domains of the ras superfamily required for guanine nucleotide binding , hydrolysis , and effector interaction .
the full - length cdna of rasd1 predicts a 280-aminoacid protein with a calculated molecular mass of 31,700da .
rasd1 selectively activates the gi / go - protein signaling pathway and appears to act as a guanine nucleotide exchange factor for gi , somewhat mimicking gpcr .
also , it has been reported that rasd1 may have a dual role in modulating the activation of ac2 ( adenylyl cyclase 2 ) signaling by concurrently blocking pkc ( protein kinase c ) and g activity2 proteins that function as activators of ac2 .
can also regulate g signaling , it may be that rasd1 interferes with multiple inputs to ac2 that function in an additive or synergistic manner for maximal ac2 activity .
although their physiological functions remain to be fully elucidated , rasd1 has been implicated in the photic and nonphotic responsiveness of the circadian clock , in tonic inhibitor of atrial natriuretic factor ( anf ) secretion and a modulator of hormone secretion in volume overload condition of heart by inhibiting protein regulation of anf release .
also rasd1 has a function to regulate neurotransmitter mediated behaviors and cancers . in previous paper
also , rasd1 is functionally classified as a guanine nucleotide exchange factor ( gef ) for gi proteins and facilitates guanosine diphosphate ( gdp ) to gtp exchange of gi proteins and triggers it to be functionally active until intrinsic gtpase activity of gi protein turns off the signal by hydrolyzing gtp to gdp .
therefore , since the function of rasd1 as a gef of gi may resemble the activators of gi proteins , we expressed rasd1 and mtrpc4 in hek293 cells and detected the effect of rasd1 on the activity of mtrpc4. rasd1 , rasd1 and rasd1 ( a constitutively active form of rasd1 ) induced large mtrpc4 current .
in contrast , expressing dominant - negative form of rasd1 , rasd1 , did not activate mtrpc4 current .
in addition , to exclude the possibility that rasd1 activates other trp channels such as mtrpc4 , htrpc5 , or mtrpc6 since amino acid sequence homology shows more than 40% similarity among those channels , we tested whether rasd1 activates these channels . however , rasd1 could not activate mtrpc4 and mtrpc6 channels and reduced htrpc5 currents .
also , we tested various ras proteins other than rasd1 to check the specificity of mtrpc4 to rasd1 . among various ras proteins , rasd1 was the only ras protein to increase mtrpc4. therefore , mtrpc4 is the unique channel activated by rasd1 and rasd1 is a unique trp channel activator among various ras proteins in ras family . the sequence homology analysis
demonstrate that rasd1 is most closely related to members of the ras superfamily of smwg proteins , with 55% amino acid homology to rap2b , 36% homology to rit1 , and 50% homology to the prototypical ras protein , h - ras .. recently , several nucleotide sequences predicting proteins with high degrees of homology to human rasd1 ( genebank no .
af009246 ) , and rat rasd1(genebank no.bc099136 ) , which share 98% homology with human rasd1 .
bc026377 ) , which share 62% homology with rasd1 ( fig . 1 ) . ras superfamily proteins such as rasd1 , rasd2 , rit1 , h - ras and rap2b have highly conserved gtp binding pocket ( 14 ) domains and an effector loop which participates in protein - protein interactions with other signaling molecules and is necessary for full biological activity .. the presumed structure of the rasd1 contains several characteristic ras superfamily motifs including the phosphate / magnesium binding regions gxxxxgk(s
/ t ) ( p - loop ) ( 1 ) , dxxg ( 2 ) , and the guanine base binding loops nkxd ( 3 ) and exsak ( 4 ) .
the motif regions g-1 to g-3 which are characteristic of gtpases are present in rasd1 .. the c terminus possesses a typical caax motif , an important biochemical feature of a ras superfamily .
caax motif undergoes enzymatic posttranslational farnesylayion or prenylation , which related to its subcellular localization by promoting the translocation of the rasd1 to the plasma membrane .. we tested whether rasd2 and rit1 can activate trpc4 without any activator like rasd1 ( fig . 2 ) .
since rasd1 proteins were tagged with cyan fluorescent protein ( pecfp ) proteins at the c - terminus , pecfp was transfected as a negative control for rasd1 . as shown in fig . 2b
, pecfp alone did not affect mtrpc4 current ( 1.39 0.24 pa / pf , n = 20 ) .
rasd2 , a constitutively active form of rasd2 , induced large mtrpc4 current ( fig . 2 ;
a mutation of serine at position 40 to valine ( s40v ) made rasd2 unable to hydrolyse gtp to gdp , hence holding it constitutively active .
rit1 , a constitutively active form of rit1 , induced large mtrpc4 current ( fig . 2 ;
a mutation of glutamine at position 79 to leucine ( q79l ) made rit1 unable to hydrolyse gtp to gdp , hence holding it constitutively active .
the following sequences were aligned by using multiple sequence alignment by florence corpet : human rasd1 ( genebank no .
af009246 ) , rat rasd1 ( genebank no.bc099136 ) , human rasd2 ( genebank no .
bc026377 ) , rat rasd2 ( genebank no.af134409 ) , human rit1 ( genebank no.u71203 ) , mouse rit1 ( genebank no.u712505 ) , human h - ras ( genebank no .
ay373386 ) , rat h - ras ( genebank no.m13011 ) , human rap2b ( genebank no .
regions defining the gtp - binding and hydrolysis domain ( 1-4 ) are boxed and annotated with their identifying consensus sequences .
consensus abbreviations : b , basic residue ; j , polar residue ; o , hydrophobic residue ; x , any residue .
the three conserved gdp / gtp binding motifs ( g1-g3 ) , and phosphate - magnesium binding motifs ( pm1-pm3 ) are boxed .
the red colors below the sequence indicate amino acid identity , whereas the blue color indicates similarity .
( a ) all panels indicate i - v relationship of currents measured from hek293 cells expressing mouse trpc4-ecfp channel and constitutively active rasd2 or rit1 proteins .
we studied trpc4 channel activity with cs - rich extracellular solution ( cs ) since trpc4 has greater permeability to cs than na .
the comparison between pecfp and rasd2 or rit1 was carried out with student 's t - test .
statistical significance was denoted by an asterisk ( * ) at p < 0.05 .
the following sequences were aligned by using multiple sequence alignment by florence corpet : human rasd1 ( genebank no .
af009246 ) , rat rasd1 ( genebank no.bc099136 ) , human rasd2 ( genebank no .
bc026377 ) , rat rasd2 ( genebank no.af134409 ) , human rit1 ( genebank no.u71203 ) , mouse rit1 ( genebank no.u712505 ) , human h - ras ( genebank no .
ay373386 ) , rat h - ras ( genebank no.m13011 ) , human rap2b ( genebank no .
regions defining the gtp - binding and hydrolysis domain ( 1-4 ) are boxed and annotated with their identifying consensus sequences .
consensus abbreviations : b , basic residue ; j , polar residue ; o , hydrophobic residue ; x , any residue .
the three conserved gdp / gtp binding motifs ( g1-g3 ) , and phosphate - magnesium binding motifs ( pm1-pm3 ) are boxed .
the red colors below the sequence indicate amino acid identity , whereas the blue color indicates similarity .
( a ) all panels indicate i - v relationship of currents measured from hek293 cells expressing mouse trpc4-ecfp channel and constitutively active rasd2 or rit1 proteins .
we studied trpc4 channel activity with cs - rich extracellular solution ( cs ) since trpc4 has greater permeability to cs than na .
the comparison between pecfp and rasd2 or rit1 was carried out with student 's t - test .
next , to characterize the activation mechanism of gi proteins between rasd1 and mtrpc4 channels , we used pertussis toxin ( ptx )
. ptx has been widely used as a reagent to characterize the involvement of heterotrimeric g proteins in signaling .
this toxin catalyzes the adp ribosylation of subunits of gi family , and this modification prevents the occurrence of the receptor - g protein interaction . because the population of functional gi is strongly reduced by ptx , we examined whether ptx antagonizes the action of rasd1 .
rasd1-activated mtrpc4 currents from rasd1-expressing cells at 60 mv were significantly reduced when ptx was pretreated .
however , when gtps was added to internal solution as a positive control for the activation , ptx could not reduce gtps activated mtrpc4 currents from rasd1-expressing cells . from these results
, we reasoned that rasd1 requires certain population of functional gi proteins to activate mtrpc4 channels .
muscarinic acetylcholine receptor type 2 ( m2r ) , for example , is strong mtrpc4 channel activator , and functional gi proteins are quintessential for its action onto mtrpc4 channels . in a sense , if rasd1 and m2r both exist , the competitive action for functional gi protein would be expected .
therefore , we expressed both rasd1 and m2r in hek293 cells and tested how the mtrpc4 channel current results . without rasd1 ,
m2r strongly activated mtrpc4 channels in response to extracellular carbachol ( 100 m ) .
when rasd1 was co - expressed , however , m2r - activated mtrpc4 current was significantly reduced which clearly demonstrates competitive action between rasd1 and m2r .
therefore , activation signaling of rasd1 to mtrpc4 involves gi protein , and certain population of functional gi protein is essential for activation of mtrpc4 by rasd1 .
there are several types of gi proteins in hek293 cells in which the level of expression decreases following order gi2 > gi3 > gi1 .
to verify the subtype of gi proteins , first , we tested the effect of the subtype of wild - type ( wt ) gi proteins on mtrpc4 currents .
wt gi2 or gi3 protein itself increased the mtrpc4 currents in hek293 cells without activator gtps .
mtrpc4 currents were increased by activating endogenous gi proteins in hek cells via expressed rasd1 .
therefore , we co - expressed mtrpc4 channel , rasd1 , and wt gi protein subtypes to test further effect of expressed wt gi protein subtypes based on the endogenous gi protein subtypes in hek cells on mtrpc4 currents by rasd1 .
mtrpc4 currents by rasd1 with gi1 protein , gi2 protein or gi3 protein showed no difference compared with that by rasd1 protein alone , or expression of wt gi2 or gi3proteins alone .
because rasd1 fully activated endogenous gi proteins in hek cells , mtrpc currents may not be further increased by co - expressing gi proteins with rasd1 .
however , rasd1-activated mtrpc4 currents were increased dramatically by co - expressing gi1 protein with rasd1compared with that by expression of wt gi1 protein alone .
these result suggested that either rasd1 activates gi1 protein more potently or rasd1 is expressed at the low level in hek cells .
next , we tested the effect of dominant negative gi proteins on rasd1-activated mtrpc4 currents .
dominant negative gi1 and gi3 significantly reduced rasd1-activated mtrpc4 currents , but gi2 did not affect . therefore , gi1 and gi3 proteins are crucial for activation of mtrpc4 by rasd1 with more potent effect on gi2 protein .
since rasd1 is gef of gi proteins , it removes gdp from subunit and changes gdp to gtp .
although classical pathway of heterotrimeric g protein describes subunit mostly , the release of g subunit is equally important .
therefore , we tested the involvement of g subunits in rasd1 activation of mtrpc4. when g1 and g2 proteins or g1 and g7 proteins were co - expressed with mtrpc4 channels , neither of them was able to activate mtrpc4 channels .
also , co - expression of -adrenergic receptor kinase ( ark ) that scavenges free g proteins did not reduce rasd1-activated mtrpc4 currents .
furthermore , g ( holds g proteins as a heterotrimer making g proteins to be insensitive to activating signaling ) and g ( keeps g dimers in free form ) mutant did not induce significant difference in rasd1 activation of mtrpc4. from these results , g signaling can be excluded from possible signaling candidates of rasd1 . in previous study , we experimented about the relation between various small g protein ( ras , rho , rab proteins ) and trpc4 ion channel .
it is known that rasd2 can bind g and gi and rit can also bind go .
since trpc4 can be activated by gi and go , trpc4 could activated by not only rasd1 and rasd2 acting gi but also rit acting on go . taken together , rasd1 selectively activated trpc4 channels , and it was the only ras protein among ras protein family that can activate trpc4 channels .
for this to occur , it was found that certain population of functional gi1 and gi3 proteins are essential .
together , the results indicate that activation of gi subunits by rasd1 is the primary mechanism for activating trpc4 .
this raised the question of whether activation of the channels requires direct interaction of rasd1 with the gi subunits .
, hek cells were transfected with rasd1-cfp and gi1 , and their association was analyzed by co - immunoprecipitation .
key prerequisites for our experiments of interaction of rasd1 and g proteins were strategic attachment of cfp or yfp to g protein . to study g protein subunit interactions in cell ,
cfp or yfp were inserted into the bc - loop within the -helical domain of the gi , a domain that has been used previously to insert various sequences into g-subunits .
there was no significant fret of rasd1 with trpc4 , trpc5 , or g. whether gi1(wt)-yfp or gi3(wt)-yfp has more fret efficiency than gi2(wt)-yfp or not needs further studies .
( b ) fret efficiency was measured in hek293 cells rasd1-cfp , gi2 wt - yfp , gi2 ql - yfp , gq wt - yfp , gq ql - yfp , yfp - mtrpc4 , mtrpc4-cfp , yfp - mtrpc5 , yfp - gg , and gyfp - g. among these proteins , rasd1 bound gi2 wt , gi2 ql , and gq wt .
( b ) fret efficiency was measured in hek293 cells rasd1-cfp , gi2 wt - yfp , gi2 ql - yfp , gq wt - yfp , gq ql - yfp , yfp - mtrpc4 , mtrpc4-cfp , yfp - mtrpc5 , yfp - gg , and gyfp - g. among these proteins , rasd1 bound gi2 wt , gi2 ql , and gq wt .
dexamethasone ( a synthetic glucocorticoid ) is a widely used immunosuppressant and insulin secretion regulator . in a previous research ,
mtrpc4 current was significantly increased with dexamethasone . with these results on hands , we tested whether rasd1 activation by dexamethasone is sufficient to generate trpc4-like current in ins-1 cells , since both rasd1 and trpc4 are detectable in pancreatic -cells .
100 nm dexamethasone was sufficient to generate cationic current in ins-1 cells , and the i - v curve resembled that of trpc4 , suggesting that dexamethasone activates trpc4 in ins-1 cells . also , inhibition of rasd1 with small interfering rna ( sirna ) blunted dexamethasone - induced trpc4 current in ins-1 cells , indicating that dexamethasone activates trpc4 via rasd1 in ins-1 cells . though rasd1 is functionally classified as a guanine nucleotide exchange factor ( gef ) for gi proteins , lellis - santos et al .
suggested that dexamethasone induced a rapid and sustained increase in rasd1 mrna expression in min6 cells ( 3.5- and fold15- compared with control after 24 and 48 h of dexamethasone treatment ) .
therefore , we think that dexamethasone triggers trpc4-like cationic current in ins-1 cells at least via increasing protein expression level of rasd1 . whether dexamethasone enhances the gef activity of rasd1 or not needs more experiments .
the finding of trpc4 current in pancreatic -cell in response to dexamethasone was interesting since similar study regarding leptin and glucose regulation of trpc4 was recently published .
it is the most important and widely studied hormones in the control of energy balance .
leptin is a 16 kda protein mostly secreted from adipose tissue which has a critical role regulating body weight and energy homeostasis .
leptin signals via the leptin receptors ( leprs ) , which exists as several isoforms .
these receptors are expressed in the brain and in peripheral tissues . in the hypothalamus leptin acts as an anorexigenic hormone regulating the melanocortine / neuropeptide y system to reduce food intake ,
however , circulating leptin levels are increased in obese humans , suggesting that obesity may be either a result or a cause of leptin resistance .
leptin - deficient and leptin receptor - deficient mice exhibit thymic atrophy and are immunodeficient .
leptin is also considered a proinflammatory adipokine because of the actions it exerts in several cells of the immune system , including monocytes / macrophages , dendritic cells , neutrophils , eosinophils , basophils , natural killer cells , and lymphocytes .
suggested that a janus 2 tyrosine kinase ( jak 2)-dependent pathway via stimulation of pi3 kinase and plcgamma1 activated trpc channels .
therefore , we think that leptin through a jak2pi3 kinase plcgamma pathway may activate trpc4 channels in ins-1 cells . in this study , leptin is shown to activate trpc4 channel and ca influx through trpc4 activates ampk that triggers translocation of atp - dependent k channel ( katp ) to the plasma membrane to hyperpolarize the resting membrane potential in pancreatic -cell .
another study reported that dexamethasone increased the protein expression of rasd1 in pancreatic -cells and diminished insulin secretion .
these studies indicated that rasd1 and trpc4 could be common molecular candidates in controlling insulin secretion in -cells ( fig .
figure 4.schematic diagram for the signaling pathway involved in dexamethasone - induced trpc4 channel activity .
rasd1 is classified as a guanine nucleotide exchange factor ( gef ) for gi proteins .
dexamethasone enhanced rasd1 that promotes gdp to gtp exchange in gi proteins and triggers it to be functionally active until the intrinsic gtpase activity of gi proteins turns off the signal by hydrolyzing gtp to gdp .
leptin binds to its receptor ( lrb ) to activate jak2 , which phosphorylates irs proteins and activates pi3 kinase .
rasd1 is classified as a guanine nucleotide exchange factor ( gef ) for gi proteins .
dexamethasone enhanced rasd1 that promotes gdp to gtp exchange in gi proteins and triggers it to be functionally active until the intrinsic gtpase activity of gi proteins turns off the signal by hydrolyzing gtp to gdp .
leptin binds to its receptor ( lrb ) to activate jak2 , which phosphorylates irs proteins and activates pi3 kinase .
the trpc subfamily consists of 7 proteins designated as trpc1 to 7 , which can be further divided into 4 subgroups based on their sequence homology and functional similarities : ( 1 ) trpc1 , ( 2 ) trpc4 and trpc5 , ( 3 ) trpc3 , trpc6 and trpc7 , and ( 4 ) trpc2 .
molecules downstream of plc , such as ip3 and dag , and pip2 hydrolysis have been suggested as activators and as an activation mechanism for trpc channels , respectively .
trpc4 channel consist of 6 transmembrane domains , intracellularly located amino and carboxyl termini and a putative pore - forming region . within the trpc family ,
trpc4 and trpc5 represent a structurally distinct subgroup that is characterized by its ability to form homo- and heteromultimeric channels with each other as well as with trpc1 . trpc4 and trpc4 are the most abundantly expressed and functionally characterized .
trpc4 channel lacks a domain of 84 amino acids in the c - terminal region containing a putative binding site for calmodulin ( cam ) binding and inositol 1 , 4 , 5-triphosphate ( ip3 ) receptors .
the deleted regions of trpc4 is also responsible for the channel activity by pi(4 , 5)p2 .
trpc4 has the 4 short amino acid sequence motifs ( m1-m4 ) that show typical features of trp .
m1 motif is discovered upstream of tm1 and is conserved in this form throughout the trpc subfamily .
this channel contains a coiled - coil domain , 4 ankyrin - like repeats , and predicted multimerization domain in the n - terminus as potential protein - protein interaction motifs .
trpc4 is expressed in diverse organs and cell types including the dendrites and soma of various types of neurons , smooth muscles , the cardiovascular system , including endothelial and cardiac cells , skeletal muscle cells , the myometrium , the kidney , and immune cells .
trpc4 channels differ extremely in their permeability and pharmacological modulation , other biophysical properties , and mode of activation depending on the cellular environment .
activation of trpc4 channels by agonists induced ca entry directly or indirectly via depolarization and activation of voltage - gated ca channels .
trpc4 channel was reported that this channel , as well as phospholipase c 1 and 2 interact with the first pdz domain of nherf , regulatory factor of the na / h exchanger .
the c - terminal pdz ( dlg , zo-1 , psd-95 ) motif of trpc4 has been implicated in the control of channels surface expression and localization .
the pdz deletion motif ( t - r - l ) of trpc4 dramatically reduced the plasma membrane levels of the channel , which led the authors to suggest that the interaction between htrpc4 and nherf is required for the retention and stabilization of htrpc4 channels in the cell membrane .
caveolin-1 ( cav-1 ) is associated with dynamic protein complex consisting of trpc4 , trpc1 and ip3rs .
the loss of cav-1 blunted the localization of trpc4 and agonist - stimulated complex formation .
trpc4 channel has been proposed to be activated by gq / phospholipase c signaling , release of intracellular castores , or vesicular translocation to the membrane .
gq - plc signaling mediated activation of plc increased ip3 that binds to the ip3 receptor located on the endoplasmic reticulum and released intracellular ca . a conformational change of trpc channel bound to ip3 receptors , which bind to the c - terminal end of the trpc channels via the ip3/calmodulin receptor binding domain . among trp channels , trpc4 channels are unique in that they activated by trivalent cations gd and la independently of gpcr .
mutation and modeling demonstrated that the k715 and r716 regions are important for the interaction between gi2 and trpc4 channel .
thus , we suggest that the regions 700720 containing the k715 and r716 regions are important for the interaction between gi2 and trpc4 channel .
recently , ml204 was identified as a blocker of both recombinant and endogenous trpc4 channel that lacks activity on most voltage - gated channels and other trps .
selectivity for block of trpc4 channels was examined in fluorescent and electrophysiological experiments against closely related trpc channels and more distantly related trpv , trpa and trpm channels , and against non - trp ion channels .
ml204 afforded good selectivity ( fold19- ) against trpc6 channels and more modest selectivity against trpc3 and trpc5 ( fold9- ) channels .
little or no block of trpv , trpa , trpm or voltage - gated ion channels was observed .
ml204 exhibited properties useful for a variety of in vitro investigations . ml204 decreased the increase in intracellular ca mediated by trpc4 after activation of -opioid receptors . recently , receptor - operated or / and store - operated mechanisms contribute to the activation of trpc4 channels is especially important for the activation mechanism of epidermal growth factor ( egf ) on trpc4 .
trpc4 mediated ca signaling pathway evoked by egf was identified specifically in sub - confluent , proliferating clusters of human microvascular endothelial cells .
direct mechanism is that 2 tyrosine residues of the c - terminus in human trpc4 , tyr-959 and tyr-972 were phosphorylated following egf receptor ( egfr ) stimulation of cos-7 cells .
this phosphorylation was mediated by src family tyrosine kinases ( stks ) , with fyn appearing to be the dominant kinase .
pka activation in turn has a negative feedback effect on egf - induced stimulation of the mapk cascade at the level of raf-1 in the erk limb of this superfamily .
similar study regarding leptin and glucose regulation of trpc4 was recently published . in this study , leptin is shown to activate trpc4 channel and ca influx through trpc4 activates ampk that triggers translocation of atp - dependent k channel ( katp ) to the plasma membrane to hyperpolarize the resting membrane potential in pancreatic -cell .
leptin activates trpc4 in pomc , and kisseptin - secreting hypothalamic neuron . through pomc ,
however , the exact mechanism how leptin activate trpc4 at the molecular level , via tyrosine phosphorylation of trpc4 like egf or jak2-pi3k - pip3-plcgamma1 pathway , needs more experiments .
the physiological role of these channels was recently established , demonstrating that trpc4 and trpc6 are the molecular candidates for non - selective cation channels activated by the muscarinic receptor stimulation ( micat ) in visceral smooth muscle cells .
studies in knock - out showed that trpc4 plays and important role in vascular physiology .
trpc4 mice have markedly reduced store- and receptor - induced endothelial ca entry and impaired endothelium - dependent vasorelaxation .
knockout of trpc4 strongly reduced acetylcholine - activated non - selective cation currents in visceral smooth muscle cells that are involved in the regulation of gastric motility .
airway tracheal and bronchial smooth muscles express trpc4 and trpc5 that are recognized as possible candidates in the pathogenesis of asthma and chronic obstructive pulmonary disease ( copd ) .
recently , single - nucleotide polymorphisms ( snps ) in the trpc4 gene are associated with human diseases . for example , the trpc4 mutation of snp in the trpc4 gene is associated with a reduced risk of myocardial infarction .
this mutation leads to an increase in receptor - operated cation currents and ca entry .
this mechanism underlying protection is an improved endothelial function , but this needs more investigation . in other study ,
photoparoxysmal responses ( ppr ) characterized by abnormal visual sensitivity of the brain to photic stimulation which is generally related to idiopathic generalized epilepsies ( ige ) are related to various snps in the trpc4 gene .
this relation was not significant after corresponding corrections , this trend toward relation of trpc4 variants and ppr / ige is of especial interest and deserves further investigation since it could be shown that trpc4 is involved in numerous functions related with epilepsy .
rasd1 was first discovered as a dexamethasone inducible monomeric ras protein in att-20 mouse corticotroph cells in the year 1998 and is expressed at high concentrations in brain and at lower concentrations in heart , liver , kidney , skeletal muscle , pancreas , and placenta .
the upregulation of rasd1 expression by glucocorticoids was due to glucocorticoid response element ( gre ) in the 3'- flanking region of the human rasd1 gene .
rasd1 has all of the conserved domains of the ras superfamily required for guanine nucleotide binding , hydrolysis , and effector interaction .
the full - length cdna of rasd1 predicts a 280-aminoacid protein with a calculated molecular mass of 31,700da .
rasd1 selectively activates the gi / go - protein signaling pathway and appears to act as a guanine nucleotide exchange factor for gi , somewhat mimicking gpcr .
also , it has been reported that rasd1 may have a dual role in modulating the activation of ac2 ( adenylyl cyclase 2 ) signaling by concurrently blocking pkc ( protein kinase c ) and g activity2 proteins that function as activators of ac2 .
can also regulate g signaling , it may be that rasd1 interferes with multiple inputs to ac2 that function in an additive or synergistic manner for maximal ac2 activity .
although their physiological functions remain to be fully elucidated , rasd1 has been implicated in the photic and nonphotic responsiveness of the circadian clock , in tonic inhibitor of atrial natriuretic factor ( anf ) secretion and a modulator of hormone secretion in volume overload condition of heart by inhibiting protein regulation of anf release .
in previous paper , we reported that gi proteins are potent activators of mtrpc4 channels .
also , rasd1 is functionally classified as a guanine nucleotide exchange factor ( gef ) for gi proteins and facilitates guanosine diphosphate ( gdp ) to gtp exchange of gi proteins and triggers it to be functionally active until intrinsic gtpase activity of gi protein turns off the signal by hydrolyzing gtp to gdp .
therefore , since the function of rasd1 as a gef of gi may resemble the activators of gi proteins , we expressed rasd1 and mtrpc4 in hek293 cells and detected the effect of rasd1 on the activity of mtrpc4. rasd1 , rasd1 and rasd1 ( a constitutively active form of rasd1 ) induced large mtrpc4 current .
in contrast , expressing dominant - negative form of rasd1 , rasd1 , did not activate mtrpc4 current .
in addition , to exclude the possibility that rasd1 activates other trp channels such as mtrpc4 , htrpc5 , or mtrpc6 since amino acid sequence homology shows more than 40% similarity among those channels , we tested whether rasd1 activates these channels . however , rasd1 could not activate mtrpc4 and mtrpc6 channels and reduced htrpc5 currents . also , we tested various ras proteins other than rasd1 to check the specificity of mtrpc4 to rasd1 . among various ras proteins ,
rasd1 was the only ras protein to increase mtrpc4. therefore , mtrpc4 is the unique channel activated by rasd1 and rasd1 is a unique trp channel activator among various ras proteins in ras family . the sequence homology analysis demonstrate that rasd1 is most closely related to members of the ras superfamily of smwg proteins , with 55% amino acid homology to rap2b , 36% homology to rit1 , and 50% homology to the prototypical ras protein , h - ras .. recently , several nucleotide sequences predicting proteins with high degrees of homology to human rasd1 ( genebank no .
af009246 ) , and rat rasd1(genebank no.bc099136 ) , which share 98% homology with human rasd1 .
bc026377 ) , which share 62% homology with rasd1 ( fig . 1 ) . ras superfamily proteins such as rasd1 , rasd2 , rit1 , h - ras and rap2b
have highly conserved gtp binding pocket ( 14 ) domains and an effector loop which participates in protein - protein interactions with other signaling molecules and is necessary for full biological activity .. the presumed structure of the rasd1 contains several characteristic ras superfamily motifs including the phosphate / magnesium binding regions gxxxxgk(s
/ t ) ( p - loop ) ( 1 ) , dxxg ( 2 ) , and the guanine base binding loops nkxd ( 3 ) and exsak ( 4 ) .
the motif regions g-1 to g-3 which are characteristic of gtpases are present in rasd1 .. the c terminus possesses a typical caax motif , an important biochemical feature of a ras superfamily .
caax motif undergoes enzymatic posttranslational farnesylayion or prenylation , which related to its subcellular localization by promoting the translocation of the rasd1 to the plasma membrane .. we tested whether rasd2 and rit1 can activate trpc4 without any activator like rasd1 ( fig . 2 ) . since rasd1 proteins were tagged with cyan fluorescent protein ( pecfp ) proteins at the c - terminus , pecfp was transfected as a negative control for rasd1 . as shown in fig . 2b
, pecfp alone did not affect mtrpc4 current ( 1.39 0.24 pa / pf , n = 20 ) .
rasd2 , a constitutively active form of rasd2 , induced large mtrpc4 current ( fig . 2 ; 11.1 2.23 pa / pf ; n = 10 ) . a mutation of serine at position 40 to valine ( s40v ) made rasd2 unable to hydrolyse gtp to gdp , hence holding it constitutively active
rit1 , a constitutively active form of rit1 , induced large mtrpc4 current ( fig . 2 ; 9.86 4.82 pa / pf ; n = 7 ) .
a mutation of glutamine at position 79 to leucine ( q79l ) made rit1 unable to hydrolyse gtp to gdp , hence holding it constitutively active .
this constitutively active rit1 induced strong morphological change ..
figure 1.sequence alignment of several forms of rasd1 , rasd2 , and rit1 proteins .
the following sequences were aligned by using multiple sequence alignment by florence corpet : human rasd1 ( genebank no .
af009246 ) , rat rasd1 ( genebank no.bc099136 ) , human rasd2 ( genebank no .
bc026377 ) , rat rasd2 ( genebank no.af134409 ) , human rit1 ( genebank no.u71203 ) , mouse rit1 ( genebank no.u712505 ) , human h - ras ( genebank no .
ay373386 ) , rat h - ras ( genebank no.m13011 ) , human rap2b ( genebank no .
regions defining the gtp - binding and hydrolysis domain ( 1-4 ) are boxed and annotated with their identifying consensus sequences .
consensus abbreviations : b , basic residue ; j , polar residue ; o , hydrophobic residue ; x , any residue .
the three conserved gdp / gtp binding motifs ( g1-g3 ) , and phosphate - magnesium binding motifs ( pm1-pm3 ) are boxed .
the red colors below the sequence indicate amino acid identity , whereas the blue color indicates similarity .
figure 2.rasd2 and rit1 activates trpc4 channel .
( a ) all panels indicate i - v relationship of currents measured from hek293 cells expressing mouse trpc4-ecfp channel and constitutively active rasd2 or rit1 proteins .
we studied trpc4 channel activity with cs - rich extracellular solution ( cs ) since trpc4 has greater permeability to cs than na .
the comparison between pecfp and rasd2 or rit1 was carried out with student 's t - test .
statistical significance was denoted by an asterisk ( * ) at p < 0.05 .
the following sequences were aligned by using multiple sequence alignment by florence corpet : human rasd1 ( genebank no .
af009246 ) , rat rasd1 ( genebank no.bc099136 ) , human rasd2 ( genebank no .
bc026377 ) , rat rasd2 ( genebank no.af134409 ) , human rit1 ( genebank no.u71203 ) , mouse rit1 ( genebank no.u712505 ) , human h - ras ( genebank no .
ay373386 ) , rat h - ras ( genebank no.m13011 ) , human rap2b ( genebank no .
regions defining the gtp - binding and hydrolysis domain ( 1-4 ) are boxed and annotated with their identifying consensus sequences .
consensus abbreviations : b , basic residue ; j , polar residue ; o , hydrophobic residue ; x , any residue .
the three conserved gdp / gtp binding motifs ( g1-g3 ) , and phosphate - magnesium binding motifs ( pm1-pm3 ) are boxed .
the red colors below the sequence indicate amino acid identity , whereas the blue color indicates similarity .
( a ) all panels indicate i - v relationship of currents measured from hek293 cells expressing mouse trpc4-ecfp channel and constitutively active rasd2 or rit1 proteins .
we studied trpc4 channel activity with cs - rich extracellular solution ( cs ) since trpc4 has greater permeability to cs than na .
the comparison between pecfp and rasd2 or rit1 was carried out with student 's t - test .
next , to characterize the activation mechanism of gi proteins between rasd1 and mtrpc4 channels , we used pertussis toxin ( ptx ) . ptx has been widely used as a reagent to characterize the involvement of heterotrimeric g proteins in signaling .
this toxin catalyzes the adp ribosylation of subunits of gi family , and this modification prevents the occurrence of the receptor - g protein interaction . because the population of functional gi is strongly reduced by ptx , we examined whether ptx antagonizes the action of rasd1 .
rasd1-activated mtrpc4 currents from rasd1-expressing cells at 60 mv were significantly reduced when ptx was pretreated . however , when gtps was added to internal solution as a positive control for the activation , ptx could not reduce gtps activated mtrpc4 currents from rasd1-expressing cells . from these results
, we reasoned that rasd1 requires certain population of functional gi proteins to activate mtrpc4 channels .
muscarinic acetylcholine receptor type 2 ( m2r ) , for example , is strong mtrpc4 channel activator , and functional gi proteins are quintessential for its action onto mtrpc4 channels . in a sense , if rasd1 and m2r both exist , the competitive action for functional gi protein would be expected .
therefore , we expressed both rasd1 and m2r in hek293 cells and tested how the mtrpc4 channel current results . without rasd1 ,
m2r strongly activated mtrpc4 channels in response to extracellular carbachol ( 100 m ) .
when rasd1 was co - expressed , however , m2r - activated mtrpc4 current was significantly reduced which clearly demonstrates competitive action between rasd1 and m2r . therefore , activation signaling of rasd1 to mtrpc4 involves gi protein , and certain population of functional gi protein is essential for activation of mtrpc4 by rasd1 .
there are several types of gi proteins in hek293 cells in which the level of expression decreases following order gi2 > gi3 > gi1 . to verify the subtype of gi proteins ,
first , we tested the effect of the subtype of wild - type ( wt ) gi proteins on mtrpc4 currents .
wt gi2 or gi3 protein itself increased the mtrpc4 currents in hek293 cells without activator gtps .
mtrpc4 currents were increased by activating endogenous gi proteins in hek cells via expressed rasd1 .
therefore , we co - expressed mtrpc4 channel , rasd1 , and wt gi protein subtypes to test further effect of expressed wt gi protein subtypes based on the endogenous gi protein subtypes in hek cells on mtrpc4 currents by rasd1 .
mtrpc4 currents by rasd1 with gi1 protein , gi2 protein or gi3 protein showed no difference compared with that by rasd1 protein alone , or expression of wt gi2 or gi3proteins alone .
because rasd1 fully activated endogenous gi proteins in hek cells , mtrpc currents may not be further increased by co - expressing gi proteins with rasd1 .
however , rasd1-activated mtrpc4 currents were increased dramatically by co - expressing gi1 protein with rasd1compared with that by expression of wt gi1 protein alone .
these result suggested that either rasd1 activates gi1 protein more potently or rasd1 is expressed at the low level in hek cells .
next , we tested the effect of dominant negative gi proteins on rasd1-activated mtrpc4 currents .
dominant negative gi1 and gi3 significantly reduced rasd1-activated mtrpc4 currents , but gi2 did not affect .
therefore , gi1 and gi3 proteins are crucial for activation of mtrpc4 by rasd1 with more potent effect on gi2 protein .
since rasd1 is gef of gi proteins , it removes gdp from subunit and changes gdp to gtp .
although classical pathway of heterotrimeric g protein describes subunit mostly , the release of g subunit is equally important .
therefore , we tested the involvement of g subunits in rasd1 activation of mtrpc4. when g1 and g2 proteins or g1 and g7 proteins were co - expressed with mtrpc4 channels , neither of them was able to activate mtrpc4 channels .
also , co - expression of -adrenergic receptor kinase ( ark ) that scavenges free g proteins did not reduce rasd1-activated mtrpc4 currents .
furthermore , g ( holds g proteins as a heterotrimer making g proteins to be insensitive to activating signaling ) and g ( keeps g dimers in free form ) mutant did not induce significant difference in rasd1 activation of mtrpc4. from these results , g signaling can be excluded from possible signaling candidates of rasd1 . in previous study , we experimented about the relation between various small g protein ( ras , rho , rab proteins ) and trpc4 ion channel .
it is known that rasd2 can bind g and gi and rit can also bind go .
since trpc4 can be activated by gi and go , trpc4 could activated by not only rasd1 and rasd2 acting gi but also rit acting on go . taken together , rasd1 selectively activated trpc4 channels , and it was the only ras protein among ras protein family that can activate trpc4 channels .
for this to occur , it was found that certain population of functional gi1 and gi3 proteins are essential .
together , the results indicate that activation of gi subunits by rasd1 is the primary mechanism for activating trpc4 .
this raised the question of whether activation of the channels requires direct interaction of rasd1 with the gi subunits .
, hek cells were transfected with rasd1-cfp and gi1 , and their association was analyzed by co - immunoprecipitation .
key prerequisites for our experiments of interaction of rasd1 and g proteins were strategic attachment of cfp or yfp to g protein . to study g protein subunit interactions in cell ,
cfp or yfp were inserted into the bc - loop within the -helical domain of the gi , a domain that has been used previously to insert various sequences into g-subunits .
there was no significant fret of rasd1 with trpc4 , trpc5 , or g. whether gi1(wt)-yfp or gi3(wt)-yfp has more fret efficiency than gi2(wt)-yfp or not needs further studies .
( b ) fret efficiency was measured in hek293 cells rasd1-cfp , gi2 wt - yfp , gi2 ql - yfp , gq wt - yfp , gq ql - yfp , yfp - mtrpc4 , mtrpc4-cfp , yfp - mtrpc5 , yfp - gg , and gyfp - g. among these proteins , rasd1 bound gi2 wt , gi2 ql , and gq wt .
( b ) fret efficiency was measured in hek293 cells rasd1-cfp , gi2 wt - yfp , gi2 ql - yfp , gq wt - yfp , gq ql - yfp , yfp - mtrpc4 , mtrpc4-cfp , yfp - mtrpc5 , yfp - gg , and gyfp - g. among these proteins , rasd1 bound gi2 wt , gi2 ql , and gq wt .
dexamethasone ( a synthetic glucocorticoid ) is a widely used immunosuppressant and insulin secretion regulator . in a previous research ,
, we tested whether rasd1 activation by dexamethasone is sufficient to generate trpc4-like current in ins-1 cells , since both rasd1 and trpc4 are detectable in pancreatic -cells .
100 nm dexamethasone was sufficient to generate cationic current in ins-1 cells , and the i - v curve resembled that of trpc4 , suggesting that dexamethasone activates trpc4 in ins-1 cells .
also , inhibition of rasd1 with small interfering rna ( sirna ) blunted dexamethasone - induced trpc4 current in ins-1 cells , indicating that dexamethasone activates trpc4 via rasd1 in ins-1 cells .
though rasd1 is functionally classified as a guanine nucleotide exchange factor ( gef ) for gi proteins , lellis - santos et al . suggested that dexamethasone induced a rapid and sustained increase in rasd1 mrna expression in min6 cells ( 3.5- and fold15- compared with control after 24 and 48 h of dexamethasone treatment ) .
therefore , we think that dexamethasone triggers trpc4-like cationic current in ins-1 cells at least via increasing protein expression level of rasd1 . whether dexamethasone enhances the gef activity of rasd1 or not needs more experiments .
the finding of trpc4 current in pancreatic -cell in response to dexamethasone was interesting since similar study regarding leptin and glucose regulation of trpc4 was recently published .
it is the most important and widely studied hormones in the control of energy balance .
leptin is a 16 kda protein mostly secreted from adipose tissue which has a critical role regulating body weight and energy homeostasis .
leptin signals via the leptin receptors ( leprs ) , which exists as several isoforms .
these receptors are expressed in the brain and in peripheral tissues . in the hypothalamus leptin acts as an anorexigenic hormone regulating the melanocortine / neuropeptide y system to reduce food intake ,
however , circulating leptin levels are increased in obese humans , suggesting that obesity may be either a result or a cause of leptin resistance .
leptin - deficient and leptin receptor - deficient mice exhibit thymic atrophy and are immunodeficient .
leptin is also considered a proinflammatory adipokine because of the actions it exerts in several cells of the immune system , including monocytes / macrophages , dendritic cells , neutrophils , eosinophils , basophils , natural killer cells , and lymphocytes .
qiu et al . suggested that a janus 2 tyrosine kinase ( jak 2)-dependent pathway via stimulation of pi3 kinase and plcgamma1 activated trpc channels .
therefore , we think that leptin through a jak2pi3 kinase plcgamma pathway may activate trpc4 channels in ins-1 cells . in this study
, leptin is shown to activate trpc4 channel and ca influx through trpc4 activates ampk that triggers translocation of atp - dependent k channel ( katp ) to the plasma membrane to hyperpolarize the resting membrane potential in pancreatic -cell .
another study reported that dexamethasone increased the protein expression of rasd1 in pancreatic -cells and diminished insulin secretion .
these studies indicated that rasd1 and trpc4 could be common molecular candidates in controlling insulin secretion in -cells ( fig .
figure 4.schematic diagram for the signaling pathway involved in dexamethasone - induced trpc4 channel activity .
rasd1 is classified as a guanine nucleotide exchange factor ( gef ) for gi proteins .
dexamethasone enhanced rasd1 that promotes gdp to gtp exchange in gi proteins and triggers it to be functionally active until the intrinsic gtpase activity of gi proteins turns off the signal by hydrolyzing gtp to gdp .
leptin binds to its receptor ( lrb ) to activate jak2 , which phosphorylates irs proteins and activates pi3 kinase .
rasd1 is classified as a guanine nucleotide exchange factor ( gef ) for gi proteins .
dexamethasone enhanced rasd1 that promotes gdp to gtp exchange in gi proteins and triggers it to be functionally active until the intrinsic gtpase activity of gi proteins turns off the signal by hydrolyzing gtp to gdp .
leptin binds to its receptor ( lrb ) to activate jak2 , which phosphorylates irs proteins and activates pi3 kinase .
we showed 1 ) that rasd1 small g protein activates trpc4 channel via gef for gi proteins .
three ) rasd1 interacts with gi protein rather than trpc4 or g. four ) in pancreatic cell line , ins-1 , rasd1 induced trpc4-like current . in the previous studies , we reported that gi / o proteins are essential for the activation of trpc4 .. related to gi protein , rasd1 and homologs also activated trpc4 by inducing gtp exchange for gi proteins .
interestingly , ptx did not inhibit rasd1-induced current in the presence of intracellular gtps . in the beginning
however , we could not obtain the clear evidence showing the mutant responding to gtps but not gi proteins .
any mutant that did not respond to gi2 protein also did not respond to gtps . on the contrary ,
-spectrin domain ( 730758 amino acids ) deleting mutant showed the response to gi protein but not to gtps , suggesting that gi protein - dependent pathway might be different from more general activation pathways by gtps including gi protein pathways .
we need trpc4 mutants which respond to gtps but not to gi proteins for clear distinction between gi protein pathway and gtps dependent pathway or to find other activation pathways independent from gi protein pathways .
it will also be important to test whether trpc4 currents can be recorded simultaneously with intracellular calcium concentration to see the relationship of trpc4 with calcium itself . finally , as was done for girk , we should study further the binding site for intracellular calcium itself , the effect of polyamine and mg on trpc4 in the inside - out patch mode , the roles of ptdins(4,5)p2 and pkc in the desensitization and the role of g proteins in channel assembly and trafficking in addition to their known role in cellular signaling . clinically , the effect of glucocorticoid is important in care of hospitalized diabetes mellitus ( dm ) patients .
it is known that long - term prescription of dexamethasone could lead to insulin resistance and decrease in insulin secretion .
if the patient has been treated with dexamethasone as an anti - inflammatory drug or immunosuppressant , diabetes mellitus as his / her underlying disease could occur as a paradoxical effect of the drug .
the relationship among rasd1 , trpc4 , and insulin secretion might suggest new therapeutic agent for this particular clinical situation .
however , the exact mechanism how leptin activates trpc4 at the molecular level , via tyrosine phosphorylation of trpc4 like egf or jak2-pi3k - pip3-plcgamma1 pathway , needs more experiments .
it is well known that insulin secretion from pancreatic -cells is increased by parasympathetic nervous system .
when we tried to activate trpc4 channel with carbachol , however , carbachol did not activate trpc4 current in ins-1 cells ( fig .
showed that acetylcholine ( ach ) acts on different receptor subtypes producing both a stimulatory ( m1 , m3 ) and an inhibitory ( m2 , m4 ) action on insulin release .
. also showed that mutant mice selectively lacking the m3 muscarinic acetylcholine receptor subtype in pancreatic -cells display impaired glucose tolerance and greatly reduced insulin release .
in contrast , transgenic mice selectively overexpressing m3 receptors in pancreatic -cells show a profound increase in glucose tolerance and insulin release . moreover , these mutant mice are resistant to diet - induced glucose intolerance and hyperglycemia .
suggested that -cell m3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis . on the other hand , miguel et al
. showed an important functional cooperation between the cholinergic neurotransmitter ach and the incretin hormone glp-1 on insulin secretion mediated through the m3 muscarinic receptor subtype .
however , the insulinotropic action of ach was associated with a paradoxical inhibitory effect on glp-1 stimulated camp production , achieved through a novel ptx- and pirenzepine - sensitive m1 muscarinic receptor activated pathway .
these results suggest that intracellular ca and camp are important for the insulinotropic action of ach . considering the role of trpc4 in m2 and m3 muscarinic receptor signaling ,
there is still the possibility for trpc4 to be involved in the insulinotropic action of ach in native pancreatic -cells .
one hundred micromolars of carbachol induced trpc4-like current in ins-1 cells measured by perforated patch clamp configuration . under carbachol stimulation , trpc4-like current
black arrow heads ( cs ) and red arrow heads ( cs + cch ) indicate the time points where corresponding i - v curves were obtained .
one hundred micromolars of carbachol induced trpc4-like current in ins-1 cells measured by perforated patch clamp configuration . under carbachol stimulation , trpc4-like current
black arrow heads ( cs ) and red arrow heads ( cs + cch ) indicate the time points where corresponding i - v curves were obtained .
this work was supported by a national research foundation of korea ( nrf ) grant funded by the korea government ( msip ) ( # 2014r1a5a2009936 , 20100019472 and 2012r1a2a1a01003073 ) .
j. m. , and k. h. were supported by the graduate program of bk plus program from the korea government ( msip ) .
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trpc4 is important regulators of electrical excitability in gastrointestinal myocytes , pancreatic -cells and neurons .
much is known regarding the assembly and function of these channels including trpc1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events .
further , they are one of the best - studied targets of g protein - coupled receptors and growth factors in general and gi / o and gq protein coupled receptor or epidermal growth factor and leptin in particular . however , our understanding of the roles of small g proteins and leptin on trpc4 channels is still rudimentary .
we discuss potential roles for rasd1 small g protein and leptin in channel activation in addition to their known role in cellular signaling .
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the main orthodontic malocclusions that require maximum anchorage for premolar extraction are severe crowding and protrusion .
extraction of permanent teeth to set the occlusion to class i is a routine approach in orthodontic treatment .
achieving maximum or absolute anchorage is the most important strategy among the systems minimizing the anchorage loss during space closure .
recently , temporary anchorage devices ( tads ) such as miniscrews , mini - implants , and miniplates have been introduced in orthodontic practice as absolute anchorage mechanics .
studies showed that bone anchorage treatments showed less anchorage loss than tooth anchorage mechanics . over the past 60 years
, skeletal anchorage systems showed a variety of design , material , and placement locations .
miniscrews are applied for many treatment modalities such as the corrections in the anteroposterior dimension such as retraction , protraction , distalization , and in the vertical dimension such as openbites , vertical control in high - angle patients , intrusion and extrusion of the teeth , deepbite , and canted occlusal planes .
the purpose of the study is to determine the anchorage potential of tads for canine retraction during space closure and compare and evaluate the amount of anchorage loss and the movement of molars during canine retraction in class i dentoalveolar protrusion .
this retrospective study was designed to evaluate the effects of tads as an absolute anchorage compared to conventional molar - anchored canine retraction .
a total of fifty patients were selected having a class i malocclusion with a treatment indication of bilateral maxillary first premolar extraction ; and they were divided into two groups . the first group consisted of 25 patients with a mean age of 18.7 years ( ranged 1422 years ; 16 girls and 9 boys ) to whom tads were applied as an anchorage mechanic between the attached gingiva of the upper second premolar and first molar teeth .
the second group consisted of 25 patients with a mean age of 19.4 years ( ranged 1323 years ; 14 girls and 11 boys ) , in whom conventional molar anchorage was reinforced with a transpalatal arch ( tpa ) .
the first molar bands were soldered to a transpalatal bar constructed on the plaster models , and the tpa is applied to the upper first molar teeth .
all the patients were treated by the same orthodontist with 0.022 roth - prescribed brackets . among the patients who have completed their fixed orthodontic treatment , the inclusion criteria were as follows : ( 1 ) patients without any craniofacial deformity and systemic diseases , ( 2 ) having a malocclusion requiring first premolar extraction , ( 3 ) maximum anchorage need for space closure , ( 4 ) angle class i malocclusion with anterior protrusion with an anb angle of maximum 4 and minimum of 1. the treatment schedules of all the selected patients were inspected , and the patients who received the standardized treatment protocol were chosen for this study . according to the treatment protocol
, tads had been placed between the upper second premolar and first molar teeth on the attached gingiva following leveling and aligning of the dental arch .
ti coil springs had been applied between the upper canine bracket and tad on the 0.019 0.025 stainless steel arch to provide a continuous force of 150 g on a bracket slot of 0.022 with roth 's prescription .
tpas had been constructed on the plaster models and applied after the initial leveling and aligning of the upper arch .
the landmarks and the cephalometric points , planes , and measurements are shown and described in figure 1 .
lateral cephalometric analysis : ( 1 ) the distance between the upper molar and pterygoid vertical plane ( u6 pterygoid vertical plane distance ) ; ( 2 ) the distance between the upper canine and pterygoid vertical plane ( u3 pterygoid vertical plane distance ) ; ( 3 ) upper molar inclination ( u6 - frankfort horizontal angle ) ; ( 4 ) upper molar inclination ( u6 - pp angle ) ; ( 5 ) upper canine inclination ( u3 - frankfort horizontal angle ) ; ( 6 ) upper canine inclination ( u3 - pp angle ) ; ( 7 ) upper molar extrusion ( u6 - frankfort horizontal distance ) ; ( 8) upper molar extrusion ( u6 to pp distance ) ; ( 9 ) upper canine extrusion ( u3 - frankfort horizontal distance ) ; ( 10 ) upper canine extrusion ( u3 - pp distance ) ; ( 11 ) ans - xi - pm ; ( 12 ) , go - gn - sn ; ( 13 ) fma pre- and post - treatment measurements were performed twice 2 weeks apart by the same examiner via the same digitizing software . paired sample t - test
was applied to measure the difference between the two measurements of the randomly selected twenty cephalograms , no significant difference was found ( p > 0.05 ) , and the values ranged between 0.86 and 0.94 in the accepted limits .
the normality of data was checked in two groups and the data were nonsignificant , thus a parametric statistical analysis was performed .
the pre- and post - treatment comparisons within the same group were performed via paired sample t - test .
the comparisons of tad and tpa groups were analyzed via independent sample t - test via statistical package for social sciences ( spss ) version 22.0 ( ibm corp , ny , usa ) .
the landmarks and the cephalometric points , planes , and measurements are shown and described in figure 1 . lateral cephalometric analysis : ( 1 ) the distance between the upper molar and pterygoid vertical plane ( u6 pterygoid vertical plane distance ) ; ( 2 ) the distance between the upper canine and pterygoid vertical plane ( u3 pterygoid vertical plane distance ) ; ( 3 ) upper molar inclination ( u6 - frankfort horizontal angle ) ; ( 4 ) upper molar inclination ( u6 - pp angle ) ; ( 5 ) upper canine inclination ( u3 - frankfort horizontal angle ) ; ( 6 ) upper canine inclination ( u3 - pp angle ) ; ( 7 ) upper molar extrusion ( u6 - frankfort horizontal distance ) ; ( 8) upper molar extrusion ( u6 to pp distance ) ; ( 9 ) upper canine extrusion ( u3 - frankfort horizontal distance ) ; ( 10 ) upper canine extrusion ( u3 - pp distance ) ; ( 11 ) ans - xi - pm ; ( 12 ) , go - gn - sn ; ( 13 ) fma
pre- and post - treatment measurements were performed twice 2 weeks apart by the same examiner via the same digitizing software .
paired sample t - test was applied to measure the difference between the two measurements of the randomly selected twenty cephalograms , no significant difference was found ( p > 0.05 ) , and the values ranged between 0.86 and 0.94 in the accepted limits .
the normality of data was checked in two groups and the data were nonsignificant , thus a parametric statistical analysis was performed .
the pre- and post - treatment comparisons within the same group were performed via paired sample t - test .
the comparisons of tad and tpa groups were analyzed via independent sample t - test via statistical package for social sciences ( spss ) version 22.0 ( ibm corp , ny , usa ) .
the pretreatment statistics of differences among the groups concerning the lateral cephalometric measurements is shown in table 1 .
after treatment , the difference between the values was 2.4 mm , indicating a significant difference ( p < 0.01 ) .
similarly , the upper molar angle relative to the frankfort horizontal ( fh ) plane ( p < 0.05 ) and palatal plane ( p < 0.01 ) showed a significant difference after treatment when two groups were compared .
the canine distalization showed a similar range of movement between the two groups concerning the inclination relative to fh and palatal plane that shows an insignificant difference ( p > 0.05 ) .
the net difference for vertical movement of the maxillary first molar in relation to fh plane was 0.9 mm , and 1.2 mm relative to the palatal plane , however the differences were not significant ( p > 0.05 ) .
the extrusion of the canine following distalization had similar values relative to fh and palatal plane when comparing the two groups , indicating an insignificant difference ( p > 0.05 ) .
the vertical facial measurements ( lower facial height , go - gn - sn , and mandibular plane angle ) showed no significant differences after treatment among the groups .
comparison of temporary anchorage device and transpalatal arch groups at t0 comparison of treatment changes between t0 and t1
the main objective of this study was to evaluate the dental and skeletal effects and the efficiencies of tads and tpas for canine retraction movement .
the mean measurements at the beginning of the treatment for both groups are given in table 1 .
the measurements of the two groups showed similar values indicating that the groups are harmonious .
analysis of the differences between the two groups showed the differences between absolute anchorage of tads and reinforced anchorage mechanics ( tpa ) .
the results of this study agree with the results of the studies suggesting the placement of tads for absolute anchorage .
thiruvenkatachari et al . and herman et al . compared the amount of anchorage loss observed in the first molars reinforced with and without tads during canine retraction and stated that no anchorage was lost on the side where tad was placed .
similarly , in our study , no anchorage loss was seen in the tad group , but a mean 2.4 mm of mesial movement was observed in the tpa group .
garfinkle et al . evaluated the effectiveness of tads in a controlled clinical trial to provide anchorage for the closure of canine spaces in premolar extraction cases and reported that no anchorage was lost with the system ; in addition , the stability of the miniscrews was constant up to 250 g force applied , showing a similarity with these results .
several studies have investigated the relationship between optimum force magnitude and rate of canine retraction .
storey and smith have reported an optimum force of 150200 g for retraction , iwasaki et al . suggested that an 18 g force could be an effective force for tooth movement ; likewise , ricketts advocated a 75 g force and lee recommended a 150200 g as the optimum force for canine retraction . since in general , light forces are thought to be more biologic and less painful , in our study , 150 g force was applied to be effective for canine retraction .
benson et al . have compared two groups with midpalatal implants and reinforced anchorage with headgear , revealing that the skeletal and dental points moved mesially with an average of 0.5 mm in headgear group and no movement in midpalatal implant group .
the findings of the anchorage loss in the study of sharma et al . showed an anchor loss of 4 mm in tpa anchorage systems .
they have evaluated the maxillary molar distance as the indicator of anchorage loss , whereas in this study , the amount of molar mesialization , tipping , and extrusion was also inspected in the tpa group .
storey and smith showed that with conventional orthodontic mechanics , 550% of the total extraction space can be taken up by an anchor unit made up of the first molar and second premolar for canine retraction , showing a similarity to our results .
tpa is not a convenient maximum anchorage device and does not provide a significant protection on the anteroposterior position , inclination , and extrusion of the maxillary first molars for canine retraction following extraction .
these results showed that the mean anchorage loss was 2.4 mm ; therefore , tpa did not adequately reinforce the posterior anchorage in maximum anchorage conditions .
it would be beneficial to evaluate the effectiveness of different approaches of achieving orthodontic space closure using tad - supported anchorage .
although the treatment duration can not be reduced by tads , the better posterior anchorage and greater retraction of the upper anterior teeth are achieved compared to the conventional anchorage systems .
the conventional anchorage systems caused the molars to extrude , leading the vertical facial height to increase .
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objective : the purpose of this study was to compare the dental and skeletal effects of canine retraction using conventional anchorage reinforcement systems and comparing them with the usage of tads.materials and methods : the sample consisted of 50 patients having class i malocclusions with bimaxillary protrusion indicated for first premolar extraction , and allocated into two groups .
the first group consisted of 25 patients with a mean age of 18,7 years ( min:14 , max:22 years , 16 girls and 9 boys ) that tads were applied as an anchorage mechanic between attached gingiva of upper second premolar and first molar teeth .
the second group consisted of 25 patients with a mean age of 19,4 years ( min:15 , max:23 years , 14 girls and 11 boys ) that conventional molar anchorage with transpalatal arch ( tpa ) was applied for the anchorage mechanics against canine retraction.results:the results showed that mean mesial movement and the tipping of the first molars in tad group between t0 - t1 were insignificant ( p > 0,05 ) , however in the tpa group were significant ( p<0,01 ) .
vertical movement of the molars were not significant when two groups were compared ( p>0,05).conclusion : although tpa is a useful appliance , it does n't provide an effective anchorage control on anteroposterior movement maxillary first molar teeth concerning first premolar extraction treatment .
tads are more convenient to provide absolute anchorage during maxillary canine retraction in contrast to transpalatal arch .
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pollutants in groundwater aquifers may constitute a significant human health risk .
a large variation in response may result among human populations experiencing the same level and duration of exposure to pollutants .
variability in response , as a result of exposure to a carcinogenic contaminant such as trichloroethylene ( tce ) , can be represented by a distribution function of safe doses .
spatial variability in aquifer characteristics and contaminant transport parameters requires the use of stochastic transport models to quantify variability in exposure concentrations .
a second moment method is used to evaluate the probability of exceeding safe dose levels for a contaminated aquifer .
the name of this method stems from the fact that the formulation is based on the first and second moments of the random variables . with this method ,
the probability is a function of the variability of contaminant concentration ( which incorporates variability in hydrogeologic parameters such as hydraulic conductivity ) and the variability in response in the human population . in this manner ,
the severity of the health risk posed by a contaminated aquifer and the evaluation of appropriate strategies and technologies for aquifer remediation are a function of contaminant concentrations and human health risks .
the applicability and limitations of this method are demonstrated with data on groundwater contaminated by tce at hill air force base , utah.imagesfigure 1.figure 2.figure 3.figure 4 .
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ectodermal dysplasia ( ed ) comprises of a large , heterogeneous group of inherited disorders in which two or more structure derived from embryonic ectoderm are affected , i.e. , skin , hair , nails , nerve cells , sweat glands , part of eye and ear as well as dentition .
thurman published the first case report in 1848 while the term ectodermal dysplasia was coined by weech in 1929 .
the frequency of different eds in a given population is highly variable with a prevalence of seven cases per 10 000 births .
the most common eds are x - linked recessive hypohidrotic ed ( christ - siemens - touraine syndrome ) and hidrotic ed ( clouston syndrome ) .
hidrotic type of ed is more severe form presented with sensitivity to heat , frequent high fevers and is associated with more dental defects .
significant oral findings include hypodontia ( 80% ) , loss of occlusal vertical dimension , protuberant lips , and lack of normal alveolar ridge development .
affected males have agenesis of primary and permanent teeth , whereas heterozygous females have agenesis of permanent teeth .
tooth malformations such as conical teeth and taurodontism of the molars are common findings , whereas oligodontia is a key feature in hypohidrotic ed .
this case report emphasizes on the prosthetic management of ed by enhancing the appearance and function through full mouth rehabilitation with a long - span fixed partial denture designed using a custom made broadrick flag .
a 17-year - old female patient reported to the department of prosthodontics , sreemookambika institute of dental sciences , kulasekharam , tamilnadu , with the complaint of absence of upper and lower front teeth .
extraoral examination showed mild facial dysmorphism , depressed nasal bridge , and protuberant lips [ figure 1 ] .
extraoral examination intraoral examination revealed dry mucosa with partial anodontia and conical - shaped canines and premolars .
occlusal examination revealed class i molar occlusal relation on left quadrants , whereas there was no occlusion on right quadrant [ figure 2 ] .
radiographic examination showed wide pulp chambers in all the teeth with a thin layer of dentin and enamel [ figure 3 ] .
the provisional diagnosis was ed and the treatment plan was ruled out to be orthodontic correction of occlusal relation followed by esthetic rehabilitation of the partially edentulous situation using long - span bridges in both maxillary and mandibular arch .
intraoral examination radiographic examination orthodontic correction was done after which [ figure 4 ] a diagnostic impression ( zelgan2002 ) was made and face bow transfer [ figure 5 ] was done .
an interocclusal ( virtual bite registration paste ) record was used to articulate casts in amman girbachartex semi adjustable ap type articulator .
a custom made broadrick flag was fabricated using autopolymerizing acrylic resin ( dpiself cure powder , the bombay burmah trading corporation , mumbai ) [ figure 6 ] .
this was attached to the upper member of the articulator using a resin jig and occlusal plane as well as curve of spee was determined .
a curve of spee is an arc of curve that passes through the cusp tip of mandibular teeth to condyle ; it is possible to locate the center of curve on the flag using a compass . for this , two reference points were taken into consideration , the distal slope of mandibular canine as anterior reference point and the distal inclines of the distobuccal cusp of last molar as the posterior reference point .
the arms of the compass were spread at four inches and were placed on the posterior reference point and an arc was drawn over the flag .
the procedure was repeated by placing the compass on the anterior reference point and another arc was drawn on the flag to obtain an intersection point which is the proposed center of the curve of spee .
the compass is placed over this intersection point on the flag and a curve is scribed onto the occlusal surface of the lower teeth which represents the actual occlusal plane of the patient .
this arc was transferred to the patient 's mouth during tooth preparation using an acrylic stent .
conservative tooth preparation was done in all teeth with knife edge finish line except the second molars which had an open apex .
master impression was made using putty wash technique ( aquasil ) followed by facebow transfer and articulation .
after orthodontic correction custom made broadrick flag temporization was done using tooth color acrylic resin bridge [ figure 6 ] ( opi the bombay burman trading corporator ltd ) .
casting was done , followed by verification of the metal coping in the patient 's mouth [ figure 7 ] . porcelain shade selected prior to tooth preparation was applied to form a long - span 12-unit maxillary and 11-unit mandibular bridge [ figure 8 ] .
the bridges were cemented using gic type i ( gc , gold label glass ionomer , luting , lining cement gc corporation tokyo , japan ) .
occlusion was verified using articulating paper ( unident instruments ( india ) pvt ltd ) .
oral hygiene instructions were given and a periodic review was done every three months [ figure 9 ] .
prosthodontic management of an ed patient includes fabrication of complete or partial removable prostheses , over dentures with or without tooth preparation , or overdenture with or without attachments , long - span fixed partial denture prosthesis , and dental implants.[47 ] the placement of implants in growing children is not recommended in the maxilla , where the implants can be submerged by the downward growth of investing tissues .
implant placement can be considered in anterior mandible as the growth of anterior mandible is completed by 3 years of age .
a limiting factor is that it is necessary to remake the prostheses to accommodate changes in occlusal plane as the person grows .
hence , removable of denture is the suggested treatment modality for young patients due to rapid growth of the jaws , while the underdeveloped ridges may affect the retention and stability .
if fever teeth are present , a tooth supported over denture or an overdenture with attachment can be advised .
once the full growth is attained , a fixed partial denture or and endosseous implant therapy can be performed in these patients .
lack of bone density , knife edge mandible ridge which requires bone grafting for the placement of implants precluded the use of implants in the present case scenario . in this case scenario ,
the treatment philosophy followed is the full mouth rehabilitation using simultaneous restoration of both the arches as advocated by bailey , grubb , linkow , sendax , and kazis . full mouth rehabilitation with long - span fixed partial denture resulted in improvement of the function , esthetics , and the psychosocial confidence of the patient .
the challenge to restorative dentist when restoring posterior dentition is to obtain the most suitable position and orientation of the posterior occlusal scheme where the natural curve of spee is deranged .
broadrick flag is a simple tool which provides a close approximation of patients original occlusal curve , thereby permitting an ideal occlusal reconstruction .
a full mouth rehabilitation therapy was provided for a patient suffering from ed which restored the form , function , esthetics , and confidence of the affected individual .
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ectodermal dysplasia ( ed ) represents a group of patients with mild to severe congenital and developmental anomalies .
dentists are the first person to identify ed in young patients .
the impairment is not just the form and function but extends to the social outlook as well as the psychology of the affected individual .
this case report describes management of ed with a long - span fixed partial denture fabricated using a broadrick flag .
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more than 90% of ingested foreign objects will pass uneventfully through the gastrointestinal tract [ 1 , 2 , 3 , 4 , 5 ] .
only 10% of such cases require the endoscopic removal of the foreign body , while only 1% require a surgical procedure [ 1 , 2 , 3 , 4 , 5 ] .
however , foreign objects longer than 6 cm can not negotiate the physiological narrowing of the pylorus and always remain in the stomach .
these objects should be endoscopically removed as soon as possible to avoid pressure necrosis , gastrointestinal perforation or bleeding . however , in rare cases , large foreign bodies which have been swallowed can also remain in the stomach over a long period of time without any significant symptoms .
in such cases endoscopic extraction is always the first therapeutic option . if endoscopic removal fails or if there is evidence of obstruction or perforation , laparoscopic gastrotomy should be performed .
a 36-year - old caucasian male with a previous history of heroin abuse and chronic hepatitis c was admitted to the department of internal medicine , division of gastroenterology , university hospital split because of hematemesis and melena . on admission , the patient reported purposefully swallowing a lighter which he had double wrapped in cellophane 17 months before in a police station ( where he was being questioned under the suspicion of smuggling narcotics ) . at that moment he refused the suggested endoscopic or surgical extraction of the foreign body .
a plain abdominal x - ray study confirmed the presence of a large foreign body in the left upper abdominal quadrant ( fig .
esophagogastroduodenoscopy revealed the presence of an ulcer in the angular part of the stomach ( forrest iii ) as well as a large foreign body wrapped in dark - colored cellophane in the middle of the stomach .
nevertheless , a gastroscopy was performed the following day and the foreign body was successfully removed endoscopically using a polypectomy snare .
the extracted foreign body was a lighter ( 8 cm long ) which was double wrapped in cellophane ( fig .
the patient was treated with an adequate diet , pantoprazole and 2 u of packed red blood cells .
he was dismissed from the hospital after a week in good clinical condition , with the discharge medication being pantoprazole 40 mg administered orally to be taken daily for 1 month .
a control upper endoscopy after 1 month indicated full scarred healing of the gastric ulceration .
the most common causes of gastrointestinal foreign bodies are food boluses or accidental swallowing of other objects [ 1 , 2 , 3 , 4 ] . psychiatric patients and prisoners
may purposefully swallow a wide variety of bizarre objects while drug smugglers may swallow condoms filed with narcotics .
this case report describes a very rare case of a lighter retained in the stomach over a period of 17 months which was safely extracted from the stomach endoscopically by snare extraction .
it is also important to note that some elements of the ingested lighter may have very different toxic profiles depending on their chemical form .
for example , lighter fluid is toxic because it contains hydrocarbons including benzene , butane , hexamine and propane . given that our patient double wrapped the swallowed lighter in cellophane , this prevented the significant corrosion of the lighter in an acidic environment , and thus merely a gastric ulcer occurred which in this case was probably primarily due to chronic mechanical pressure .
to the best of our knowledge , this is the first case report which discusses a lighter as a chronic stomach foreign body .
although the majority of swallowed indigestible foreign bodies pass through the gastrointestinal tract without complications , foreign objects longer than 6 cm , such as a lighter , after reaching the stomach can not negotiate the pylorus and may become entrapped [ 1 , 2 , 3 , 4 , 5 ] . in such cases , the object should be removed endoscopically as soon as possible to avoid pressure necrosis , gastric perforation or bleeding .
however , clinical presentation may also include vague symptoms such as fever and abdominal pain , or the symptoms may be extremely subtle over a longer period of time .
the signs and symptoms caused by foreign body ingestion also vary according to the interval between ingestion and presentation .
plain radiographs are always required for patients with a known or suspected swallowed foreign body and have an influence on further management .
flexible endoscopy is considered the first choice for the management of gastric foreign bodies due to its efficacy , low morbidity and reduced costs compared to surgical treatment [ 1 , 2 , 3 , 4 , 5 ] .
the removal of large foreign bodies from the stomach is influenced by the patient 's clinical condition as well as the technical abilities of the endoscopist [ 1 , 2 , 3 , 4 , 5 , 6 ] .
however , caution as well as extensive experience of the endoscopist is required [ 1 , 2 , 3 , 4 , 5 , 6 ] . if endoscopic removal fails or if there is evidence of obstruction or perforation , surgical gastrotomy should be performed .
a swallowed lighter trapped in the stomach over a long period of time is undoubtedly a special clinical challenge . trapped long and rigid foreign objects in the stomach
are associated with an increased risk of impaction , perforation and bleeding . according to this ,
objects longer than 6 cm should be removed endoscopically whenever possible . in cases where endoscopic removal fails
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the majority of ingested foreign bodies will pass safely through the gastrointestinal tract , but long and rigid foreign bodies are associated with an increased risk of gastrointestinal impaction , perforation and bleeding . however , large foreign bodies which have been swallowed can remain trapped in the stomach over a longer period of time without any significant symptoms .
this case report describes the case of a 36-year - old man addicted to heroin who purposefully swallowed a lighter ( double wrapped in cellophane ) and presented with hematemesis and melena 17 months after the ingestion of the foreign body .
the lighter was successfully removed via flexible endoscopy using a polypectomy snare . swallowed long and
rigid foreign bodies trapped in the stomach over a long period of time always represent a special clinical and endoscopic challenge . in cases where endoscopic removal fails
, a laparoscopic surgical approach may be an alternative .
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during this study in 2002 , we screened almost all patients who were suspected clinically to have a tick - borne rickettsiosis and who were hospitalized in the infectious diseases department of khabarovsk municipal hospital no.10 .
five to 20 days before onset of the disease , most patients reported tick bites or tick exposure . some had typical eschars on the skin without a history of a tick bite .
the illness was acute , febrile , and usually involved a macular or maculopapular rash .
patients with clinically evident features of tick - borne encephalitis ( neurologic signs ) or lyme disease ( erythema migrans ) were excluded from the study . in total , samples from 65 patients were investigated , 64 blood samples and 17 skin biopsy results . paired sera from most of the patients were tested with conventional serologic methods .
the first serum sample was taken immediately after admission to the hospital , usually 19 days after the onset of the disease .
buffy coats were separated from fresh blood specimens immediately after collection at patient s admission .
dna was extracted from the buffy coat and skin biopsies samples with qiamp dna blood mini kit and dneasy tissue kit ( qiagen , tokyo , japan ) according to manufacturer s instructions .
we used the nested polymerase chain reaction ( pcr ) method for primary detection of the rickettsial dna in human samples .
the glta ( citrate synthase ) gene was chosen as the target for amplification because of its genus specificity and conservativeness .
the primer cs1d ( 16 ) , which is used traditionally to amplify the full - length glta gene , is complementary to the ultimate 5-end of the open reading frame for this gene of r. prowazekii .
since we expected to find dna of the spotted fever group rickettsiae , we modified the primer to be completely homologous to the corresponding portion of the gene of r. conorii ( table 1 ) .
r. conorii has been chosen as a template to design primer cs2d as the only spotted fever group rickettsia having a completely sequenced genome at the time of investigation .
comparison of these two primers indicated that the newly designed primer ( cs2d ) was at least 100 times more sensitive than cs1d in the amplification of the serially diluted dna of r. montanensis , which was used as a positive control ( data not shown ) .
primers cs877f and cs1258r were used in the nested pcr assay . to amplify the full - length of the glta gene , we used pcr followed by hemi - nested pcr using the product of the first pcr as a template .
two conservative regions of the outer membrane protein a ( ompa ) gene were amplified ( base positions 91680 and 3,6086,789 with respect to the sequence published for r. rickettsii ) by using previously described primers ( 17 ) .
initial amplification of the 3,182-bp longer region was achieved with primers 190 - 3588f and 190 - 5044r and with primers 190 - 4859f and 190 - 6808r .
products of this reaction were used later in the nested and hemi - nested pcr reactions with corresponding primers .
the ompb gene was amplified from clinical samples and dna by using standard primers ( 18 ) , except for the ultimate forward primer 120-m59 , which was found to produce nonspecific amplicons with the human dna in clinical samples .
as with the ompa , the dna from clinical samples was amplified in two steps . in the first step , we amplified two halves of the gene , with a small overlapping region with primers 120-m36 with 120 - 2988 and 120 - 2788 with 120 - 4879 .
the products of these reactions were used as templates for each of the specific nested or heminested reactions for seven regions .
amplification has been carried out by routine methods in a final volume of 50 l with appropriate negative controls ( 10,17 ) . in all reactions , as a positive control , we used a mixture of dna of the following microorganisms : anaplasma phagocytophilum , neorickettsia sennetsu , wolbachia pipientis , francisella tularensis , bartonella henselae , borrelia garinii , coxiella burnetii , and r. montanensis .
we found that the cocktail of dna of tick - borne or phylogenetically close to tick - borne bacteria is convenient to use in pcr with potentially polyinfected human samples .
we checked all samples for other possible tick - borne bacterial and pathogens with the following primers under conditions suggested in published references : sl primers for ospa gene of borrelia ( 19 ) ; bhcs.781p and bhcs.1137n primers for bartonella citrate synthase ( 20 ) ; he1 , he3 , and he4 primers for 16s rdna of ehrlichia chaffeensis ( 21 ) ; ge3a , ge10r , ge9f , and ge2 primers for 16s rrna gene of a. phagocytophilum ( 22 ) ; and p3708 and p4257 primers for the gene encoding p44 protein of a. phagocytophilum ( 23 ) [ data not shown ] .
the pcr products were purified for dna sequencing with the qiaquick pcr purification kit ( qiagen ) and then directly sequenced by using pcr primers .
sequencing reactions were carried out with a d - rhodamine terminator cycle dna sequencing kit ( applied biosystems , foster city , ca ) according to the manufacturer s instructions .
sequencing reaction products were resolved by electrophoresis with an abi prism 377 sequencer ( applied biosystems ) .
the results obtained were processed into sequence data with autoassembler software ( applied biosystems ) .
the sequences of the glta , both regions of the ompa gene , and the ompb genes were aligned by using the software genetix - win 5.1 ( software development co. , ltd . ,
sequences in the ompb genes of r. heilongjiangensis and r. hulinensis were not available in the genbank database so we amplified and sequenced the gene .
the sequences used for comparison were obtained from the genbank database , aligned , and then corrected manually to preserve codon alignment and conserved motifs .
the phylogenetic tree was calculated by using neighbor - joining method with mega2 version 2.1 software ( available from : http://megasoftware.net ) .
two serologic tests were performed in khabarovsk plague control station : immunofluorescence studies with a combined antigen consisted of two local strains of b. garinii and one local strain of b. afzelii , and an enzyme - linked immunosorbent assay detected antibodies against tick - borne encephalitis virus ( 24 ) . for further investigations ,
sera were dried on blotting papers as described previously ( 25 ) and transported to marseilles , where microimmunofluorescence testing ( 26 ) was performed by using in - house prepared antigens of r. heilongjiangensis ( strain 054 , atcc vr-1524 ) , r. hulinensis ( strain hl-93 , atcc vr-1527 ) , r. sibirica ( strain 246 , atcc vr-151 ) , r. conorii ( strain moroccan atcc vr-141 ) , c. burnetii ( strain nine mile , atcc vr-615 ) , orientia tsutsugamushi ( strains gilliam , karp , kato , and kawazaki ) , e. chaffeensis ( strain arkansas ) , a. phagocytophilum ( strain webster ) , bartonella henselae ( houston-1 , atcc-49882 ) , and b. quintana ( strain oklahoma ) .
antigens were applied by pen point to 18-well microscope slides , dried for 30 min , and fixed .
appropriate positive- and negative - control serum samples were tested on each slide together with twofold dilutions of patients serum samples made in 3% nonfat dry milk in phosphate - buffered saline ( pbs ) .
slides were incubated in a moist chamber for 30 min at 37c , washed twice in pbs and once in distilled water ( 10 min each ) ; reactive antibodies showed fluorescein isothiocyanate
conjugated goat anti - human chain and -chain immunoglobulins ( biomrieux , marcy letoile , france ) .
after the conjugate was added , slides were incubated for 30 min at 37c , washed in two pbs for 10 min and for 5 min in distilled water , and mounted in buffered glycerol .
endpoints for each antigen were the lowest concentrations of serum that definitely conferred fluorescence on bacteria .
nucleotide sequences obtained during this study were deposited in genbank under the following numbers : ay260451 for ompb gene of r. heilongjiangensis , strain 054 ; ay260452 for ompb gene for r. hulinensis ; ay280712 for ompb gene ; ay280711 for previously tandemly repeated region portion of ompa gene ; ay280710 for another portion ; and ay280709 for glta gene of rickettsia spp . found in this study .
during this study in 2002 , we screened almost all patients who were suspected clinically to have a tick - borne rickettsiosis and who were hospitalized in the infectious diseases department of khabarovsk municipal hospital no.10 .
five to 20 days before onset of the disease , most patients reported tick bites or tick exposure . some had typical eschars on the skin without a history of a tick bite .
the illness was acute , febrile , and usually involved a macular or maculopapular rash .
patients with clinically evident features of tick - borne encephalitis ( neurologic signs ) or lyme disease ( erythema migrans ) were excluded from the study . in total , samples from 65 patients were investigated , 64 blood samples and 17 skin biopsy results . paired sera from most of the patients were tested with conventional serologic methods .
the first serum sample was taken immediately after admission to the hospital , usually 19 days after the onset of the disease .
buffy coats were separated from fresh blood specimens immediately after collection at patient s admission .
dna was extracted from the buffy coat and skin biopsies samples with qiamp dna blood mini kit and dneasy tissue kit ( qiagen , tokyo , japan ) according to manufacturer s instructions .
we used the nested polymerase chain reaction ( pcr ) method for primary detection of the rickettsial dna in human samples .
the glta ( citrate synthase ) gene was chosen as the target for amplification because of its genus specificity and conservativeness .
the primer cs1d ( 16 ) , which is used traditionally to amplify the full - length glta gene , is complementary to the ultimate 5-end of the open reading frame for this gene of r. prowazekii .
since we expected to find dna of the spotted fever group rickettsiae , we modified the primer to be completely homologous to the corresponding portion of the gene of r. conorii ( table 1 ) .
r. conorii has been chosen as a template to design primer cs2d as the only spotted fever group rickettsia having a completely sequenced genome at the time of investigation .
comparison of these two primers indicated that the newly designed primer ( cs2d ) was at least 100 times more sensitive than cs1d in the amplification of the serially diluted dna of r. montanensis , which was used as a positive control ( data not shown ) .
primers cs877f and cs1258r were used in the nested pcr assay . to amplify the full - length of the glta gene , we used pcr followed by hemi - nested pcr using the product of the first pcr as a template .
two conservative regions of the outer membrane protein a ( ompa ) gene were amplified ( base positions 91680 and 3,6086,789 with respect to the sequence published for r. rickettsii ) by using previously described primers ( 17 ) .
initial amplification of the 3,182-bp longer region was achieved with primers 190 - 3588f and 190 - 5044r and with primers 190 - 4859f and 190 - 6808r .
products of this reaction were used later in the nested and hemi - nested pcr reactions with corresponding primers .
the ompb gene was amplified from clinical samples and dna by using standard primers ( 18 ) , except for the ultimate forward primer 120-m59 , which was found to produce nonspecific amplicons with the human dna in clinical samples .
as with the ompa , the dna from clinical samples was amplified in two steps . in the first step , we amplified two halves of the gene , with a small overlapping region with primers 120-m36 with 120 - 2988 and 120 - 2788 with 120 - 4879 .
the products of these reactions were used as templates for each of the specific nested or heminested reactions for seven regions .
amplification has been carried out by routine methods in a final volume of 50 l with appropriate negative controls ( 10,17 ) . in all reactions , as a positive control , we used a mixture of dna of the following microorganisms : anaplasma phagocytophilum , neorickettsia sennetsu , wolbachia pipientis , francisella tularensis , bartonella henselae , borrelia garinii , coxiella burnetii , and r. montanensis .
we found that the cocktail of dna of tick - borne or phylogenetically close to tick - borne bacteria is convenient to use in pcr with potentially polyinfected human samples .
we checked all samples for other possible tick - borne bacterial and pathogens with the following primers under conditions suggested in published references : sl primers for ospa gene of borrelia ( 19 ) ; bhcs.781p and bhcs.1137n primers for bartonella citrate synthase ( 20 ) ; he1 , he3 , and he4 primers for 16s rdna of ehrlichia chaffeensis ( 21 ) ; ge3a , ge10r , ge9f , and ge2 primers for 16s rrna gene of a. phagocytophilum ( 22 ) ; and p3708 and p4257 primers for the gene encoding p44 protein of a. phagocytophilum ( 23 ) [ data not shown ] .
the pcr products were purified for dna sequencing with the qiaquick pcr purification kit ( qiagen ) and then directly sequenced by using pcr primers .
sequencing reactions were carried out with a d - rhodamine terminator cycle dna sequencing kit ( applied biosystems , foster city , ca ) according to the manufacturer s instructions .
sequencing reaction products were resolved by electrophoresis with an abi prism 377 sequencer ( applied biosystems ) .
the results obtained were processed into sequence data with autoassembler software ( applied biosystems ) .
the sequences of the glta , both regions of the ompa gene , and the ompb genes were aligned by using the software genetix - win 5.1 ( software development co. , ltd .
sequences in the ompb genes of r. heilongjiangensis and r. hulinensis were not available in the genbank database so we amplified and sequenced the gene .
the sequences used for comparison were obtained from the genbank database , aligned , and then corrected manually to preserve codon alignment and conserved motifs .
the phylogenetic tree was calculated by using neighbor - joining method with mega2 version 2.1 software ( available from : http://megasoftware.net ) .
two serologic tests were performed in khabarovsk plague control station : immunofluorescence studies with a combined antigen consisted of two local strains of b. garinii and one local strain of b. afzelii , and an enzyme - linked immunosorbent assay detected antibodies against tick - borne encephalitis virus ( 24 ) . for further investigations ,
sera were dried on blotting papers as described previously ( 25 ) and transported to marseilles , where microimmunofluorescence testing ( 26 ) was performed by using in - house prepared antigens of r. heilongjiangensis ( strain 054 , atcc vr-1524 ) , r. hulinensis ( strain hl-93 , atcc vr-1527 ) , r. sibirica ( strain 246 , atcc vr-151 ) , r. conorii ( strain moroccan atcc vr-141 ) , c. burnetii ( strain nine mile , atcc vr-615 ) , orientia tsutsugamushi ( strains gilliam , karp , kato , and kawazaki ) , e. chaffeensis ( strain arkansas ) , a. phagocytophilum ( strain webster ) , bartonella henselae ( houston-1 , atcc-49882 ) , and b. quintana ( strain oklahoma ) .
antigens were applied by pen point to 18-well microscope slides , dried for 30 min , and fixed .
appropriate positive- and negative - control serum samples were tested on each slide together with twofold dilutions of patients serum samples made in 3% nonfat dry milk in phosphate - buffered saline ( pbs ) .
slides were incubated in a moist chamber for 30 min at 37c , washed twice in pbs and once in distilled water ( 10 min each ) ; reactive antibodies showed fluorescein isothiocyanate
conjugated goat anti - human chain and -chain immunoglobulins ( biomrieux , marcy letoile , france ) .
after the conjugate was added , slides were incubated for 30 min at 37c , washed in two pbs for 10 min and for 5 min in distilled water , and mounted in buffered glycerol .
endpoints for each antigen were the lowest concentrations of serum that definitely conferred fluorescence on bacteria .
nucleotide sequences obtained during this study were deposited in genbank under the following numbers : ay260451 for ompb gene of r. heilongjiangensis , strain 054 ; ay260452 for ompb gene for r. hulinensis ; ay280712 for ompb gene ; ay280711 for previously tandemly repeated region portion of ompa gene ; ay280710 for another portion ; and ay280709 for glta gene of rickettsia spp . found in this study .
serum samples from 11 were available for serologic studies , and clinical and epidemiologic data have been analyzed for 13 patients , including all patients with investigated serum samples .
ten of 17 samples of dna extracted from skin eschars and seven of 64 samples of dna extracted from buffy coats were positive in the nested pcr for the glta gene .
in one patient , both the skin biopsy and the buffy coat were positive and had the same dna sequence . because we had limited amounts of extracted dna
, we attempted to amplify both the ompa and ompb genes from six samples ( three skin biopsies and three blood samples ) , which were previously positive in nested pcr with primers for citrate synthase gene .
all positive samples were also screened by pcr for other possible bacterial tick - borne pathogens and were found to be negative .
results from testing , serologic or pcr , that suggested double infection were excluded from the study . clinical picture of the disease was analyzed in patients with r. heilongjiangensis infection to describe the disease associated with this organism .
all 17 nested pcr amplicons of amplified glta gene were directly sequenced and showed 100% homology .
six amplicons of ompa and ompb genes of corresponding samples were also identical with each other .
we obtained full - length glta gene sequence , 590 bp and 3,182 bp ( excluding primer sequences ) of 5- and 3-regions of the ompa gene , respectively , and a 4,852-bp length sequence of the ompb gene .
a blast search showed that all sequences were completely homologous to correspondent genes of r. heilongjiangensis .
figure 2 shows the phylogenetic relationships of this rickettsia and other species based on the analysis of both concatenated portions of the ompa gene .
the tree was made using mega 2.1 software after alignment of both portions of ompa genes obtained from genbank and sequenced during this study by genetix - win 5.1 software .
the numbers present at nodes of the tree represent the number of bootstrap replicates of 100 that display the indicated sequence groupings .
serum samples from 11 of 16 patients were studied ; data are presented in tables 2 and 3 .
none of the samples had serologic evidence of any other nonrickettsial acute , tick - borne disease .
one previously vaccinated patient had a low titer of immunoglobulin ( ig ) g against tick - borne encephalitis virus , and two had low titers to both b. henselae and b. quintana ( data not shown ) . in 9 of 11 available
paired sera from pcr - positive patients , we found serologic evidence for acute rickettsial infection , e.g. , the presence of igm antibodies , seroconversion , or a fourfold rise in antibody titer against rickettsial antigens . in samples from two patients , igg and igm antibody titers to r.
the same titers were present against r. conorii and r. heilongjiangensis . in samples from one patient
levels of antibodies against r. sibirica in all cases , except one , were lower than against r. heilongjiangensis . in four of nine cases , titers were identical against antigens of r. hulinensis and r. sibirica .
absence of specific antibodies against rickettsiae in patients 2 and 3 could be explained either by early treatment with a specific antimicrobial drug or probable errors in serum sample collection and treatment .
pcr , polymerase chain reaction ; ig , immunoglobulin ; ni , not investigated .
pcr , polymerase chain reaction ; ig , immunoglobulin ; ni , not investigated .
epidemiologic , clinical , and laboratory data available in 13 of 16 pcr - positive patients are given in table 4 . before the onset of the disease in the summer 2002 ( from june to august ) , all patients had a history of tick bite , tick exposure , or a stay in an epidemiologically suspected location . after an incubation period of 4 to 7 days , the patient had a sudden onset with fever , but no specific symptoms appeared during the first several days . in 12 patients ,
twelve patients had a primary lesion ( eschar ) at the site of tick attachment ( figure 3 ) .
the eschar consisted of a necrotic central region ( 50150 mm in diameter ) surrounded by infiltrated and inflamed tissue ( 70400 mm in diameter ) , and a zone of hyperemia ( 250500 mm in diameter ) .
the eschars were found on the waist and buttocks region ( four ) ; lower extremities ( two ) ; upper extremities and axillar region ( two ) ; and back , chest , neck , and abdomen ( one case each ) . in two patients
initial conventional treatment at home , with a combination of antipyretics , analgesics , and antibacterial therapy ( oral penicillins ) did not result in improvement ; 59 days after the onset of symptoms , all patients were admitted to the hospital .
they received oral doxycycline for 14 days and antihistamine therapy , and clinical symptoms resolved within 2 to 3 days .
laboratory tests in the hospital showed elevated levels of serum transaminases ( alanine aminotransaminase and aspartate aminotransaminase ) in 46% and 15% of patients , respectively , and these remained elevated even during convalescence but were normal at follow - up at 3 to 4 weeks .
esr , erythrocyte sedimentation rate ; a1at , alanine aminotransferase ; asat , aspirate aminotransferase .
. we successfully amplified four portions of three different rickettsial genes from human blood and skin samples , which suggested the presence of this microorganism .
we did not amplify any of these genes in samples from healthy donors or from patients suffering from other infectious diseases ( negative controls ) .
the glta gene is conservative , especially among rickettsia species ( 16 ) , and ompa and ompb genes encode main surface proteins of rickettsiae .
the identity of sequenced pcr products among patients , clinical data , and epidemiologic data suggest the focality of this rickettsiosis .
we studied paired sera from patients for reactivity with antigens of several species in the rickettsia genus , including r. heilongjiangensis and r. hulinensis .
seroconversion , presence of igm , or increasing antibody titers were observed in most patients . in seven cases ,
sera reacted at higher titers with antigen of r. heilongjiangensis when compared with r. sibirica , the only currently identified tick - borne rickettsia in the russian far east . in 77% ,
although serologic cross - reactions are common among rickettsiae of the spotted fever group ( 26 ) , the finding of lower titers against the phylogenetically more closely related r. hulinensis than the relatively more distant r. conorii was unexpected .
epidemiologic evidence of tick bite or exposure , rash , primary lesion ( eschar ) at the site of tick bite , and rapid recovery after doxycycline treatment support a rickettsial cause for the disease .
some peculiarities were noticed when signs were compared with infection caused by r. sibirica in the central siberia .
seasonal peak of infections is in the end of june and july . for siberian tick typhus ,
the rash that accompanies tick - borne rickettsiosis in the russian far east is less obvious , and the disease apparently affects older people than siberian tick typhus .
only 1 of 13 patients was > 45 years of age . generally , the disease is mild , with no serious complications or death recorded .
recently , dna of the rickettsia described in our report was amplified from h. concinna ticks collected in siberia ( s. shpynov , unpub .
. the preferable method may be pcr on eschar biopsy samples because this technique has high sensitivity and probability of finding rickettsial dna ( 27 ) .
serologic studies of samples from russian far east area should include tests with antigens of r. heilongjiangensis .
results of our studies showed that acute febrile tick - borne disease caused by r. heilongjiangensis is prevalent in the russian far east .
molecular biology approaches enabled us to identify the cause of an acute disease and to detect its bacterial origin .
as no evidence of r. sibirica human infection was found in our study , further investigations are needed clarify its role in human pathology in the russian far east , especially the khabarovsk region .
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an acute tick - borne rickettsiosis caused by rickettsia heilongjiangensis was diagnosed in 13 patients from the russian far east in 2002 .
we amplified and sequenced four portions of three rickettsial genes from the patients skin biopsy results and blood samples and showed that the amplified rickettsial genes belong to r. heilongjiangensis , which was recently isolated from dermacentor sylvarum ticks in nearby regions of china .
this rickettsia , belonging to subgroup of r. japonica , was previously suggested to be pathogenic for humans on the basis of serologic findings .
we tested serum samples with different rickettsial antigens from 11 patients and confirmed increasing titers of immunoglobulin ( ig ) g and igm to spotted fever group rickettsiae , including r. heilongjiangensis .
clinical and epidemiologic data on these patients shows that this disease is similar to other tick - borne rickettsioses .
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the indian subcontinent is classified as an intermediate hepatitis b virus ( hbv ) endemic ( hbsag carriage 2 - 7% ) zone and has the second largest global pool of chronic hbv infections .
india has a population of more than 1.2 billion with 5.7 ( reduced to 2.5 ) million human immunodeficiency virus ( hiv ) positive , 43 million hbv positive and 15 million hcv positive persons .
the risk of transfusion transmission of these viruses may be alarming due to high seroprevalence of hiv , anti - hcv , and hbsag ( 0.5% , 0.4% , and 1.4% , respectively ) among blood donors .
safety assessment of the blood supply , the quality of screening procedures and the risk of transfusion transmitted infectious diseases ( ttis ) in any country can be estimated by review and analysis of the records of blood donors for screening procedures and the prevalence of serological markers of infectious diseases .
transfusion medicine , apart from being important for the medical treatment of each patient , also has a great public health importance worldwide .
after the introduction of the blood banks and better storage techniques , it became more widely used .
blood is one of the major sources of transmission of hepatitis b , hepatitis c , hiv , syphilis , and many other diseases . discovery of these hazards brought a dramatic change in attitude of physicians and patients about transfusion of blood .
it is mandatory to test each donor 's blood for syphilis by a venereal disease reference laboratory ( vdrl ) , and for hbsag , anti - hcv , and anti - hiv . in july 1989 ,
consequent to the reports of high seroprevalence in commercial blood donors , mandatory screening of blood and blood products for hiv antibodies was initiated by indian national aids control origination ( naco ) .
the objective of this study is to estimate the seroprevalence of transfusion transmitted infections among voluntary blood donors at a rural tertiary healthcare teaching hospital in western maharashtra
. this knowledge might give us the idea of disease burden of the society and the basic epidemiology of these diseases in the rural community .
a retrospective hospital record - based study was conducted at the blood bank of a rural tertiary care teaching hospital in maharashtra , india .
data were collected for a period of 2 years from january 2009 to december 2010 .
sera of civilian residents from various localities and of different age groups , who donated blood voluntarily was screened for hiv , hbsag , hcv , and syphilis .
exclusion criteria for blood donation were current history of medication , recent history of having undergone a surgical procedure , serious illness , previous blood transfusions , weight < 50 kg , age < 18 and > 60 years , pregnant and lactating women .
the outcome variable was serological status of the selected individual , whether positive or negative for any ttis , which was determined from a blood sample .
five milliliter blood each was collected from subjects into plain , sterile bottle following informed consent .
two kits were used based on who recommendation of two different testing strategies involving enzyme - linked immunosorbent assay ( elisa ) and/or simple or rapid assays for surveillance .
samples were analyzed for antibodies to hiv 1 and 2 , hbsag and hcv by elisa .
any serum found reactive by the first assay was retested using a second assay based on different antigen preparations and/or different test principle using the anti - hiv test , hbsag which is an immunochromatographic sandwich assay and hcv by the anti - hcv test .
the validity of the test is assured as per the given criterion and the results were computed .
the data entry was carried out using microsoft office excel worksheet and percentage and proportions for each variable was calculated . the chi - square test and
the outcome variable was serological status of the selected individual , whether positive or negative for any ttis , which was determined from a blood sample .
five milliliter blood each was collected from subjects into plain , sterile bottle following informed consent .
two kits were used based on who recommendation of two different testing strategies involving enzyme - linked immunosorbent assay ( elisa ) and/or simple or rapid assays for surveillance .
samples were analyzed for antibodies to hiv 1 and 2 , hbsag and hcv by elisa .
any serum found reactive by the first assay was retested using a second assay based on different antigen preparations and/or different test principle using the anti - hiv test , hbsag which is an immunochromatographic sandwich assay and hcv by the anti - hcv test .
the validity of the test is assured as per the given criterion and the results were computed .
the data entry was carried out using microsoft office excel worksheet and percentage and proportions for each variable was calculated . the chi - square test and
in the present study , out of total 5661 voluntary blood donors , 5394 ( 95.28% ) were males and 267 ( 4.72% ) were females which shows predominance of males as compared to females for the two studied years [ table 1 ] .
distribution of voluntary blood donors in the study population the prevalence of hbsag , anti - hcv , vdrl , and anti - hiv among voluntary blood donors in the study population is showed in table 2 .
the overall seroprevalence of hbv and hcv was 1.09% and 0.74% respectively , while the prevalence of vdrl and hiv was 0.07% .
the highest prevalence was observed for hbv followed by hcv , syphilis and hiv in decreasing order .
prevalence of hbsag , anti - hcv , vdrl , and anti - hiv among voluntary blood donors in the study population all infections summed together the highest prevalence ( 55.75% ) was within the age group 21 - 30 years , followed by 36.28% within the age group 31 - 40 years , with the lowest prevalence was observed with in the age group of < 20 years ( 1.76% ) and 51 ( 0.88% ) [ table 3 ] .
distribution of blood donors with transfusion transmitted infections according to the age with respect to the individual ttis , it is observed that the prevalence of hbv was highest within at the age groups < 20 years ( 100% ) , hcv within the age group between 31 and 40 years ( 43.90% ) , syphilis within the age group between 41 and 50 years ( 16.66% ) and hiv between 21 and 30 years ( 6.34% ) .
the difference of the prevalence of transfusion transmitted diseases among different age groups was statistically not significant ( p>0.05 ) .
as mentioned in table 4 that the prevalence of hepatitis b and syphilis was higher among males as compared to females ( 58.09% and 3.80% ) respectively , while for hepatitis c , it was higher among females as compared to males ( 62.50% ) , the difference of prevalence by sex was statistically significant ( p<0.05 ) .
blood transfusion is an integral and life - saving procedure of modern medicine , but simultaneously it carries the risk of transmitting the life threatening transfusion transmissible infectious .
hiv , hepatitis b , and hepatitis c are major public health problems in developing countries .
they are transmitted parenterally , vertically , or through high - risk sexual behaviors and can cause fatal acute and chronic life - threatening disorders .
screening of blood is now mandatory for many diseases and is undertaken routinely in blood banks .
transmission of ttis during the serologically window period still poses a threat to blood safety in environments where there is high rate of ttis .
the prevalence of ttis among the indian blood donors is reported to be ranging as follows ; hbv
0.66% to 12% , hcv 0.5% to 1.5% , hiv 0.084% to 3.87% , and syphilis 0.85% to 3% respectively .
the present study revealed seroprevalence of hbv at 1.09% among the donors which is similar to findings by chattoraj et al .
. variable results of 0.66% , 2.45% , 3.44% , 5.86% , 25% have also been reported in various other studies .
the major route of hbv transmission is parenteral and it is most infective among blood - borne viruses and chronic carrier state is associated with chronic liver disease , cirrhosis and hepatocellular carcinoma .
the estimated prevalence in this study was 0.74% , similar to that reported by the other studies 0.79% , 0.88% and 0.78% ; whereas a few studies reported much lower level of prevalence such as 1.09% , 1.57% , 2.8% , and 6.21% and a yet another set of studies reported it to be at higher levels of 0.28% and 0.50% .
transmission of hcv is primarily through blood exposure and majority of the infected persons progress to chronic infection and chance of cirrhosis and hepatocellular carcinoma is more as compared to hbv .
blood is one of the main sources of transmission of hepatitis c ; hence , donor selection is of paramount importance . in the present study ,
similar findings by gupta et al . , and tiwari et al . , reported 0.084% and 0.054% prevalence of hiv among blood donors , whereas lower seroprevalence of 0.0% and higher seroprevalence of 0.13% , 0.19% , 0.26% , 0.47% , 3.8% and 11.7% have been reported . for syphilis ,
the seroprevalence was found to be 0.07% in the present study , which was much lower than reported by other studies 0.85% and 1.2% . regarding sex ,
the study found that blood transfusion transmitted diseases are more prevalent among males than females , the difference of prevalence by sex was statistically significant ( p<0.05 ) , which was comparable to other studies . a sex - wise difference in seroprevalence might be due to differences in the risk behavior .
availability of safe blood for transfusion is a must for the recipients and the community as well .
effective control strategies including a sensitive and stringent screening of all blood donors , public awareness programs , and institution of adequate public health measures are urgently needed .
it may be possible through proper donor selection and education , uniform implementation of laboratory screening tests , and adequate supply of blood through voluntary blood donations along with restriction of donation by professional donors .
most of the previous studies were from urban areas ; this report is first of its kind from rural area of western maharashtra , india .
the major limitation of the study is that there is no previous data available from this rural area for comparison and analysis of trends .
hence , we hereby recommend for future studies to look into trends for ttis from this area .
most of the previous studies were from urban areas ; this report is first of its kind from rural area of western maharashtra , india .
the major limitation of the study is that there is no previous data available from this rural area for comparison and analysis of trends .
hence , we hereby recommend for future studies to look into trends for ttis from this area .
blood is still one of the main sources of transmission of hepatitis b , hepatitis c , hiv , and syphilis .
the majority of donors in our country are voluntary , relatives or friends , who are apparently healthy , but this study found that these diseases are prevalent among donors . hence , strict selection of blood donors with the emphasis on getting voluntary donors and comprehensive screening of donors for ttis using standard methods are highly recommended to ensure the safety of blood for recipient .
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background : blood transfusion is a life - saving measure in various medical and surgical emergencies . transfusion medicine , apart from being important for the medical treatment of each patient , also has great public health importance.objectives:the present study was conducted to estimate the prevalence of transfusion transmitted infections in voluntary blood donors at a rural tertiary care teaching hospital in western maharashtra , india.materials and methods : all voluntary donors reporting to the blood bank were screened for hbsag , hepatitis c virus ( hcv ) , hiv and syphilis by using the appropriate enzyme - linked immunosorbent assay .
hiv infection was confirmed using a standard immunoblotting technique .
hepatitis b virus ( hbv ) was tested for surface antigen ( hbsag ) and hcv by the immunechromatographic method .
the venereal disease reference laboratory ( vdrl ) test was used for estimation of syphilis infection .
the study was designed for a duration of two years between january 2009 to december 2010 .
medical reports of the donors were accessed from the hospital records and analyzed.results:a total of 5661 voluntary blood donors were screened , of which 5394 ( 95.28% ) were males and 267 ( 4.72% ) were females .
the overall seroprevalence of hbv and hcv were 1.09% and 0.74% respectively ; for hiv and syphilis the seroprevalence was estimated to be 0.07% for each.conclusion:blood is still one of the main sources of transmission of infections .
hiv , hepatitis b , hepatitis c viruses and syphilis are prevalent among voluntary donors in rural india .
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many rat strains have originated in physiology laboratories that have selectively bred animals for traits related to specific biomedical interests ( 1 , 2 ) .
rattus norvegicus is well characterized in the areas of cardiovascular and pulmonary studies , pharmacology , immunology , toxicology , nutrition , behavior and aging , as well as cancer and organ transplantation . in recent years
, some large - scale physiological projects have made phenotypic data sets from many rat strains freely available online .
those projects include the physgen program for genomic applications ( pga ) ( 3 ) ( http://pga.mcw.edu ) and the national bioresource program in japan ( nbrp ) ( 4 ) ( http://www.anim.med.kyoto-u.ac.jp/nbr/ ) .
the pga was designed to produce high - throughput phenotype data for a targeted set of inbred , consomic and mutant strains under a variety of environmental conditions .
nbrp has generated a large phenotype data set for inbred and mutant strains under baseline conditions .
the rat genome database ( rgd ) phenominer project endeavors to present , extend and expand the quantitative phenotypic data presented by those two large - scale projects .
for many years , rgd has curated and displayed qualitative phenotype data for strains , quantitative trait loci ( qtls ) and genes .
qualitative phenotypes have been described with the mammalian phenotype ( mp ) ontology ( 5 ) , which , for the most part , consists of terms describing abnormal phenotypes .
the curation of quantitative phenotype data is meant to give a finer - grained view of phenotypes to make comparison between strains more accurate . instead of comparing two rat strains knowing that each have decreased systemic arterial systolic blood pressure
as annotated with mp , quantitative annotations allow the database user to know how much lower the mean arterial blood pressure is in one strain compared with a different strain or to the same strain under different conditions . in addition , the phenominer annotations provide additional data such as animal number , animal age , the measurement method used to collect the data and more .
the quantitative phenotype annotations greatly increase the depth of the phenotype data available to the database user . to curate and display quantitative data , a number of different elements needed to be brought together .
first , user - side software development was required so the data could be presented in an efficient user - interactive manner .
second , to test that type of interface and to present a sizable initial base of data , pga ( > 30 000 records ) and nbrp data ( > 3 000 records ) were imported , formatted and made available through the phenominer user - side interface ( 2 , 6 ) .
the phenominer data are accessed through rgd s phenotypes and models portal ( http://rgd.mcw.edu/phenotypes/ ) .
users select a combination of strain , clinical measurement , measurement method and/or experimental condition to find data of interest ( 6 ) .
this idea of using a combination of terms to extract specific phenotypic data sets from the database is the basis for the name phenominer .
the third element necessary for rgd s quantitative phenotype data curation was a tool for curators to annotate phenotype data from the biomedical literature . to be able to present data that is comparable between studies and protocols , standardized language ( ontologies or structured vocabularies ) needed to be used in the annotations .
. that could be particularly useful in translational research , which relies on comparison of data between laboratory animals and humans .
although quantitative data could vary widely between species for any particular phenotype , the ontology / vocabulary terms and relative quantitative changes among different conditions would allow comparisons between species to be made .
multiple ontologies / vocabularies are used to populate five required fields and an additional optional field . during initial literature curation for phenominer ,
vigorous ontology development was required for three of the ontologies ( clinical measurement ontology , cmo ; measurement method ontology , mmo and experimental condition ontology , xco ) because the phenominer project was initiated simultaneously with the original development of these three ontologies ( 7 ) .
the three ontologies were developed to address gaps that were not being filled adequately by existing publicly available ontologies .
the cmo was developed partly as an offshoot of the development of the vertebrate trait ontology ( vt ) from the animal trait ontology ( http://www.animalgenome.org/bioinfo/projects/ato/ ) ( 8) .
the existing phenotype ontologies ( like mp or the human phenotype ontology ) were not developed for use with numerical measurement values and are not suitable for quantitative annotation .
attachment of numerical values to the cmo has been the key purpose of developing and using the cmo .
the ontology for biomedical investigations ( http://obi-ontology.org/page/main_page ) has partial overlap of terminology with the mmo and xco , but it was decided that new ontologies created in - house and specifically for methods and conditions of quantitative phenotype data would be the most efficient way to proceed . beyond the bulk loading of large data sets and the initial manual curation initiative , manual literature curation will continue , as well as the uploading of phenotype data directly to rgd by rat researchers who wish to make their data publicly available ( http://rgd.mcw.edu/wg/home/phenominer-data-upload ) . in the future , phenominer curation will be done concurrently with qtl curation to more efficiently gather data from the same journal article .
also , non - qtl rat physiology data will be curated from the literature in targeted sets , based on the phenotype category of the research .
to begin the process of literature curation of quantitative phenotype data , the appropriate literature was assembled . instead of searching pubmed for rat phenotypic literature as a starting point for manual phenominer curation ,
many qtl articles report phenotypic data for parent strains and congenic strains used in the determination of the qtls .
therefore , a significant time savings was made by having a preselected literature to be curated .
that was extremely helpful because phenominer curation is a time - consuming process , even without a literature search .
blood pressure articles were curated first , followed by cancer , diabetes , renal function and inflammatory processes .
the five required categories of curated information in a phenominer annotation are experiment name , rat strain , clinical measurement , measurement method and experimental condition .
each of those is represented by a specific ontology or vocabulary , as shown in table 1 . in the case of experiment name ,
the vt ( a collaboration among rgd , mouse genome informatics ( mgi ) ( 10 ) and animal qtl database ( qtldb ) ( 8) ) is used for physiological phenotype data , and the rgd / comparative toxicogenomics database ( ctd ) disease vocabulary ( rdo ) is used for pathological phenotype data .
the rdo disease vocabulary consists of medic ( 11 ) plus additional terms and relationships added at rgd .
it was necessary to add a disease vocabulary for a subset of experiment names because the vt does not cover pathology and many experiments could not be described appropriately without disease terms . the rat strain
finally , xco provides the term or terms to describe the condition or conditions under which the experiment was run .
the xco term may also have a numerical descriptor for quantity and/or duration of the experimental condition .
table 1required ontologies / vocabularies for phenominer annotations , urls for available download sites for those ontologies / vocabularies and relevant literature referencescuration categoryontology/ vocabularyobo file downloadsreferencesexperiment name physiologicalvtftp://rgd.mcw.edu / pub / ontology / vertebrate_trait/
http://purl.bioontology.org/ontology/vtexperiment name
pathologicalrdoftp://rgd.mcw.edu / pub / ontology / disease/(8)rat strainrsftp://rgd.mcw.edu / pub / ontology / rat_strain/
http://purl.bioontology.org/ontology/rsclinical measurementcmoftp://rgd.mcw.edu / pub / ontology / clinical_measurement/
http://purl.bioontology.org/ontology/cmo(5)measurement methodmmoftp://rgd.mcw.edu / pub / ontology / measurement_method/
http://purl.bioontology.org/ontology/mmo(5)experimental conditionxcoftp://rgd.mcw.edu / pub / ontology / experimental_condition/
http://purl.bioontology.org/ontology/xco(5)all ontologies listed here can be browsed in rgd s ontology browser ( http://rgd.mcw.edu/rgdweb/ontology/search.html ) ( 9 ) .
required ontologies / vocabularies for phenominer annotations , urls for available download sites for those ontologies / vocabularies and relevant literature references all ontologies listed here can be browsed in rgd s ontology browser ( http://rgd.mcw.edu/rgdweb/ontology/search.html ) ( 9 ) . to begin annotating a study in phenominer ( figure 1a ) ,
the curator must access the reference identification number ( i d ) in the curation tool .
the tool provides the option of importing a reference by entering a pubmed i d or an rgd i d , similar to the general rgd curation tool ( 12 ) .
if a curator enters a pubmed i d , the tool will download the corresponding abstract from pubmed , assign an rgd i d to that abstract , load the citation into the phenominer curation tool and create a study i d ( sid ) for that reference .
if an rgd i d is entered or the abstract from the entered pubmed i d is already in rgd , the curation tool will load the citation and create a sid for that reference . in the context of manual phenominer curation , a
study refers to a single published article . during the initial phase of manual phenominer literature curation ,
only pre - existing rgd ids were needed because all of the references had previously been imported from pubmed for qtl curation . once the reference is selected , the next step ( figure 2a ) is to choose
the curator must select a vt term or an rdo term , which matches the experiment being described .
create experiment page ( figure 2b ) , which features an autocomplete text box for entering an ontology / vocabulary term i d or term .
the branch icon to the right side of the term i d selection text box links to the ontology browser ( 9 ) in a separate window , providing extra help in finding the most appropriate experiment name .
multiple experiment names may be entered in succession to generate multiple experiments associated with one study . for every experiment entered
, a unique experiment i d ( eid ) is generated to track the data for both curators and end users .
( a ) the quantitative phenominer curation workflow showing the sequence that establishes first , the study , second , the experiment name ( exp ) and third , all the details of the annotation record ( rec ) .
this workflow uses the same ontologies as the phenominer curation , but uses the general curation tool because no numerical values are entered .
figure 2(a ) selected study page this page displays the citation , a reference link and other information about the study . clicking the
( b ) create experiment page this page features an autocompleting text box for entering term ids for either vertebrate trait terms ( vt ) or disease terms ( rdo ) .
the text box will accept a vt or rdo i d or any key word(s ) found in vt or rdo terms .
if a suggested term is selected , the i d is automatically entered in the selection text box and the term itself will be automatically entered in the accompanying experimental name box .
( a ) the quantitative phenominer curation workflow showing the sequence that establishes first , the
study , second , the experiment name ( exp ) and third , all the details of the annotation record ( rec ) .
this workflow uses the same ontologies as the phenominer curation , but uses the general curation tool because no numerical values are entered .
( a ) selected study page this page displays the citation , a reference link and other information about the study . clicking the add experiment link ( red arrow ) returns the
( b ) create experiment page this page features an autocompleting text box for entering term ids for either vertebrate trait terms ( vt ) or disease terms ( rdo ) . the text box will accept a vt or rdo i d or any key word(s ) found in vt or rdo terms . if a suggested term is selected , the i d is automatically entered in the selection text box and the term itself will be automatically entered in the accompanying experimental name box .
to proceed to the record ( full annotation ) creation , the curator selects the all experiments link in the menu bar . all experiments for the study being curated are listed on the
view experiments page ( figure 3 ) . to access the data entry page for records ( create record page )
add record link ( red arrow in figure 3 ) at the right end of the appropriate experiment line .
figure 3view experiments page this page lists all experiments for the particular study being curated , in this case
body mass ( eid 3558 ) and arterial blood pressure trait ( eid 3557 ) for sid:851 ( listed on left side of page ) .
view experiments page this page lists all experiments for the particular study being curated , in this case
body mass ( eid 3558 ) and arterial blood pressure trait ( eid 3557 ) for sid:851 ( listed on left side of page ) .
the create record page ( figure 4 ) is where the rest of the data for a single record is entered .
it consists of a series of autocomplete text boxes , drop - down text boxes and editable plain text boxes .
all of the data entered are associated with terms from five ontologies / vocabularies : rs , cmo , mmo , xco and the optional ma ( mouse adult gross anatomy dictionary ) ( 13 ) .
the entry boxes for the ontologies / vocabularies are autocomplete text boxes , just like the one for experiment name entry ( figure 2 ) .
each entry box also has a branch icon to its right side , for easy access to the ontology browser .
four required fields are associated with the rat strain ( figure 4a ) : animal count ,
sex. the first three fields are plain alphanumeric text boxes that take either a number or
n / a if the number is not included in the data from the reference . the
unspecified.
figure 4create record page most of the information for a manual phenominer annotation is entered here .
four ontology / vocabulary terms are required and many more optional fields allow the addition of basic experimental information . in this example , the rat strain ( a ) is bn / nhsdmcwi , the cmo term ( b ) is systolic blood pressure , the mmo term ( c ) is tail cuff plethysmography and the xco term ( d ) is nave control condition. the optional ma term goes in the site acc id text box in the measurement method section ( c ) .
create record page most of the information for a manual phenominer annotation is entered here .
four ontology / vocabulary terms are required and many more optional fields allow the addition of basic experimental information . in this example , the rat strain ( a ) is bn / nhsdmcwi , the cmo term ( b ) is systolic blood pressure , the mmo term ( c ) is tail cuff plethysmography and the xco term ( d ) is nave control condition. the optional ma term goes in the site acc id text box in the measurement method section ( c ) .
the main information to be entered in the clinical measurement section ( figure 4b ) is the
error. the value is a single measurement average generated for one rat strain by the reported experiment in the literature , accompanied by the appropriate units and error .
the units are in a drop - down list , which is updated on an ongoing basis with units found in the literature by the rgd curators .
first , many units found in the physiological literature are not found in available unit ontologies ( i.e. the units ontology or the units of measurement ontology ) ( http://bioportal.bioontology.org/ ) .
second , many units involve ratios of units , which are more easily used as pre - constructed terms in a drop - down list rather than being constructed by multiple entries in the curation software interface .
average type ( for anything other than a simple mean ) , formula ( to support a calculated measurement value ) and note ( for any information the curator may want to add for enhancement of the annotation ) . the measurement method section ( figure 4c ) is for entering information about the procedures used to make the clinical measurement .
site acc id is an optional ontology / vocabulary field , which accepts mouse adult anatomy terms ( ma ) to indicate the anatomical site of the measurement .
pi type is the type of insult ( if present ) used during the experiment to provoke a change in the measurement ( for example , an injection of a drug given to the subject to affect blood pressure measurement ) .
pi time is the post - insult time , meaning the latency period between insult and measurement .
the experimental condition section ( figure 4d ) is the only section that accepts multiple entries .
create record page allows a curator to add as many experimental conditions as necessary . because editing may warrant removing an experimental condition , a
value fields are used to describe concentrations of drug , diet supplements or other numerically definable experimental condition .
dur fields are used to indicate the duration of a condition , if applicable .
application method is a free text field to label the condition as injection , gavage or other process .
ordinality is a required field for the experimental condition , and it indicates the sequence of conditions , in case of multiple conditions . consecutive
ordinality numbers indicate simultaneous conditions . after entering data to all appropriate fields and clicking the
save button , the record is logged as an addition to that particular experiment ( eid , second entry from left in figure 5 ) .
all the data fields are presented to the curator on one line so when multiple lines are viewed , it is easy to scan for differences between the lines . with so many different fields to fill , it was important to have a versatile editing function in the curation tool . in the menu bar of any list of studies , experiments or records in phenominer
edit link , which links to a data entry page nearly identical to that used for creation of that study , experiment or record .
any field can be altered on the editing pages . not only can single records be edited , but multiple records can be edited simultaneously .
this is convenient because some phenotyping experiments have many different rat strains as subjects , with all the supporting information being identical .
that means that it is easy to change a common incorrect entry for as many records as necessary .
create new record link on the menu bar is selected , a blank template is opened . however ,
if the check box to the left of a record in the view records window is checked , the create new record feature will duplicate the selected record on the create record page .
then , a curator may change one or more fields to generate a new record , instead of re - entering all the identical data manually for the new record .
figure 5view records page a typical annotation viewed in the phenominer curation tool with all data visible on one line .
view records page a typical annotation viewed in the phenominer curation tool with all data visible on one line .
all of the phenominer studies in rgd can be accessed through the list all studies link on the menu bar at the top of all phenominer curation tool pages .
phenominer studies page ( figure 6 ) lists all the studies in the phenominer curation tool .
the columns can all be sorted by clicking on the column headings . from each line ,
the edit study page can be accessed by the hyperlinked sid , the reference abstract can be accessed from the i d in the reference column , the
view experiments page for any study can be accessed from the hyperlinked number in the experiments column and the
figure 6phenominer studies page this is a one - page view of all studies in the phenominer curation tool .
the status is color - coded il ( red ) : initial load ; ip ( dark red ) : in progress ; c ( blue ) : curated ; f ( green ) : final ; w ( black ) : withdrawn .
phenominer studies page this is a one - page view of all studies in the phenominer curation tool .
the status is color - coded il ( red ) : initial load ; ip ( dark red ) : in progress ; c ( blue ) : curated ; f ( green ) : final ; w ( black ) : withdrawn .
curation status can be set on any level ( study , experiment , record ) of the create or
the curation status is set with a drop - down menu , which indicates if the annotation is initial load , in progress , curated , final or
withdrawn. initial load is the default status , so both initial load and in progress indicate the annotations have not been completed .
when curators have completed annotations for a particular study , all those annotations are marked
curated. then an editor checks the annotations for consistency and accuracy , before changing the status to
final. all final annotations are loaded into the user - side interface so the data can be accessed through the public database .
an additional related side project involves annotating qtls qualitatively with the ontologies / vocabularies used for the quantitative phenotype annotations ( figure 1b ) .
this is done in the general curation tool because it does not need any numerical data and it is displayed on qtl report pages , instead of in the phenominer end - user interface . because most of the initial articles for phenominer manual curation contain qtls already annotated in rgd , those qtls were re - annotated in parallel with the phenominer quantitative curation .
this allowed information in the more standardized format of ontologies / vocabularies to be associated with those qtls , superseding information that was only associated with the qtls in curator notes .
this qualitative annotation component also allowed some quality control involving updates of strain nomenclature assigned to qtls and updates of disease terms assigned to qtls . moving forward
, phenominer - style qualitative curation of qtls will be incorporated into the process of regular qtl curation .
because three of the ontologies ( cmo , mmo and xco ) used in phenominer curation originated simultaneously with the phenominer curation project , ontology development controlled the pace of the curation process . internally
, rgd has implemented an ontology development process similar to the sourceforge ( http://sourceforge.net/projects/geneontology/ ) term request tracker system that the gene ontology consortium ( 14 ) has used for years .
as curators annotate articles and find data that can not be described with a current cmo , mmo or xco term , they send a specific request to a single ontology editor .
that editor is a screener who decides the correct wording for the term , the correct place for the term in the ontology and assigns any synonyms and/or definition , if necessary .
the requested terms are then reviewed by a second ontology editor who verifies or changes the wording and placement of the terms in the ontology tree .
the second editor then enters the new terms into the ontologies via obo - edit software ( 15 ) , and adds any missing synonyms and/or definitions . for xco terms that are chemical names , an xref ( cross - reference to the same or similar information ) to the appropriate chebi ( chemical entities of biological interest ) ( http://www.ebi.ac.uk/chebi/ ) database entry
xrefs for other xco , cmo and mmo terms may be added in the future to map them to other ontologies / vocabularies .
the majority of the requested terms ( > 500 ) have been for the cmo .
the requested xco terms are also specific , but because multiple clinical measurements in one reference are often made using identical conditions , the cmo term requests also outnumber the xco requests .
in addition to term requests , requests for units to accompany the values recorded for cmo and xco terms are also handled by the same process as the term requests .
the units are maintained as flat lists , which reside in drop - down menus in the clinical measurement and experimental condition sections of the
create record page of the curation tool ( figure 4b and d ) . just like the ontology terms
new term and unit requests came in waves as the curators moved through the different categories of phenotypes during the curation of the backlog of qtl literature .
even though a lag in time existed between the term / unit requests and entry of those terms / units into the ontologies / vocabularies / lists , the curators continued to make annotations using placeholder
when term / unit requests were fulfilled , annotations were revisited and the placeholder terms / units were replaced .
early in the course of the phenominer project , adjustments to the curation tool needed to be made to optimize and streamline the data entry . with so many data entry fields and various types of text boxes for those fields , it took much back and forth communication between curators and the software developer to find the best design for data entry and editing .
as with other curation tools ( 12 ) , the software developer continued improving the tool while the curators were using it until it was deemed to be as efficient as possible .
originally , all of the ontology - term entry boxes were designed as plain - text entry boxes .
it was soon apparent that entry errors could be numerous and time - consuming to correct . to address this ,
the entry boxes were changed to term - id entry boxes with an autocomplete feature for entered text .
now , only a valid term i d will be accepted in the ontology - term entry boxes , which eliminates typographical errors and invalid ids .
thus , quality control of ontology term entry is done automatically as the terms are entered .
again , the original format included plain - text entry boxes . to standardize curator entries for consistency and to eliminate typographical errors ,
the plain - text entry boxes were replaced with drop - down lists of units . because different authors may use different units for the same type of experiments , standard units had to be assigned for each cmo term .
what it meant for the curators was a restriction to units that could be converted to the standard unit .
the curation tool automatically checks each unit entry to determine if that unit can be converted to the standard unit .
if a unit can not be converted , the tool returns an error message that requests a different unit .
the editing function was also a feature that developed during the course of the initial curation efforts .
at first , only single annotations could be edited . because single experiments can have many data points that vary only slightly in their annotated descriptions
, the curators needed a way to change common data entries in many annotations simultaneously .
the developer was able to change the editing software such that bulk edits could be done with multiple annotations simultaneously , thus accelerating the editing process .
although the phenominer curation tool is in a finished stage , it is likely that future curation will drive more requests for software adjustments .
phenominer originally started as a stand - alone project with a goal to visualize the existing rat phenotype data collected in the pga project .
after the data visualization tool was completed , rgd decided to curate rat quantitative phenotype data from the biomedical literature .
because the pre - existing rgd curation tools could not work with the phenominer database schema , development of a separate curation tool was necessary .
the new curation tool uses features specifically designed for annotating quantitative experiment data as well as curation steps that differ from other curation tools at rgd .
the database schema of phenominer comprises entities at different levels corresponding to the curation software user interfaces to enter information for
study is associated with multiple experiments , and one experiment is associated with multiple
experiment record is associated with one rat sample ( rat strain i d ) , one clinical measurement , one measurement method and one experimental condition group. one experimental condition group may include multiple experimental conditions .
information of rat strain , clinical measurement , measurement method and experimental condition group are stored in individual database tables .
the flow of data from curation tool to user interface is shown in figure 7 .
more information about the technical aspects of the phenominer curation tool and end - user display will be described elsewhere ( liu , w . , de pons , j .
operations in data flow : c = create , r = read , u = update , d = delete ( api = application programming interface ) .
operations in data flow : c = create , r = read , u = update , d = delete ( api = application programming interface ) .
rgd has recently established a quantitative phenotype curation paradigm that takes advantage of seven different ontologies / vocabularies .
some of those ontologies / vocabularies were built with a focus on the phenominer project , with ongoing ontology development as the curation proceeded . beyond the phenominer project at rgd ,
the cmo , mmo and xco have been used in a human clinical data project ( cover ) ( http://cover.wustl.edu/cover/ ) and the vt and cmo have been used to annotate animal qtls in the animal qtldb ( http://www.animalgenome.org/bioinfo/projects/ato/main.html ) ( 8) .
in addition to > 33 000 bulk loaded phenominer annotations from two large - scale rat physiology projects , > 4500 phenominer annotations have been added to the database in the initial manual curation phase of the project . in the animal qtl projects only terms
are used , so quantitative animal data comparable with the phenominer data is not currently available . in the case of the human data ,
access is somewhat restricted by an e - mail request system for data from the cover project .
both the bulk - loaded , high - throughput data and manually curated literature data are available in the phenominer end - user interface ( http://rgd.mcw.edu/phenotypes/ ) where studies across many rat strains , methods and conditions can be compared .
the quantitative phenotype data can also help researchers decide what rat strain would be the best model for a particular disease or phenotype they are studying .
future additions to phenominer data will include user - uploaded data and manual curation of rat non - qtl phenotype data from biomedical literature .
this work was supported by the national heart , lung and blood institute on behalf of the national institutes of health ( hl064541 and hl094271 ) .
funding for open access charge : national heart , lung and blood institute on behalf of the national institutes of health [ hl64541 ] .
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the rat genome database ( rgd ) is the premier repository of rat genomic and genetic data and currently houses > 40 000 rat gene records as well as human and mouse orthologs , > 2000 rat and 1900 human quantitative trait loci ( qtls ) records and > 2900 rat strain records .
biological information curated for these data objects includes disease associations , phenotypes , pathways , molecular functions , biological processes and cellular components .
recently , a project was initiated at rgd to incorporate quantitative phenotype data for rat strains , in addition to the currently existing qualitative phenotype data for rat strains , qtls and genes .
a specialized curation tool was designed to generate manual annotations with up to six different ontologies / vocabularies used simultaneously to describe a single experimental value from the literature . concurrently , three of those ontologies needed extensive addition of new terms to move the curation forward .
the curation interface development , as well as ontology development , was an ongoing process during the early stages of the phenominer curation project.database url :
http://rgd.mcw.edu
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nothing we have learned about mitosis since it was discovered a century ago is as dazzling as the discovery itself
this statement reflected mazia s ( and the whole field s ) frustration with the lack of mechanistic understanding of cell division .
although voluminous phenomenological data had been gathered on the mitotic apparatus ( spindle ) , the principles governing its assembly remained elusive .
interestingly , mazia s opus major was published merely a year after the original formulation of the search and capture ( s&c ) hypothesis ( kirschner and mitchison , 1986 ) .
if mazia had read this paper ( it was not cited in his 1987 review ) , he might have changed his stance .
indeed , s&c offered the first plausible mechanism to drive spindle assembly in animal cells and signified the transition from descriptions to molecular investigations of the process .
the s&c hypothesis stems from the discovery of microtubule dynamic instability ( mitchison and kirschner , 1984 ) . in sharp contrast to other cytoskeletal filaments , the plus ends of a typical microtubule oscillate between periods of growth and shrinkage caused by the addition and loss of -tubulin subunits .
the frequency of shrinkage events increases dramatically when a cell enters mitosis , transforming the longer , more stable interphase microtubule cytoskeleton into two dynamic radial arrays nucleated by the duplicated centrosomes . during the growth phase , a microtubule tip moves over several micrometers , and its trajectory is likely to vary from one period of growth to another .
this unique behavior inspired the discoverers of dynamic instability to propose that microtubules could search for a target positioned within their reach , which would eventually be hit by a growing microtubule ( fig .
1 a ) . if the target were capable of capturing and capping this microtubule , thereby suppressing its dynamics , such a hit would result in the formation of a stable connection between the target and the centrosome .
in the context of spindle assembly , the proposed targets were kinetochores , the paired macromolecular assemblies residing at the centromere of each mitotic chromosome .
the s&c mechanism would therefore progressively incorporate multiple individual chromosomes into a common bipolar microtubule array with microtubule minus ends converging on the centrosomes and plus ends directed toward the equator ( fig .
( a ) sequence of events envisioned in the classic formulation of the s&c hypothesis .
microtubules nucleated at the duplicated centrosomes form two radial arrays ( blue and orange ) .
dynamically unstable microtubules explore space until a growing plus end encounters a kinetochore ( magenta ) .
this encounter results in the capture and partial stabilization of the microtubule ( denoted by a color change of both the microtubule and kinetochore to green ) . captured microtubules connect individual kinetochores to the centrosomes ( spindle poles ) . in this scenario
, each kinetochore ultimately becomes attached to microtubules , although the duration of spindle assembly varies significantly as a result of the stochastic nature of the process .
( b ) direct visualization of microtubule capture by kinetochores in a newt lung cell ( adapted with modifications from rieder and alexander , 1990 ) .
because of the large cell size , individual chromosomes are often positioned in areas with a low density of microtubules and remain motionless for extended periods before suddenly moving poleward ( arrows ) at a velocity that often exceeds 30 m / min .
immunofluorescence of the assembling spindle fixed immediately after initiation of the poleward movement reveals a kinetochore interacting with a single microtubule ( arrowheads ) .
note that the initial contact is not to the plus end but to the wall of the microtubule ( i.e. , lateral interaction ) .
( c ) the efficiency and fidelity of s&c depends on kinetochore orientation and the distance from centrosomes .
chromosomes positioned near the intersection of the spindle equator and spindle axis would have a reasonable chance of forming proper amphitelic attachments ( 1 ) .
chromosomes located at the periphery of the spindle have reduced chances of capturing microtubules ( 2 and 3 ) .
in contrast , chromosomes positioned near a centrosome are exposed to a high density of unipolar microtubules ( 4 and 5 ) , which promotes either erroneous attachment of both sister kinetochores to the same spindle pole ( syntelic attachment ; 4 ) or attachment of only one kinetochore ( monotelic attachment ; 5 ) .
less than four years after the formulation of the hypothesis , microtubule capture by chromosomes was directly visualized in live cells ( hayden et al . , 1990 ; rieder and alexander , 1990 ) .
these elegant studies established that the first step of a chromosome s incorporation into the spindle involves a direct contact between the kinetochore and a single microtubule ( fig .
however , as is common in cell biology , s&c was born as an intuitive cartoon rather than a testable model .
the question of whether dynamic instability constituted a searching behavior efficient enough to incorporate all of the chromosomes into a common spindle was not addressed until almost a decade after the original formulation of the hypothesis .
the first theoretical evaluation suggested that dynamically unstable microtubules would in fact find a target much faster than conventional steadily growing filaments ( holy and leibler , 1994 ) .
however , the absolute time required to find all 46 chromosomes in a human cell via completely unbiased stochastic s&c was calculated to be several times longer than the typical duration of mitosis ( wollman et al . , 2005 ) .
furthermore , the establishment of proper connections to microtubules would depend on how a chromosome is positioned within the nascent spindle ( fig .
these considerations imply the existence of additional factors that facilitate spindle assembly , and a series of such mechanisms has been identified in recent years .
a common theme emerging from these studies is that s&c depends on spatially selective biochemical pathways that promote the formation of microtubules in the vicinity of kinetochores and also differentially engage molecular motors to position chromosomes in the areas with maximal exposure to spindle microtubules .
furthermore , proper attachment of chromosomes to the spindle is assisted by orderly changes in the shape of the cell and the adaptive architecture of kinetochores .
together , these facilitating mechanisms ensure that stochastic encounters between microtubules and kinetochores result in a rapid yet low error incorporation of all chromosomes into the mitotic apparatus .
in the original formulation of s&c , radial arrays of microtubules nucleated by the duplicated centrosomes were expected to be the sole source of spindle microtubules .
however , the density of microtubule ends and therefore the probability of capture decreases rapidly as the distance between the centrosome and chromosomes increases , and a 200-nm small kinetochore has only a slight chance of being contacted by a microtubule originating from a centrosome positioned 1520 m away within the 1015-min period typical of spindle assembly ( wollman et al . , 2005 ) .
this problem can be overcome if the density of microtubules near kinetochores is increased , and multiple mechanisms have been identified that selectively promote microtubule nucleation and stabilize microtubule plus ends near the chromosomes , leading to the formation of kinetochore - attached microtubule bundles termed kinetochore fibers ( k - fibers ) .
the role of chromosome arms in mitosis was once compared with that of a corpse at a funeral ; the dna comprising the bulk of the genome provides the reason for the proceedings , but does not actively participate in the event ( mazia , 1961 ) .
however , this view was challenged by the demonstration that viral dna injected into a frog egg , or plasmid dna - coated beads incubated in metaphase egg extract , induce the formation of spindle - like structures in the absence of centrosomes or kinetochores ( karsenti et al .
the most prominent gradient is of rangtp ( discussed extensively in forbes et al . , 2015 ) .
rangtp is produced by the guanine nucleotide exchange factor for ran , regulator of chromosome condensation 1 ( rcc1 ) , which ubiquitously decorates chromatin .
the opposing activities of rcc1 and rangap result in a steep gradient of rangtp centered on chromosomes ( kalab et al . , 2002 , 2006 ) .
similar to its function in nucleocytoplasmic transport , rangtp releases cargoes from nuclear transport receptors called importins ( gruss et al . , 2001 ; nachury et al . , 2001 ; wilde et al . ,
are many proteins that function in microtubule polymerization and organization , such as tpx2 ( gruss and vernos , 2004 ) , the spindle microtubule cross - linking kinesin xctk2 , which requires a rangtp gradient for proper localization and motility ( weaver et al . , 2015 ) , and components of the nonspecific lethal ( nsl ) complex , which binds to and stabilizes k - fiber minus ends ( meunier and vernos , 2011 ; meunier et al . , 2015 )
. an important source of rangtp - induced spindle microtubules that serves to increase the density of microtubule plus ends in the vicinity of chromosomes is microtubule - templated nucleation mediated by the augmin complex , which requires the microtubule nucleator -tubulin ( petry and vale , 2015 ) and is stimulated by tpx2 ( petry et al . , 2013 ) .
in addition to being liberated from importins by rangtp , the inhibition of tpx2 is also relieved by the golgi protein gm130 , which sequesters importin- to golgi membranes ( wei et al . , 2015 ) .
thus , the rangtp gradient promotes both de novo nucleation of microtubules near kinetochores and amplification of microtubule growth toward chromosomes ( fig .
if the chromatin and kinetochores become spatially separated , the gradient can erroneously guide microtubules away from the kinetochores .
this was directly observed in mitotic cells with unreplicated genomes , where the bulk of chromatin along with its associated rangtp gradient resides in the cell periphery and astral microtubules extend from the centrosomes toward the chromatin , which becomes particularly prominent when k - fiber formation is inhibited ( oconnell et al . , 2009 ) .
( a ) rcc1 localized to chromosomes increases the local concentration of rangtp ( pink ) and promotes microtubule polymerization by liberating cargoes such as tpx2 ( yellow ) and the nonspecific lethal ( nsl ) complex ( beige ) from inhibitory interaction with importins ( blue ) .
microtubules positioned near the kinetochore are likely to be rapidly captured , which initiates formation of a nascent k - fiber with a capped minus end ( left side ) . at the kinetochore ( red ) ,
microtubule nucleation is stimulated when rangtp relieves inhibition of nucleoporins elys and nup107160 by transportin , allowing recruitment of -tubulin ring complexes ( light green ; right side ) . within the spindle ,
templated microtubule nucleation is mediated by augmin ( dark green ) and stimulated by tpx2 .
( b ) because of its rapid diffusion , rangtp forms a gradient resulting in a differential regulation of microtubule nucleation / dynamics near versus away from the chromosomes .
this , in turn , facilitates integration of chromosomes and their associated kinetochore microtubules into a common spindle .
( c ) a k - fiber formed via the kinetochore - mediated mechanism ( arrows ) grows via polymerization at plus ends , generating a larger target for astral microtubules .
direct contact ( capture ) between the elongating k - fiber and an astral microtubule ( arrowheads ) is followed by poleward transport and incorporation of the fiber s free end into the spindle .
rangtp is thought to contribute to spindle assembly in all metazoan cells , but it is most crucial in the second meiotic division of vertebrate eggs ( kalab et al . , 1999 ;
ohba et al . , 1999 ; wilde and zheng , 1999 ; dumont et al . , 2007 ) , when centrosomes are absent and the chromosomes and spindle are tiny relative to the size of the cell , making spindle assembly via unbiased s&c virtually impossible .
however , eggs contain stockpiles of cellular material including ran pathway components , and the rangtp generator rcc1 is not significantly enriched or activated on chromosomes , begging the question of why a large unbound pool of rcc1 does not obscure a chromatin - centered rangtp gradient . using xenopus laevis egg extracts , zhang et al .
( 2014 ) found that the cytoplasmic pool of rcc1 is inactive because of its association in a complex together with ran and ran binding protein 1 ( ranbp1 ) .
importantly , chromosomes still regulate microtubule nucleation and stability in the absence of a rangtp gradient ( maresca et al . ,
2009 ) as a result of a second chromatin - induced pathway mediated by the chromosome passenger complex ( cpc ; zierhut and funabiki , 2015 ) .
the kinase subunit of the cpc , aurora b , locally phosphorylates and inactivates microtubule - destabilizing proteins including mcak ( andrews et al .
spatial activation of aurora b in mitosis occurs through a kinase cascade initiated by the kinase haspin , which phosphorylates histone h3 .
phospho - h3 is bound directly by another cpc component , survivin , enriching aurora b and promoting its trans - autophosphorylation ( zierhut and funabiki , 2015 ) . a powerful nucleosome depletion and add - back approach in xenopus egg extracts demonstrated that histone h3 phosphorylation is the only target of haspin important for the spatial regulation of aurora b ( zierhut et al . , 2014 ) .
by concentrating at the centromere that underlies each kinetochore , the cpc is known to control and correct erroneous microtubule attachments to kinetochores , thereby promoting biorientation of chromosomes so that chromatids are attached to opposite spindle poles and poised for segregation ( lampson and cheeseman , 2011 ) .
active aurora b may diffuse away and phosphorylate substrates at a distance ( wang et al . ,
interestingly , another kinase of the aurora family , aurora a , is situated at the spindle poles , where erroneously attached chromosomes frequently accumulate , and generates a pole - centered phosphorylation gradient that also contributes to error correction ( chmtal et al . , 2015 ; ye et al . , 2015 ) . in the context of s&c ,
in addition , rangtp appears to act directly at kinetochores , relieving inhibition of a complex containing nuclear pore proteins and -tubulin ( bernis et al . , 2014 ;
once captured , the plus ends of microtubules that reside at the kinetochore tend to grow continuously , resulting in a steady elongation of the nascent k - fiber ( maiato et al . , 2004 ) .
as these fibers extend outwards , they provide large antennae that are rapidly discovered by the astral microtubules and are incorporated into the common spindle ( fig .
the minus end capture of preformed k - fibers is particularly evident when spindles transition from a monopolar to a bipolar configuration ( khodjakov et al . , 2003 ) .
in the s&c hypothesis of 1986 , the molecular nature of a capture event was not defined , but was assumed to dampen dynamics at the tip of the kinetochore - associated microtubule ( kirschner and mitchison , 1986 ) .
however , observations of microtubule capture in cells revealed that kinetochores initially come in contact with the wall of a microtubule ( fig . 1 b ) and that these lateral interactions are subsequently replaced by attachment to the microtubule plus end ( rieder and alexander , 1990 ; tanaka et al . , 2005 ; magidson et al . , 2011 ; kalinina et al . , 2013 ) . indeed , direct single - step capture of the tip is significantly less probable than an encounter at a random point along the length of a microtubule , particularly because microtubules are known to pivot in space , which significantly enhances their ability to search for kinetochores ( kalinina et al . , 2013 ) .
molecular mechanisms that govern conversion from lateral to end - on attachments remain poorly understood and may occur as a direct transition of the same microtubule ( gandhi et al . , 2011 ) . alternatively , lateral interactions may facilitate capture of other plus ends by orienting kinetochores favorably within the spindle ( magidson et al . , 2011 , 2015 ) .
the transition from lateral interaction to end - on attachment involves the coordinated activities of several molecular motors and microtubule depolymerases ( shrestha and draviam , 2013 ) .
in fact , a second fundamentally important property of capture revealed by live - cell observations was the activity of molecular motors residing at the kinetochore .
kinetochores were seen to initiate rapid movement toward the minus end of a captured microtubule immediately after lateral contact ( rieder and alexander , 1990 ) .
the s&c hypothesis predated the discovery of motor proteins in the spindle , and in its primitive form , the duplicated centrosomes were thought to dictate the bipolar configuration of the spindle .
it is now recognized that cytoplasmic dynein and a large family of kinesin motor proteins normally act to drive microtubule self - organization and spindle bipolarity ( gatlin and bloom , 2010 ) .
the fundamental role of molecular motors is best illustrated in egg extract systems that lack centrosomes or kinetochores ( heald et al . , 1996 ) , and upon elimination of the centrosome in vertebrate cells ( khodjakov et al .
first , they generate the bipolar microtubule array , which provides tracks for polarized chromosome movements that facilitate their biorientation .
second , plus end directed motors that function to cross - link microtubules and sort them into an antiparallel array , including kinesin-5 ( eg5/kif11 ) and kinesin-12 ( xklp2/kif15 ) , establish a spindle axis , whereas minus end directed motors , including kinesin-14 ( xctk2/hset ) and dynein , provide balancing forces and act to focus spindle poles ( fig .
microtubule ( mt)-bound motors promote bipolar spindle formation , whereas chromosome - associated motors drive proper kinetochore orientation and chromosome movement to the equator .
box 1 : motor - dependent mechanisms establish bipolarity as eg5 ( kinesin-5 ) motors slide antiparallel microtubules apart with their minus ends leading and their plus ends directed toward the spindle equator . box 2 : minus end directed motors such as dynein move microtubules poleward with their minus ends leading , thereby incorporating k - fibers into the spindle and focusing spindle poles .
box 3 : kinetochore - associated dynein transports chromosomes along astral microtubules toward the spindle poles from the periphery .
box 4 : plus end directed chromokinesins ( kinesin-4 and -10 ) eject chromosome arms outward .
box 5 : cenp - e ( kinesin-7 ) transports unattached kinetochores toward the equator along spindle microtubules .
although some motors act on spindle microtubules to organize them , others are present on kinetochores and chromosome arms and position them near the spindle equator , where conditions favor the attachment of sister kinetochores to microtubules from opposite spindle poles .
dynein , which also exists in a kinetochore - bound pool , participates in the initial capture of astral microtubules , promoting lateral attachment and the movement of chromosomes toward the minus ends at spindle poles ( yang et al . , 2007 ) .
this force is counteracted by the chromosome arm associated chromokinesins kinesin-10 ( kid / nod ) and kinesin-4 ( kif4/xklp1 ) that push the arms away from the centrosome ( wandke et al . , 2012 ) . as a result of this tug of war ,
scattered chromosomes are drawn from the cell periphery to the vicinity of spindle poles ( barisic et al . , 2014 ) .
from there , chromosomes are subsequently delivered to the spindle equator by the kinetochore - associated kinesin-7 cenp - e ( kapoor et al . , 2006 ; cai et al . ,
interestingly , cenp - e prefers the less dynamic microtubules directed toward the spindle equator that contain posttranslationally modified -tubulin lacking the c - terminal tyrosine . in vitro reconstitution experiments revealed that cenp - e dependent transport is enhanced on detyrosinated microtubules , and treatment causing ubiquitous tubulin detyrosination in cells caused chromosome transport in random directions away from spindle poles ( barisic et al . , 2015 ) .
thus , motors organize the bipolar antiparallel microtubule array and drive chromosome movements that promote their congression and biorientation and are guided by biochemical cues , including rangtp - induced gradients and -tubulin posttranslational modifications .
s&c - driven spindle assembly is profoundly affected by geometric constraints such as the size and shape of the cell and the spatial organization of spindle components at the onset of mitosis . because the length of dynamic microtubules is limited , accessory mechanisms must exist to prevent the excessive scattering of chromosomes or actively gather them within the searchable volume . in extremely large cells , such as animal oocytes ,
the chromosomes are driven into a compact group by actin filaments ( lnrt et al . , 2005 ) .
in somatic cells , a cage of intermediate filaments that surrounds the nucleus during interphase averts the dispersion of chromosomes after nuclear envelope breakdown ( mandeville and rieder , 1990 ) .
similarly , chromosomes can be confined by the remnants of the nuclear envelope that usually surround the spindle ( tsai et al . , 2006 ; ma et al . , 2009 ) .
recent work suggests that the compartmentalization of space by the residual nuclear envelope operates as a matrix that not only confines larger mitotic apparatus components like chromosomes but also creates a diffusion barrier that concentrates soluble tubulin , as well as proteins involved in the regulation of mitosis within the spindle region .
conversely , this barrier prevents the invasion of cytoplasmic organelles into the spindle compartment to avoid their steric interference that would impede interactions between kinetochores and microtubules ( schweizer et al . ,
intriguingly , the spindle matrix contains proteins such as bugz that harbor low - complexity hydrophobic sequences and undergo a temperature - dependent phase transition that promotes microtubule polymerization ( jiang et al . , 2014 ) .
therefore , the search for chromosomes normally takes place not throughout the cytoplasm but within a compact and biochemically distinct subcellular environment that promotes spindle assembly .
regulation that affects the size , shape , and composition of this compartment may indirectly yet profoundly affect the efficiency and fidelity of spindle assembly .
for example , a common feature of animal cells is that they round up during mitosis . preventing this morphological change either by perturbing cortical actin or by pure mechanical means impedes the gathering of chromosomes into a compact group near the geometric center of the cell .
this in turn limits the efficiency of s&c and leads to a higher probability of chromosome loss ( lancaster et al .
although gathering the chromosomes within the reach of spindle microtubules is necessary , it also poses a problem for s&c - driven spindle assembly . in a crowded environment , many kinetochores become inaccessible to microtubules because of occlusion by chromosome arms ( fig .
the number of kinetochores that are invisible to microtubules increases rapidly as the number of chromosomes grows .
computational analyses suggest that only 3% of kinetochores would be accessible to microtubules if 46 average - size chromosomes were randomly distributed within a typical - size spherical nuclear volume ( paul et al . , 2009 ) .
to overcome this problem , cells have developed mechanisms that shape , orient , and distribute chromosomes into spatial patterns that actively present kinetochores to the searching microtubules during prometaphase ( kitajima et al .
these mechanisms involve the interplay between a chromokinesin - mediated ejection force on the arms ( rieder and salmon , 1994 ; vanneste et al . , 2011 ) and inward - directed forces produced by the kinetochore - associated microtubule motors , which arrange the chromosomes into a toroid around the nascent spindle .
in this belt - like configuration ( fig . 4 b ) , kinetochores become exposed to a high density of microtubules , which promotes efficient capture .
subsequently , stronger and more stable end - on attachments allow chromosomes to gradually repopulate the central part of the spindle .
conditions that prevent the formation of the chromosomal belt ( e.g. , the inactivation of chromokinesins ) prolong spindle assembly and markedly increase the number of lagging chromosomes that segregate improperly during the ensuing anaphase ( magidson et al . , 2011 , 2015 ) .
( a ) the efficiency of s&c is affected by the spatial organization of chromosomes .
the arms of peripheral chromosomes ( blue ) shield kinetochores ( red ) positioned deeper inside the nucleus from astral microtubules ( green ) .
( b ) typical spatial patterns observed in mammalian cells at progressive stages of spindle assembly . at prophase ,
duplicated centrosomes ( green ) separate to opposite sides of the nucleus , and the distribution of kinetochores ( orange ) appears to be random . during early prometaphase , chromosomes form a toroid with most kinetochores residing on the surface of the nascent spindle and chromosome arms pointing outwards . upon formation of stable end - on kinetochore attachments ,
chromosomes repopulate the central part of the spindle , becoming uniformly distributed at the spindle equator at metaphase .
( c ) the ejection force of the arms ( blue arrows ) opposed by the inward forces generated at the kinetochore ( red arrow ) rotates the centromere so that sister kinetochores become preferentially oriented toward opposite spindle poles .
notice that larger kinetochores support a more significant rotation ( top ) , whereas smaller kinetochores lose contact with microtubules , dampening the inward force ( bottom ) .
note that chromosome arms ( blue ) are bent inside the prophase nucleus but become straightened by the ejection force ( prometaphase and metaphase ) .
inner kinetochores ( cenp - a ; yellow ) remain compact throughout mitosis , whereas the outer kinetochore ( cenp - f ; orange ) encircles a large part of the centromere during prometaphase and compacts after the formation of end - on attachments ( metaphase ) .
another set of geometric constraints that inevitably affect the efficiency and fidelity of s&c - based spindle assembly are the size and relative positions of sister kinetochores assembled at the centromere ( stergren , 1951 )
. intuitively , small sister kinetochores positioned on opposite sides would ensure error - free spindle assembly , as they would be sterically shielded by the centromere from capturing microtubules that emanate from the same spindle pole .
indeed , when sister kinetochores become juxtaposed , the number of syntelic attachments increases dramatically ( lonarek et al .
however , small kinetochores can not capture microtubules very efficiently and would increase the time required for spindle assembly .
interestingly , recent computational analyses suggest that the intuitive reciprocal relationship between efficiency and fidelity in s&c - driven spindle assembly is incorrect . a model that considers
the formation of end - on attachments in a spindle environment dominated by lateral microtubule interactions predicts that the enlargement of kinetochores during prometaphase would both accelerate spindle assembly and suppress the number of errors ( magidson et al . , 2015 ) . this unexpected synergy is the result of rotational alignment of centromeres with respect to the spindle axis driven by opposing forces acting at the kinetochores versus chromosome arms .
the extent of angular prealignment is less for smaller kinetochores , which are not capable of remaining in direct contact with microtubules during extensive rotations ( fig . 4 c ) .
indeed , enlargement of kinetochores during earlier stages of spindle assembly followed by their compaction upon the formation of end - on attachment ( fig .
4 d ) has been directly observed in cells ( thrower et al . , 1996 ;
hoffman et al . , 2001 ; magidson et al . , 2015 ) as well as in egg extracts ( wynne and funabiki , 2015 ) .
interestingly , computational modeling suggests that once angular chromosome alignment is attained , efficient correction of erroneous attachments will be achieved simply because of the rapid turnover of microtubules at kinetochores ( zaytsev and grishchuk , 2015 ) .
thus , dynamic changes in chromosome architecture in the context of spatial cues and constraints , together with the high turnover rate of microtubules within the spindle , promote the proper attachment of sister kinetochores to opposite spindle poles .
cell biology is a rapidly advancing field , and new observations frequently disprove mechanistic hypotheses after just a few short years . yet , nothing that we have learned about mitosis in the last 30 years , which includes the discovery of scores of factors involved in spindle assembly , has been inconsistent with the basic principles of s&c . instead , the many facilitating mechanisms elucidated over the years have been organically incorporated into the model .
it is important to emphasize that these mechanisms are often not essential : spindles form in the absence of mitotic gradients ( maresca et al . , 2009 ) , or when the function of key motors is blocked ( ganem et al . , 2005 ; gayek and ohi , 2014 ) or
as long as the minimal requirements for s&c ( i.e. , dynamic microtubules and capture by kinetochores ) are in place , a functional spindle can assemble . however , the duration of spindle assembly and the number of erroneous chromosome attachments increase dramatically in the absence of facilitating s&c mechanisms . importantly , both of these side effects compromise the fate of daughter cells : the prolongation of mitosis has been shown to halt progression through the ensuing cell cycle ( uetake and sluder , 2010 ) , and erroneous segregation of a chromosome can trigger perpetuating chromosomal instability ( thompson and compton , 2008 ) .
thus , the complexity of numerous nonessential mechanisms sustains the wonderfully simple principle of s&c .
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cell division is enacted by a microtubule - based , self - assembling macromolecular machine known as the mitotic spindle . in 1986 , kirschner and mitchison proposed that by undergoing dynamic cycles of growth and disassembly , microtubules search for chromosomes .
capture of microtubules by the kinetochores progressively connects chromosomes to the bipolar spindle .
30 years later , search and capture remains the cornerstone of spindle assembly .
however , a variety of facilitating mechanisms such as regulation of microtubule dynamics by diffusible gradients , spatially selective motor activities , and adaptive changes in chromosome architecture have been discovered .
we discuss how these mechanisms ensure that the spindle assembles rapidly and with a minimal number of errors .
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moderate to severe pain is common among patients with cancer and remains a significant challenge to practitioners , despite advances in pain management and the widespread adoption of the world health organization ( who ) guidelines for cancer pain management.1,2 the who 3-step ladder approach to cancer treatment includes the use of opioids with fixed , around - the - clock dosing in patients who do not respond to non - opioid analgesics.3,4 however , breakthrough cancer pain ( btcp ) is a common problem that is currently only minimally addressed by the who pain relief ladder.1,3 btcp has been defined as a transitory exacerbation of pain that occurs on a background of otherwise stable pain in a patient receiving chronic opioid therapy.5 it is distinguished from continuous background pain that is relatively well managed with opioids .
btcp episodes are generally moderate to severe in intensity , reach peak intensity in approximately 3 minutes , and generally last 30 minutes.57 estimates of the incidence and prevalence of btcp vary , although btcp is more common among patients with advanced disease.5 results of a survey of adult inpatients referred for evaluation and treatment to a cancer pain management service showed that of 63 patients who achieved stable opioid dosing and reported pain of moderate or less intensity , 41 ( 65% ) experienced at least one episode of btcp in the preceding 24 hours , with a median of four episodes.5 of 51 episodes of btcp , the maximum intensity occurred within 3 minutes of onset in 22 ( 43% ) episodes , and the median duration was 30 minutes.5 another study of 245 patients admitted for hospice care showed that 218 patients ( 89% ) experienced btcp , with approximately 88% of those patients experiencing more than one episode per day and 84% of episodes producing moderate , severe , or excruciating pain .
the majority ( 73% ) of btcp episodes lasted 30 minutes , with 26% lasting just 10 minutes or less.7 btcp is associated with a high level of patient dissatisfaction with treatment and also has a negative impact on patients lives.68 impairments in sleep , concentration , emotional well - being , interpersonal relationships , everyday function , and work performance are common in patients with btcp.6,8,9 for example , for some patients , the simple act of turning in bed can trigger an episode of btcp and thereby interfere with sleep.9 for other patients , even gentle hugs can bring on excruciating btcp , thus preventing physical contact with others and straining personal relationships.8 this review will focus on current management strategies for btcp , with a focus on the newest formulation of rapid - onset fentanyl : namely , fentanyl sublingual spray .
although traditional oral opioids , such as morphine , have been the mainstay in the treatment of btcp , their effectiveness is limited by a relatively slow onset of analgesia ( often 30 minutes ) , the need for hepatic first - pass metabolism ( which reduces bioavailability ) , the relatively long duration of effect ( which increases the risk for adverse events ) , and variable absorption.1013 oral opioids also are not ideal in patients with nausea , vomiting , or dysphagia.11,14,15 several rapid - onset formulations of fentanyl have been approved by the us food and drug administration ( fda ) for the treatment of btcp .
fentanyl is a synthetic , highly potent , lipophilic -opioid agonist that rapidly crosses the blood brain barrier and has a half - life ranging from 1.6 to 6 hours.13,16 the different fentanyl formulations vary in pharmacokinetic properties and ease of use , but all of them have a rapid onset and a relatively short duration of analgesia.10,17 in comparison with traditional oral opioids , the rapid - onset fentanyl formulations convey a significant advantage in the treatment of btcp , with a pharmacokinetic profile that more closely matches that of a btcp episode.10,13,17,18 with the approval of fentanyl sublingual spray by the fda in january 2012,19 six rapid - onset formulations of fentanyl are now available in the usa
transmucosal lozenge , sublingual orally disintegrating tablet , buccal tablet , buccal soluble film , pectin nasal spray , and sublingual spray ( table 1).13,17,2029 randomized , double - blind , placebo - controlled clinical trials in adults with cancer have demonstrated the efficacy of each of these rapid - onset fentanyl formulations in the treatment of btcp.10,18,26,27,2931 adverse events in these trials have been typical for opioids ( the most common being nausea , vomiting , headache , dizziness , and somnolence).18,26,27,2931 although all of the rapid - onset fentanyl formulations show efficacy in the treatment of btcp in opioid - tolerant patients , each formulation has certain advantages and disadvantages .
the oral transmucosal fentanyl citrate ( otfc ) lozenge requires effort on the part of the patient to control the administration , which results in variability in absorption and the swallowing of a significant amount of drug.13,17,26,27 any fentanyl swallowed in saliva will undergo hepatic first - pass metabolism , resulting in reduced bioavailability.13,14 in addition , one otfc unit contains 2 grams of sugar , and prolonged use may increase the risk of dental decay , including dental caries.20,32 fentanyl buccal tablets have greater bioavailability than otfc ( 65% versus 50% ) , but the tablets still require 14 to 25 minutes to disintegrate completely , and approximately 50% is swallowed in the saliva.20,22 fentanyl sublingual orally disintegrating tablet disintegrates rapidly and has an overall bioavailability of 54%.10,21 the tablet must remain under the tongue , and patients should avoid swallowing during administration.21 fentanyl buccal soluble film requires little effort on the part of the patient ; little saliva is necessary for absorption , but the film requires up to 30 minutes to completely dissolve and 49% of the dose is swallowed with saliva.10,13,23,27 fentanyl pectin nasal spray may be useful in patients with nausea and vomiting , whereby oral administration is difficult;13,33 however , some patients are less accepting of intranasal administration , and coexisting nasal problems may complicate use.14,34,35 fentanyl sublingual spray requires minimal patient effort , and little drug is swallowed in saliva.14,29,36 in a clinical trial , patients indicated that the sublingual spray formulation is convenient and easy to use.29 fentanyl sublingual spray provides a useful alternative to fentanyl intranasal spray for patients with nasal problems or those who dislike the intranasal route of administration , although it should be used with caution in patients with grade 2 mucositis unless the benefits outweigh the potential risk from possible increased exposure.25 several cancer treatment guidelines discuss options for the management of btcp that include the use of rapid - onset opioids .
the national comprehensive cancer network recommends rapid - onset opioids when exacerbations of pain are not due to inadequate around - the - clock dosing.37 the european society for medical oncology recommends use of opioids with a rapid onset and short duration of action.38 the european association for palliative care recommends oral immediate - release opioids or transmucosal fentanyl and acknowledges that the latter are preferable in some cases due to their rapid onset and shorter duration of effect.39 although each of these guidelines mentions btcp , none explicitly addresses it .
the science committee of the association for palliative medicine of great britain and ireland recently formulated guidelines specifically for the treatment for btcp , although the committee notes that their recommendations are based on limited evidence and provide only general strategies.40 nonetheless , their recommendations include the use of opioids as first - line pharmacologic treatment for btcp , noting that oral immediate - release opioids are not optimal due to their delayed onset and long duration of analgesia and rapid - onset opioid formulations , such as transmucosal ( sublingual , buccal , intranasal , pulmonary ) formulations , may be more advantageous.40 clearly , there is a need for new clinical trials on the treatment of btcp and the development of definitive , evidence - based , treatment guidelines .
btcp treatment options need to optimize the benefits while minimizing the risks for any particular patient . as with all potent opioids ,
there is little literature on abuse and misuse of the rapid - onset formulations of fentanyl used to treat btcp , although rates for abuse and addiction to rapid - onset fentanyl that have been reported in longer - term clinical trials of patients with chronic noncancer pain are low .
16,4143 however , abuse and misuse of rapid - onset fentanyl should not be overlooked when treating patients with cancer - related pain .
medical professionals need to consider the challenge of managing cancer pain or pain among survivors over a potentially longer duration , as cancer is no longer considered a terminal disease .
advancements in medical therapy have helped patients with cancer live longer , and survival rates have increased.44 all patients treated with opioids require careful monitoring of signs of misuse .
diversion is an important consideration among patients receiving palliative care in nursing facilities or from caregivers at home.42,45 in an attempt to mitigate the risk of misuse , abuse , addiction , overdose , and serious complications due to medication errors associated with use of opioids , the fda has instituted a risk evaluation and mitigation strategy ( rems ) for the class of transmucosal immediate - release fentanyl products.46 a similar class - wide rems has been implemented for all of the extended - release and long - acting opioids used to treat chronic pain.47 these rems programs were designed to educate prescribers and pharmacists on appropriate patient selection and on providing patients with information on the proper use , storage , and disposal of potent opioids to minimize risks and improve patient outcomes.46,47
fentanyl sublingual spray ( subsys , insys therapeutics , inc , chandler , az , usa ) was approved by the fda in 2012 for the treatment of btcp in adult patients who are already receiving and who are tolerant to opioids for the management of underlying persistent cancer pain.13,19,25 fentanyl sublingual spray is available in dosages of 100 , 200 , 400 , 600 , 800 , 1200 , and 1600 g.25 the 1200 g dose is provided as packages of two 600 g units , and the 1600 g dose is provided as packages of two 800 g units .
first , the sublingual mucosa is highly vascularized and is the most permeable region of the oral mucosa .
it contains a relatively large surface area with a mucosal thickness measuring about 100200 m , compared with 500800 m for the buccal mucosa.48,49 sublingual administration also bypasses hepatic first - pass metabolism , which minimizes variability in bioavailability.14,15,48 the sublingual route of administration is particularly useful in patients with nausea , vomiting , or dysphagia.15 in terms of patient preference , there is some indication that patients prefer the sublingual route of administration over other routes of administration , such as intranasal or rectal , for treatment of btcp.34 in comparison with most other opioids , fentanyl has a significantly greater degree of sublingual absorption,15 with fewer adverse effects such as unpleasant taste , burning , light - headedness , and numbness.15 fentanyl sublingual spray may produce a rapid onset of analgesia in part because the liquid formulation bypasses the need for tablet disintegration and allows for distribution of the active compound throughout the sublingual mucosa , thereby potentially maximizing the rate and extent of absorption.29 in addition , fentanyl sublingual spray is composed of the more permeable base form of fentanyl and contains menthol.25,50,51 the non - ionized base form of fentanyl favors lipophilicity and absorption through the sublingual membrane , which increases bioavailability.50,51 in fact , the base form of fentanyl has demonstrated 30% bioavailability compared with the fentanyl citrate salt formulation.50 menthol has long been recognized as a highly effective permeation enhancer for a variety of compounds and tissues , including the transmucosal mucosa.5254 fentanyl sublingual spray has the unique advantage of combining base fentanyl with menthol to achieve fast absorption and superior bioavailability , with minimal variability.50,51 published clinical trials of fentanyl sublingual spray have evaluated its pharmacokinetic properties in healthy subjects and determined its efficacy and tolerability in opioid - tolerant patients with btcp.29,36,55
a two - part , phase i , randomized , five - period crossover trial in healthy adults ( aged 1855 years ) was conducted to determine the dose - proportionality of five doses of fentanyl sublingual spray ( 100 , 200 , 400 , 600 , and 800 g ) under fasted conditions ( part a ) , and determine the effects of temperature and ph in the oral cavity on the bioavailability of fentanyl after administration of fentanyl sublingual spray 200 g ( part b).55 a total of 53 subjects ( 38 female , 15 male ) were enrolled in part a and 14 subjects ( 3 female , 11 male ) were enrolled in part b. subjects withdrew due to positive urine drug screen ( n = 2 ) , positive urine alcohol screen ( n = 1 ) , and withdrawal of consent ( n = 9 ) .
the mean standard deviation ( sd ) age of subjects was 31 10 years in part a and 32 10 years in part b.55 mean 36-hour plasma fentanyl concentrations across five different doses are shown in figure 1.55 for all doses of fentanyl sublingual spray , mean plasma concentration of fentanyl was detectable within 5 minutes , the first sample point , and was approximately 27.3% , 60.6% , and 86.6% of the mean maximum plasma concentration ( cmax ) within 5 , 10 , and 20 minutes of administration , respectively .
mean plasma concentration of fentanyl reached a plateau approximately 30 minutes after administration , which was maintained for 6090 minutes.55 mean cmax increased and median time to maximum plasma concentration ( tmax ) decreased with increasing doses of fentanyl sublingual spray .
linear dose proportionality was demonstrated across the dose range of 100800 g of fentanyl sublingual spray , as determined by linear regression of cmax and area under the concentration - time curve ( auc ) from time zero extrapolated to infinity ( auc ) ( figure 2 ) .
pretreatment of the oral cavity with a cold or hot beverage or a low- or high - ph beverage did not alter the absorption of fentanyl sublingual spray significantly , as determined by assessing cmax and auc under different temperature and acidity conditions.55 fentanyl sublingual spray was generally well tolerated .
treatment - emergent adverse events were reported by 31 of 53 subjects ( 58.5% ) in part a and 9 of 14 subjects ( 64.3% ) in part b.55 the most commonly reported adverse events were nausea ( n = 17 ) , vomiting / intermittent vomiting ( n = 12 ) , and somnolence ( n = 8) in part a and nausea ( n = 10 ) , vomiting ( n = 3 ) , and headache ( n = 3 ) in part b.55 a second pharmacokinetic study was conducted as a single - dose , randomized open - label , three - way crossover trial in healthy adults ( aged 1855 years ) to compare the rate of absorption and systemic bioavailability of fentanyl sublingual spray 400 g with otfc lozenge 400 g.36 intravenous fentanyl citrate 100 g was used as an independent treatment , and a 7-day washout period separated treatments . of 40 enrolled subjects ( eight female , 32 male ) , 29 completed all three treatment periods .
reasons for withdrawal were noncompliance ( n = 8) , violation of entry criteria ( n = 2 ) , and withdrawn consent ( n = 1 ) . the mean sd age of subjects who completed the study was 35 10 years.36 compared with otfc , fentanyl sublingual spray was associated with a higher cmax and greater mean fentanyl plasma concentration from 5 minutes through 6 hours after administration ( figure 3).36 in addition , tmax was achieved more rapidly after dosing with fentanyl sublingual spray than with otfc ( median 1.5 hours versus 2.0 hours ; p < 0.05 ) .
five minutes after administration , mean plasma concentration of fentanyl was 19.0% of cmax for fentanyl sublingual spray and below the lower limit of quantification of the assay ( < 0.025 ng / ml ) for otfc ( figure 4 ) .
ten minutes after administration , mean plasma concentration of fentanyl was 53.7% of cmax with fentanyl sublingual spray and 6.1% of cmax with otfc ( figure 4 ) .
the systemic bioavailability , calculated using auc or auc from time zero to last quantifiable concentration ( auclast ) , was 76% and 72% , respectively , after fentanyl sublingual spray administration compared with 51% and 54% , respectively , after otfc administration.36 both fentanyl sublingual spray and otfc were well tolerated , with 15 of 40 subjects ( 37.5% ) reporting adverse events.36 only five of 26 reported adverse events were considered to be probably or possibly related to treatment ( two reports of glossodynia and one report each of dizziness , headache , and dry throat).36 overall , the results of these studies demonstrate linear dose proportionality of fentanyl sublingual spray across the dose range of 100800 g , indicating an increase in the rate of absorption and bioavailability of fentanyl with increasing doses .
fentanyl was detectable in the plasma within 5 minutes after administration , with a sustained elevation in fentanyl plasma concentration starting approximately 30 minutes after administration that was maintained for 6090 minutes.55 alterations in the temperature and acidity of the oral cavity did not affect the pharmacokinetic properties of fentanyl sublingual spray and thus would not alter the effective dosage .
furthermore , fentanyl sublingual spray achieved greater mean plasma concentrations as early as 5 minutes after administration and had greater bioavailability ( 76% versus 51% ) than otfc lozenge .
a randomized , multicenter , double - blind , placebo - controlled clinical trial evaluated the efficacy and tolerability of and patient satisfaction with fentanyl sublingual spray in adult ( aged 18 years ) patients with cancer who were receiving opioids for the treatment of cancer - related or treatment - related pain of moderate intensity or less , were opioid - tolerant , and experienced one to four episodes of btcp daily.29 a 26-day open - label titration phase was followed by a 26-day double - blind treatment phase , with adverse events evaluated during the trial and for an additional 30 days thereafter .
a total of 130 patients ( 69 female , 61 male ) were enrolled in the study , with a mean sd age of 56 12 years .
thirty - two patients withdrew from the study during the titration phase , and three withdrew during the doubleblind phase .
the most common reasons for withdrawal were patient decision ( n = 16 ) , adverse events ( n = 7 ) , and titration failure ( n = 3).29 ninety - eight patients ( 75.4% ) were successfully titrated during the titration phase , and the median effective dose of fentanyl sublingual spray was 800 g.29 the mean standard error ( se ) summed pain intensity difference score at 30 minutes after administration ( spid30 ) , the primary efficacy measure , was 640.3 47.8 in patients receiving fentanyl sublingual spray compared with 399.6 40.8 in patients receiving placebo ( p < 0.0001).29 fentanyl sublingual spray was significantly more effective than placebo in reducing pain intensity , as measured by mean pain intensity difference and spid scores , from 5 minutes through 60 minutes , the last time period assessed ( p < 0.05 for all time points ) ( figure 5 ) . the mean se total pain relief score at 30 minutes ( totpar30 ) , a secondary efficacy measure , was also significantly greater with fentanyl sublingual spray than with placebo ( 78.3 2.1 versus 61.0 2.2 ; p < 0.0001 ) .
pain relief scores and totpar scores were significantly higher with fentanyl sublingual spray than with placebo from 5 minutes through 60 minutes ( p < 0.0001 for all time points).29 patient satisfaction was assessed using the treatment satisfaction questionnaire for medication ( tsqm ) , a validated assessment tool.29,56,57 treatment satisfaction on all four tsqm domains ( effectiveness , side effects , convenience , overall satisfaction ) improved from the start of the titration phase ( baseline ) to the double - blind treatment phase ( figure 6).29 notably , of the 95 patients who completed the double - blind phase , 90 ( 94.7% ) chose to continue in an open - label extension phase of the trial.29 seventy - eight patients ( 60.0% ) reported adverse events during the titration phase , and 47 ( 48.0% ) reported adverse events during the double - blind phase.29 the most common adverse events were nausea ( n = 17 ) , somnolence ( n = 11 ) , dizziness ( n = 10 ) , and vomiting ( n = 10 ) during the titration phase and nausea ( n = 7 ) , hyperhidrosis ( n = 5 ) , and peripheral edema ( n = 5 ) during the doubleblind phase.29 adverse events considered to be probably related to treatment occurred in 33 subjects ( 25.4% ) in the titration phase and two subjects ( 2.0% ) in the doubleblind phase.29 the results of this clinical trial demonstrate the efficacy and tolerability of fentanyl sublingual spray for the treatment of btcp in opioid - tolerant adult patients .
fentanyl sublingual spray showed significant reduction in pain at 5 minutes after administration , with pain relief lasting at least 60 minutes , the last time point evaluated . importantly ,
more than 75% of patients were successfully titrated from their prior btcp medication to fentanyl sublingual spray , and nearly all were satisfied with treatment during the double - blind phase , as indicated by their opting to continue on therapy .
effective management of btcp episodes should be based on current knowledge of how quickly this exacerbation of pain can occur , with the goal of limiting its intensity and duration .
several rapid - onset formulations of fentanyl have demonstrated efficacy and tolerability in the treatment of btcp in opioid - tolerant patients .
fentanyl sublingual spray , the newest of these products , has the characteristics of an ideal btcp treatment it has proven efficacy , rapid onset ( with significant measurable effects within 5 minutes of administration ) , a relatively short duration of action , a tolerable adverse event profile , is easily administered , and may improve overall patient satisfaction with btcp treatment .
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breakthrough cancer pain ( btcp ) is defined as a transient exacerbation of pain that arises in patients with otherwise controlled persistent pain .
btcp typically has a rapid onset and relatively short duration , but it causes a significant amount of physical and psychological distress for patients .
several rapid - onset fentanyl formulations have been introduced in the usa to replace traditional oral opioids for the treatment of btcp : a transmucosal lozenge , a sublingual orally disintegrating tablet , a buccal tablet , a buccal soluble film , a pectin nasal spray and , the newest formulation to enter the market , a sublingual spray .
this article reviews the six rapid - onset formulations of fentanyl approved in the usa for the management of btcp with emphasis on describing the published literature on fentanyl sublingual spray .
the different fentanyl formulations vary in pharmacokinetic properties and ease of use , but all have a rapid onset and a relatively short duration of analgesia .
fentanyl sublingual spray has demonstrated absorption within 5 minutes of administration , with fentanyl plasma concentrations increasing over the first 30 minutes and remaining elevated for 6090 minutes in pharmacokinetic studies in healthy subjects .
fentanyl sublingual spray shows linear dose proportionality , and changes in the temperature or acidity of the oral cavity do not alter its pharmacokinetic properties . in patients with btcp ,
statistically significant pain relief is measurable at 5 minutes after administration of fentanyl sublingual spray , when compared with placebo , with significant pain relief lasting at least 60 minutes after administration .
adverse events are typical of opioid treatment and are considered mild to moderate in intensity . in summary ,
fentanyl sublingual spray provides rapid onset of analgesia and is a tolerable and effective treatment for btcp .
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hepatocellular tumors deriving from monoclonal proliferation of hepatocytes are classically divided in benign hepatocellular adenoma ( hca ) and malignant hepatocellular carcinoma ( hcc ) .
hcas are rare tumors most frequently - developed in women before menoaupose and after a long - term use of oral contraception .
other risk factors such as glycogen storage diseases and androgen intake are also classically associated with hca development .
hca could be complicated frequently by hemorrhage and more rarely by malignant transformation in hcc [ 2 , 3 ] . for a long time , hca was considered as a benign monoclonal proliferation of hepatocytes driven by oestrogen exposition [ 4 , 5 ] .
this new classification linked specific risk factor , clinical history , and histological features to each molecular subgroup of hca [ 69 ] .
in addition , this genotype / phenotype classification has been validated by several groups worldwide demonstrating its robustness and its wide reproductibility in clinical practice [ 1015 ] . in this paper
we aimed to describe how genomic analyses enabled us to identify the different hca molecular subgroups and their specific molecular defects .
in 2002 , we identified hnf1a , as the first driver gene inactivated by mutation in hepatocellular adenomas .
hnf1a codes for the hepatocyte nuclear factor 1 a , a transcription factor involved in hepatocyte differentiation and metabolism control .
previously , in 1996 , yamagata and collaborators had identified germline mutations of hnf1a as the causal alteration of the specific diabetes named mody 3 for maturity onset diabetes of the young type 3 . in mody3 patients ,
one allele of hnf1a is inactivated in all cells of the organism showing the pivotal role of hnf1a partial inactivation in glucose homeostasis dysregulation . in hca tumor cells ,
we described complete hnf1a inactivation by mutation of both alleles in 35% to 45% of the cases ( table 1 ) . in most of the cases , both mutations occurred in tumor cells and were of somatic origin .
however , in 10% of hca inactivated for hnf1a , one mutation was germline , and consequently , we identified mody3 patients developing hca [ 19 , 20 ] .
these patients could also have adenomatosis , a rare condition defined of more than 10 adenomas in the liver [ 21 , 22 ] . in this line
, these results have revealed for the first time hnf1a as a tumor suppressor gene in addition to its role in metabolism regulation .
we further showed that hnf1a inactivation induces in hepatocyte dramatic alteration in metabolic pathways and epithelial - mesenchymal transition that can participate to tumor development [ 23 , 24 ] .
in addition the of environmental factor and germline hnf1a - mutations , others genetic features could predispose to the occurrence of hnf1a mutated adenomas . in this line
, we identified heterozygous germline mutations of cyp1b1 in a subset of patients with h - hca .
all patients with these mutations have a decrease enzymatic activity of the cytochrome p450 cyp1b1 .
because cyp1b1 is involved in the metabolism of estrogens , it suggests that development of h - hca could be promoted by a defect in this pathway in relation with exposure to oral contraception . at the pathological level ,
we further showed that the homogeneous accumulation of lipids in tumor hepatocytes was related to an increase of fatty acid synthesis induced by hnf1a inactivation .
h - hca can be easily diagnosed using pathological examination because these adenomas are characterized by a constant and specific lack of fabp1 expression in the tumor hepatocytes [ 12 , 27 ] .
the wnt / catenin pathway is a pivotal oncogenic pathway involved in solid and haematopoietic tumors .
ctnnb1 , the gene coding for -catenin , is activated by somatic mutation in a large number of different tumor types like medulloblastoma or breast cancer .
moreover , it is the most frequently mutated oncogene in hepatocellular carcinoma ( from 20 to 40% of the cases ) .
ctnnb1-activating mutations target few serine and threonine amino acids in -catenin , residues that are physiologically phosphorylated by the apc / gsk3/axin complex inducing degradation of -catenin by the proteasome .
ctnnb1 mutations impaired the phosphorylation by the apc / gsk3b / axin complex and led to the translocation of -catenin into the nucleus [ 28 , 30 ] . in this condition ,
mutations activating -catenin are described in 10 to 15% of hca ( table 1 ) [ 6 , 33 ] .
furthermore , -hca are often characterized by pseudoglandular formation , cell atypia , and cholestasis at the pathological level . using immunochemistry
, we showed that -hcas are characterized by a strong cytoplasmic expression of glutamine synthase and nuclear expression of -catenin in tumor hepatocytes .
however , despite a good specificity , these markers have a lack of sensitivity for the diagnosis of -hcas and hca should be screened for ctnnb1 mutations [ 12 , 27 , 35 , 36 ] , when glutamine synthase and -catenin markers are not informative .
importantly , we showed that hca with activating mutations of -catenin have a high risk of malignant transformation in hcc [ 6 , 36 , 37 ] .
moreover , distinguishing hca from well - differentiated hcc developed on normal liver could be challenging .
consequently , all hca harboring a mutation of -catenin should be surgically resected in order to avoid the risk of malignant transformation . in this context , the performance of immunohistochemical marker developed to discriminate high - grade dysplastic nodules from very early hcc ( like glutamine synthase , glypican 3 or hsp70 ) on cirrhosis remains poorly explored to differentiate hca from very well differenciated hcc on normal liver and should be used with caution .
a recent study has shown that the combination of glypican 3 and hsp70 has a good specificity ( 100% ) but an insufficient sensitivity ( 43% ) to distinguish hca from well - differenciated hcc [ 38 , 39 ] .
however , the small numbers of tumors analyzed preclude the generalization of these markers in clinical practice and required additional studies .
another concept is that some hepatocellular tumors will remain borderline tumors between hca and hcc despite histological analysis by an expert pathologist . in this grey zone ,
screening for ctnnb1 mutation should be mandatory to detect hca with a potent risk of malignant transformation and borderline lesion between hca and hcc that should be resected . in the physiological point of view
, the most important breakthrough has been performed by the identification of the so - called inflammatory hca and dissection of il6/jak / stat pathway [ 40 , 41 ] .
ihcas are characterized by the activation of jak / stat and interferon i and ii pathway [ 40 , 42 ] .
this subtype of adenomas exhibited strong pathological hallmark : inflammatory infiltrates , dystrophic arteries , and sinusoidal dilatation .
inflammatory hca exhibited a cytoplasmic overexpression of saa and crp , two proteins of the acute phase of inflammation , in the tumor hepatocytes ( table 1 ) [ 12 , 15 ] .
peripheral inflammatory syndrome can regress after resection of the tumor , and it could be considered as a paraneoplastic syndrome
ihca occurred more frequently in patients with high alcohol consumption and obesity , two conditions associated with chronic cytokine production [ 6 , 46 ] .
we also described an ihca transformed in hcc mutated for both gp130 ( il6st ) and -catenin ( ctnnb1 ) and developed on the background of castleman disease . in this rare disease
it underlined again the possible role of chronic cytokine production ( obesity , high alcohol consumption , and castleman disease ) as a predisposing factor to inflammatory hca occurrence .
recently , we deciphered the molecular alterations leading to the activation of inflammatory pathway in the tumor hepatocytes .
we described the oncogenes that explain the hepatocytes proliferation and the inflammatory phenotype ( oncogene - induced inflammation ) .
led to the constitutive activation of the jak / stat pathway in the absence of the il6 ligand [ 42 , 48 ] .
interestingly , these hcc are developed in normal liver and could be derived from hca .
these mutations explained the uncontrolled activation of jak / stat pathway and the observed phenotype in 6% of the ihca .
gnas gene coded for alpha subunit of gs protein and is a well - known oncogene in pituitary and thyroid adenomas .
mutations of gnas gene impaired the gtpase activity of alpha subunit and led to its permanent activation by an unregulated binding of gtp .
as a consequence , cyclic amp accumulates in the cells . in adenoma , we described a crosstalk between cyclic amp and jak / stat pathway that explained the mild inflammatory phenotype in gnas - mutated hca . in this line
mccune - albright syndrome is an orphan disease due to somatic postzygotic mosaic gnas mutation .
this genetic disorder is characterized by pituitary and thyroid adenomas , fibrous bone dysplasia , and
finally , 10% of hcas have no known genetic alterations or specific histological phenotype ( table 1 ) .
in the canonical point of view , malignant hepatocellular tumors ( hcc ) arise on chronic liver disease , mainly cirrhosis or chronic hbv infection , whereas hepatocellular benign tumors are developed on normal liver .
first , hcc could develop on normal liver , and predisposing genetic factor and genetic drivers involved in tumor initiation remain poorly described .
a simple clinical observation supports the fact that hca is not a stochastic and isolated tumorgenic event : 40% of patients with hca have multiple hca in the liver suggesting an individual predisposition to develop this rare disease . also , several genetic disease and environmental factors favored hepatocytes proliferation and benign tumors initiation . moreover , since several decades , the major hca risk factors , oestrogen and androgen consumptions , have been identified as classical genotoxins [ 5456 ] .
association between estrogen exposure and hca occurrence was first described in the seventies when oral contraception was of widespread use in western countries [ 4 , 55 , 57 ] .
nevertheless , estrogen exposure due to oral contraception is frequent , but hca occurrence is rare ( around 3/100,000 ) .
more recently , the use of a third generation of oral contraceptive with lower dose of estrogen could have modified the epidemiology of hca .
in addition , the incidence of hca in eastern countries , where oral contraception is not frequently used , remains to be evaluated .
differences in incidence and molecular subtypes of hca between eastern and western countries could help to understand the role of estrogen exposure and other risk factors like obesity and alcohol consumption in the development of benign liver tumors [ 46 , 59 ] .
when we analyzed the spectrum of mutations of hnf1a in hca , we also showed that hnf1a somatic mutations were frequently caused by g to t transversion suggesting a genotoxic exposure at the origin of the mutations .
causes of this genotoxic signature remain to be elucidated , and the role of oestrogen exposition in this genotoxic damage should be further analyzed .
a hypothesis is that hca development could be favored by both a genetic predisposition in combination with an exposure to different genotoxic agents . in this line ,
predisposing genetic factors like hnf1a germline mutation related to mody3 diabetes and gnas mosaic somatic mutations related to mccune albright disease are strong risk factors of adenomas occurrence [ 19 , 50 ] .
moreover , patients with glycogenosis type ia defined by germline inactivating mutation of glucose-6 phosphatase have a huge risk to develop multiple hca during their followup [ 6163 ] .
all these data underlined that hepatocellular benign tumors are often developed on a predisposing abnormal liver background .
this hypothesis could be called benign tumorigenic field effect as a mirror of the carcinogenic field effect
the benign tumorigenic field effect is a conjunction between genetic ( hnf1a germline mutation , gnas mosaic postzygotic mutation , and others unknown modifier genes ) and environmental factors ( oestrogen and androgen expositions ) [ 20 , 56 , 57 , 60 , 64 ] .
in addition , we showed a role of cyp1b1 , a cytochrome p450 unit involved in detoxification of catechol estrogens , in the occurrence of hca .
we identified a germline cyp1b1-inactivating mutation in 12.5% of patients developing hnf1a - inactivated hca .
moreover , when analyzing the spectrum of somatic mutations in hnf1a , we identified a genotoxic signature typical of molecule inducing adduct to dna at guanine .
thus , a combined genetic predisposition and genotoxic effect could explain the frequent occurrence of multiple hca in the same patient , and despite that the surrounding nontumor liver appears to be mainly histologically ( sub)normal , the liver is tumorigenic .
a long path has been walked in the area of hepatocellular benign tumors since edmonson described the association between hca and oral contraception .
now , the discovery of genetic drivers of hca has refined our knowledge of the life history of hca from risk factors and clinical features to the risk of malignant transformation
thus , in addition to activating mutations of -catenin , other genetic alterations leading to full malignant transformation have to be deciphered
. finally , several driver genes of benign tumorigenesis are still unknown , especially in the group of inflammatory hca without known driver mutations and unclassified hca .
ultimately , these genetic alterations will constitute therapeutic target for biotherapy that will be used in unresectable hca or in other malignancies harboring the same genetic events .
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hepatocellular adenomas ( hcas ) are benign tumors developed in normal liver most frequently in women before menopause .
hcas lead to diagnostic pitfalls and several difficulties to assess the risk of malignant transformation in these young patients .
recent advances in basic knowledge have revealed a molecular classification related to risk factors , pathological features , and risk of transformation in hepatocellular carcinoma .
three major molecular pathways have been identified altered in specific hca subgroups that are defined by either ( 1 ) inactivation of hepatocyte nuclear factor 1a ( hnf1a ) transcription factor , ( 2 ) activation of the wnt/-catenin by ctnnb1 mutations , or ( 3 ) activation of the il6/stat3 pathway by somatic mutation of il6st , gnas , or stat3 . here , we will review the different molecular classes of hca .
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atrial fibrillation ( af ) is the most common sustained arrhythmia in adults , and a high percentage of these patients present with paroxysmal af .
flecainide , a class ic anti - arrhythmic drug is the most effective drug to maintain sinus rhythm in this subgroup of population though the drug has potential pro - arrhythmic effects , including qt interval prolongation and ventricular tachycardia like torsade de pointes ( tdp ) .
furthermore , the h1 receptor antagonists are the most commonly prescribed drugs for the symptomatic treatment of pruritus . despite having low number of adverse effects , the h1 receptor antagonists have cardiotoxic effects and they have been associated with qt interval prolongation and tdp .
the objective of this article is to describe the synergistic effect of flecainide and h1 receptor antagonists in prolonging qt interval and inducing ventricular arrhythmias .
a 65-year - old female patient was admitted to our institution with palpitations , progressive dyspnea accompanied by orthopnea and paroxysmal nocturnal dyspnea . the patient had a history of hypertension , dyslipidemia , diabetes mellitus , asthma , and paroxysmal af , and
electrocardiogram demonstrated af at 137 beats per minute , narrow qrs , and absence of repolarization abnormalities .
the lung auscultation showed crepitant rales in both lower lung lobes , and the chest radiography showed signs of vascular redistribution .
transthoracic echocardiography ( tte ) showed left and right ventricles of normal size and contractility , mild dilatation of the left atrium ( 44 mm ) , and no other relevant abnormalities . by treatment with parenteral anticoagulation and diuretics ,
the patient 's condition was stabilized ; congestive symptoms were controlled , spontaneously returning to sinus rhythm .
after stabilization , bisoprolol ( 2.5 mg/12 h ) and flecainide ( 50 mg/12 h ) were initiated in order to maintain sinus rhythm . during hospitalization , the patient developed a pruriginous rash on lower extremities [ figure 1 ] that required treatment with oral h1 receptor antagonists loratadine ( 10 mg/24 h ) and hydroxyzine ( 25 mg/24 h ) , prolonging the hospital stay of the patient .
rash in lower extremities on the fourth day of concomitant therapy with flecainide and h1 receptor antagonists , the patient had cardiac arrest secondary to tdp because of prolongation of qt interval that degenerated into ventricular fibrillation requiring cardiac defibrillation for six times , orotracheal intubation , and advanced cardiopulmonary resuscitation support , and the pulse recovered after 15 min [ figures 24 ] .
cranial computerized tomography scan revealed no intracranial bleeding or ischemic stroke and coronary angiography showed no significant coronary lesions .
the patient presented a good recovery , being extubated 72 h after the cardiac arrest without neurologic sequelae .
the patient presented again with af and we opted for rate control strategy [ figure 5 ] , initiating treatment with beta - blockers ( bisoprolol 2.5 mg/12 h ) for obtaining adequate heart rate .
electrocardiogram during cardiac arrest : torsade de pointes electrocardiogram during cardiac arrest : ventricular fibrillation electrocardiogram immediately after cardiac arrest : sinus rhythm with qt interval prolongation electrocardiogram after patient stabilization : atrial fibrillation with absence of qt interval prolongation
tdp is a polymorphic ventricular tachycardia syndrome that occurs in the context of long prolongation of the qt interval .
this occurs because of the slowing of repolarization due to blockade of the delayed rectifier potassium current ( ikr ) producing prolongation of action potential .
this slowing of repolarization induces bradycardia and qt prolongation , predisposing to tdp . in accordance with clinical guidelines , flecainide is the preferred anti - arrhythmic drug in order to maintain sinus rhythm in patients with no structural heart disease because of its high rate of preserving sinus rhythm and low rate of adverse events in comparison with alternative drugs .
nevertheless , flecainide , as an anti - arrhythmic drug presents with pro - arrhythmic effects previously identified in cast trial and experimental models of ischemia . an increase in the rate of sudden death in patients taking flecainide was reported in the cast trial , principally related to qt interval prolongation and tdp .
the h1 antagonist receptors have sedative and anti - cholinergic effects , and they even have potential cardiotoxic effects , including arrhythmic complications .
however , the h1 receptor antagonists can cause malignant ventricular arrhythmias like tdp due to qt interval prolongation .
the interaction between h1 receptor antagonists and flecainide presents an exceptional occurrence and it has been very rarely reported .
although the management of these interactions is not reflected in clinical practice guidelines , the guidelines recommend that patients receiving flecainide or other class ic anti - arrhythmic drugs must undergo strict control of qt interval .
in addition , patients receiving h1 receptor antagonists should stop using class ic anti - arrhythmic drugs because of pruriginous rashes and be treated with alternative anti - arrhythmic drugs with neutral effects on the qt interval .
furthermore , in rare cases where patients necessarily require continuing the treatment with flecainide , h1 receptor antagonists could be replaced by corticosteroids .
the concomitant administration of flecainide and h1 receptor antagonists seems to have a synergistic effect in qt interval prolongation and subsequent tdp .
the concurrent administration of h1 receptor antagonists to patients receiving class ic anti - arrhythmic drugs should be avoided in order to reduce arrhythmic risk in these patients .
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a high percentage of patients having atrial fibrillation ( af ) presents with paroxysmal af .
flecainide , the prototypic class ic anti - arrhythmic drug is the most effective drug to maintain sinus rhythm in this subgroup of patients , though the drug has potential pro - arrhythmic effects .
furthermore , the h1 receptor antagonists are the most commonly prescribed drugs for the symptomatic treatment of pruritus . despite having low number of adverse effects ,
the h1 receptor antagonists have cardiotoxic effects .
flecainide and h1 receptor antagonists present arrhythmic complications including qt interval prolongation and torsade de pointes ( tdp ) .
the case presented here is a 65-year - old female who was diagnosed of atrial fibrillation and presented with rashes in lower extremities .
the patient was treated using flecainide and h1 receptor antagonists ( loratadine and hydroxyzine ) that prolonged qt interval and induced tdp .
the concomitant administration of flecainide and h1 receptor antagonists seems to have a synergistic effect in qt interval prolongation and subsequent tdp .
the concurrent administration of h1 receptor antagonists to patients receiving class ic anti - arrhythmic drugs should be avoided in order to reduce arrhythmic risk in this population .
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born and raised in italy , i worked as an undergraduate assistant in milan , studying dna replication in budding yeast .
the lab was small and tight - knit , and the work of undergraduate students was valued and rewarded . energized by this experience , i decided to move edinburgh to pursue my dream : being a full - time researcher .
i did nt know exactly what i wanted to do , everything sounded exciting , but understanding the organization and stability of genomes particularly intrigued me .
an opportunity arose to work in a basic biology laboratory that studied chromatin , so , in 1998 , i joined the group of robin allshire at the medical research council human genetics unit in edinburgh , scotland , for my doctoral work .
i ll never forget the time i was walking to the lab from my apartment in the lovely comely bank neighborhood on yet another rainy day . out of the blue
, i heard a strange crashing noise coming from the road , the sound of something large and soft hitting the cobblestones .
a swan had attempted to land on the slick , shiny , wet surface of the road , mistaking it for water .
my apartment was near a park with a swan pond , and this was the second time i had to call the local police and wait for them to come and rescue an injured bird , warning drivers to slow down and maneuver carefully around it .
the police were accustomed to getting these calls from residents in the area , but when i called the lab to let people know i was running late , yet again because of a fallen swan , my lab mates would nt stop laughing .
it was a very exciting time for chromatin biology , as the importance of novel histone modifications was just beginning to emerge .
my project focused on understanding the chromatin requirements for the silencing of pericentric heterochromatin in the fission yeast . because of previous work in budding yeast , we had predicted that silencing mechanisms in other organisms would be mediated by the hypoacetylation of the histone h4 n - terminus .
i had just embarked on mutating individual histone h4 lysines when thomas jenuwein s lab discovered that mouse suvar39h had histone h3 lysine 9 methyltransferase activity ( rea et al .
, 2000 ) . knowing that suvar39h proteins are required for silencing in the fission yeast saccharomyces pombe and flies , i quickly shifted my attention to histone h3 and showed that mutating lysine 9 completely abolished silencing , providing direct evidence that this residue is the key target of this family of methyltransferases and highlighting the remarkable resemblance between the heterochromatin of s. pombe and that of more complex organisms ( mellone et al . , 2003 ) .
seeing how much we understand today about heterochromatin and silencing , i cherish having been part of the early work and marvel at how much progress the field has made since then . working with
robin allshire taught me to be critical of my data , to connect concepts in a deep and mechanistic manner , and to think about my experiments in the context of the bigger picture .
learning to write about my work in a different language felt overwhelming , but he painstakingly edited my thesis and papers , showing me the importance of editing and revisions . working on a basic biology problem was exciting , and the broad implications of the potential findings motivated me .
i also realized how much we did nt know about the basic principles of chromatin dynamics and their impact on chromosome inheritance . toward the end of my phd work ,
i became interested in the centromere - specific histone h3 , called cenp - a , which had been hypothesized to epigenetically
i performed experiments that manipulated both canonical histone gene dosage and centromere size , and we determined that cenp - a chromatin was plastic , that is , it had the ability to spread and shrink ( castillo , mellone , et al . , 2007 ) .
my exposure to fluorescence microscopy in the allshire lab made me realize how much i loved being able to visualize the structures that fascinated me most : chromosomes and mitotic spindles .
the idea that centromeres might be defined epigenetically through the presence of cenp - a , rather than by specific dna sequences ( karpen and allshire , 1997 ) , got me completely hooked on centromeres .
i became very interested , perhaps even obsessed , with understanding how cenp - a becomes specifically localized to centromeres and maintained there across cell generations . for my postdoc
, i decided that i wanted to study this process using a genetic system that would also afford beautiful cytology . in 2003 ,
i joined the laboratory of gary karpen , which uses drosophila to study centromeres and heterochromatin , at the lawrence berkeley national laboratory and the university of california , berkeley .
the novel high - throughput rna interference ( rnai ) technology that was being developed by norbert perrimon at the drosophila rnai screening center at harvard seemed like an ideal entry point to identify the largely unexplored regulators of centromere chromatin assembly using a cell - imaging screen .
this collaborative genome - wide rnai screen ( which involved sylvia erhardt along with myself in the karpen lab and craig betts in aaron straight s lab at stanford ) identified critical factors of centromere function at a time when very little was known about the cenp - a deposition pathway .
these factors included centromere - associated proteins and cell cycle regulators ( erhardt , mellone , betts , et al .
, 2008 ) , which i was able to later pursue in my own research group .
he gave me freedom and responsibility ; supported me unconditionally through two maternity leaves ; and worked closely with me on developing grants , papers , and research seminars .
his relentless encouragement and faith in my abilities were instrumental in my decision to seek a faculty position .
in 2009 , my husband , two young daughters , and i uprooted ourselves to move to connecticut , the land of broccoli fields , as my good friend from italy calls it , due to the appearance of its tree - covered landscape from an airplane .
the hilarious photo on my university of connecticut i d , taken on my first day as a faculty member , captured my initial emotions : is this really happening ?
what if i ca nt do this ? the initial few months were challenging ; the gorgeous old university campus was a maze of brick buildings and trees , and i kept getting lost . at uconn
i was once again surrounded by large birds , but instead of swans , it was the plentiful canada geese that pass through campus every year in the thousands .
i missed lunches with my fellow postdoc friends from berkeley , needing a good laugh or to commiserate about difficult experiments .
fortunately , it was nt long before i mastered my new campus s geography and made new friends , both at uconn and outside work .
while it seemed i had less time to socialize , i soon found colleagues and friends who supported me and laughed with me .
when i started my lab , our understanding of the mechanisms of centromeric chromatin assembly was very limited .
as a first step in my independent research program , i followed up on experiments started in the karpen lab and elucidated the functional cell cycle dynamics of cenp - a and its effectors from our rnai screen cyclin a , cal1 , and cenp - c ( mellone et al . , 2011 ) .
an inherent challenge of dissecting the centromere assembly pathway is that a mutation or knockdown of centromere components often results in centromere disruption and subsequent cellular lethality . to overcome this
, we used genomic engineering in drosophila s2 cells , implementing an inducible tethering system ( laci / laco ) to dissect the stepwise formation of cenp - a chromatin without compromising viability .
this was critical in providing the first experimental evidence that cal1 is a cenp - a assembly factor required for centromere establishment ( chen , dechassa , et al . , 2014 ) .
our studies showing that centromeric chromatin can be established de novo in flies are consistent with the occurrence of neocentromeres ( centromeres that form at new locations ) in humans .
but many fundamental questions remain : how are canonical nucleosomes reorganized to make room for cenp - a during its deposition ? are cells able to detect the presence of a spurious centromere and to inactivate it ?
recent graduate students in my lab have taken on some of these challenges , showing that cenp - a deposition is directly coupled to and requires transcription ( chen et al . , 2015 ) and that cenp - a evolves in concert with its chaperone ( rosin and mellone , 2016 ) , but much exciting work remains to be done not only to inform mechanistic models for centromere specification but also to explore the genomic landscape and evolution of these fascinating chromosomal structures .
as with most new science faculty , i have experienced the ups and downs of staffing a functional laboratory , obtaining funding , publishing , and teaching effectively .
in fact , many times i felt like those swans in edinburgh confused , disappointed , and alone but just like those swans , who ultimately made it safely back to their pond ( in one case after some rehabilitation ) i have always had somebody to turn to for help and support when things did nt go as planned ( by the way , they rarely do ) . despite the highs and lows , doing research for a living remains a highly rewarding and creative career that offers both great flexibility ( especially helpful when also raising a family ) and the fortunate opportunity to encounter new ideas , meet new people , and travel to new places along the way .
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it is an honor to become a part of the talented group of cell biologists who have received this award before me . while running a research group certainly has its ups and downs , i love being a faculty member and am continuously excited by the prospect of scientific discoveries yet to be made .
i have benefited from the support of many people over the years and hope to be able to do the same for others through my mentoring and teaching .
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superficial spreading melanoma ( ssm ) is the most common one in all types of melanoma .
we report a patient having acral ssm , which developed in a long - standing melanocytic nevus during pregnancy .
a 37-year - old housewife with fitzpatrick skin phototype-3 presented with a brownish - black patch over the hypothenar area of left palm , where she had a small acquired melanocytic nevus since childhood . for the last 4 months ,
there were three moles on her trunk , two on upper back , and one on waist .
there was no history of morphological changes in theses moles and in acral nevus during previous pregnancy .
no history of anorexia , weight loss , cough , hemoptysis , seizures , and bone pain was present .
dermatologic examination revealed a brownish - black plaque over the left palm about 5 cm 3 cm in size , with an irregular blurred margin over medial aspect [ figure 1a ] .
dermoscopy ( hanse : hvs - cm500pc ) of the lesion revealed shades of brown , black , and gray color with parallel ridge pattern , [ figure 1b and c ] which supported the clinical diagnosis of malignant melanoma .
( c ) dermoscopy of lesion showing different shades of color left epitrochlear and axillary lymph nodes were not palpable .
ultrasonography of whole abdomen was done , which was normal except the incidental finding of an intrauterine normal live fetus of 5 months duration as the patient did not know about her pregnancy .
x - ray chest and computerized tomography scan abdomen were not done to avoid risk to the fetus .
on the basis of clinical and dermoscopic findings , a differential diagnosis of atypical melanocytic nevus and acral lentiginous melanoma ( alm ) were made .
wide local excision with 2 cm safe margin from the lesion 's border and down to palmer fascia till muscle exposure [ figure 2a and b ] was done under left brachial block .
the defect was repaired with split - thickness skin graft taken from left thigh [ figure 2c and d ] .
histopathology of the lesion revealed hyperkeratosis , acanthosis , and pagetoid extension of atypical melanocytes up to the stratum corneum [ figure 3a ] .
papillary dermis was packed with an atypical melanocytic cell with prominent melanin pigments [ figure 3b ] .
( a and b ) wide local excision of melanoma down to palmer fascia exposing the muscles .
( c and d ) wound repair with split thickness skin grafting - status after 1- and 2-week , respectively ( a ) pagetoid look of superficial spreading melanoma ( h and e , 10 view ) .
( b ) atypical melanocytes with prominent pigment ( h and e , 40 view ) .
( c ) immunohistochemistry showing hmb-45 positive cell this case was unique in terms of development of ssm on existing melanocytic nevus on the acral site .
the description of melanoma as a disease entity is attributed to laennec , who published an article on the melanoses in 1812 .
world over the incidence of malignant melanoma is on the rise , but it is still much lower in asians ( 0.20.5/100,000 population per annum ) as compared to the white population ( 13.540.0/100,000 population per annum ) .
risk factors for melanoma include white race , familial history , congenital mole , immunosuppression , genetic coding , photosensitivity , increased sun exposure , and increased number of nevocytic nevi .
there are distinct patterns of clinical and histopathological appearance that led to the distinction of histogenetic types of melanoma put forward in the first classification system proposed by mcgovern et al .
the classification employed histopathology , anatomical site , and degree of sun damage to distinguish four types of melanoma - ssm , lentigo maligna melanoma ( lmm ) , alm , and nodular melanoma . in ssm
, radial growth clinically leads to a well - circumscribed polycyclic patch with variegating shades of brown , gray , and black , in which a subsequent nodule develops .
histopathologically , the telltale sign of ssm is the presence of large melanocytes arranged as small aggregates or nests , that display marked upward scatter within the epidermis , a pattern referred to as pagetoid because it resembles the intraepidermal spread of breast cancer in the epidermis of nipple .
the most common sites for ssm are the female leg and the male back followed by the head and neck , and then the anterior trunk .
by contrast , lmm and alm display a lentiginous growth pattern , in which the melanocytes are arranged as solitary units along the basilar epidermis without pagetoid growth .
the association with melanocytic nevi , either directly adjacent to melanoma or elsewhere on the body is a characteristic feature of nonchronic sun damage melanoma .
the nevi arise primarily in the first two decades of life , with the melanomas starting to appear later , consistent with a model in which they require additional genetic alteration to occur in the form of braf mutation .
the well - known abcde acronym ( a - asymmetry , b - border , c - color , d - diameter , and e - elevation / evolving ) for melanoma detection was developed in 1985 and continues to be a useful tool for the physicians .
dermoscopy increases not only sensitivity but also specificity for the diagnosis of skin cancer in general and melanoma in particular .
dermoscopy of acral melanoma has characteristic findings such as parallel ridge pattern ( pigment localized to the ridges ) and multiple shades of brown , black , red , white , gray , and blue colors which differentiate it from desmoplastic nevus and other melanocytic nevi .
the gold standard for diagnosing melanoma is based on a histopathologic evaluation of the biopsy specimen .
all patients with suspected malignant melanoma should have an excision biopsy of the lesion carried out with a margin of 12 mm of clinically normal skin .
they will confirm the melanocytic nature of the lesion , but can not differentiate between benign and malignant melanocytic lesions .
s100 , gp100/hmb-45 , tyrosinase , mart-1/melan - a , and hmw_maa are melanocyte differentiation markers .
the standard therapy is wide local excision to prevent the local recurrence due to persistent disease by complete excision with histopathologically confirmed tumor - free margins . at present , if the diagnosis is of a level 1 or in situ melanoma , a margin of only 1 cm of surrounding normal skin is considered adequate .
patients with thicker tumors are more likely to develop lymph node recurrence if a 1-cm margin is used , and , therefore , the recommended excision margin is 23 cm were anatomically possible .
the histological state of the sentinel lymph node ( sln ) is the most relevant prognostic factor for the overall survival of melanoma patients .
the significance of this prognostic factor was emphasized by the inclusion of sln excision into the present american joint committee on cancer classification scheme .
concern about the effect of pregnancy on outcome from melanoma began in 1951 when pack and scharnagel reviewed 32 cases of melanoma diagnosed during pregnancy reporting extremely poor prognosis due to the rapid development of metastases .
a large study based on a swedish cancer registry of 185 women diagnosed with melanoma during pregnancy and 5348 women of the same childbearing age diagnosed with melanoma while not pregnant , showed no evidence of an adverse effect of pregnancy on survival , before or after diagnosis .
furthermore , placental and fetal metastasis of melanoma is a well - described phenomenon so that examination of the placenta at delivery in patients with poor prognosis disease is sensible .
acral , superficial spreading melanoma ( ssm ) is very rare in patients of indian origin .
the newer drugs for the treatment of melanoma are ipilimumab , trametinib , and vemurafenib . wide local excision varies from 1 to 3 cm based on breslow thickness , and sln assessment is the standard way of managing ssm .
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we are reporting a case of superficial spreading melanoma ( ssm ) on left palm of a 37-year - old pregnant housewife .
she had a small acquired melanocytic nevus on her left palm since childhood , which changed its consistency and color in the last 4 months .
dermoscopy of the lesion indicated malignant changes .
the lesion was managed surgically using split - thickness skin graft .
the histopathology report was suggestive of ssm with positive hmb-45 cells .
ssm is very rare on the acral site , and it is very difficult to differentiate it from acral lentiginous melanoma .
the rarity of the site ( acral nonchronic sun damage ) with evolution during pregnancy and importance of management approach are reasons for publishing this case .
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we describe a novel form of homeostatic synaptic plasticity in multisensory neurons in the optic tectum of xenopus laevis tadpoles .
individual tectal neurons are known to receive converging inputs from multiple sensory modalities .
we show that long - term alterations in either visual or mechanosensory activity in vivo result in homeostatic changes specific to each sensory modality .
in contrast to typical forms of homeostatic synaptic plasticity , such as synaptic scaling , we show that this type of plasticity occurs in a pathway - specific manner more reminiscent of hebbian - type plasticity .
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multiple sclerosis ( ms ) is a neurodegenerative disease characterized by chronic inflammation , demyelination , and scarring of the central nervous system .
symptoms include weakness , fatigue , sensory loss , vertigo , lack of coordination , impotence or sexual dysfunction , urinary incontinence , optic atrophy , dysarthria , and mental problems .
the average age at onset of ms is 30 years , and the disease runs its course for the remainder of the patient s life frequently causing disability of varying degrees .
the prevalence of ms varies with both geography and ethnic background with women twice as likely to be afflicted as men .
the disease substantially interferes with daily activities and family , social and working life , disturbs emotional well - being , and reduces quality of life ( qol ) .
other symptoms related to ms are weakness , paresthesia , visual changes , spasticity , cognitive dysfunction , ataxia , pain and fatigue .
fatigue remains one of the most common and debilitating symptoms in ms and is quoted as one of the single most disabling symptoms .
in contrast , 96% of ms patients experience fatigue , 88% of whom report fatigue as a moderate to high problem .
suggested etiologies of fatigue in ms have included an increase in nervous system conduction time , alterations in the function of monoamine transmitters , deconditioning , reduction in melatonin levels , increase of cytokines or immune system dysregulation , neuromuscular transmission defects , psychological factors , increased energy demands for muscle activation due to spasticity , central brain neuronal injury , damage to the brainstem reticular activating system , respiratory muscle weakness and sleep disturbances . in response to the presence of these symptoms , the study of self - efficacy is very crucial .
social cognitive theory yielded the concept of self - efficacy as the perceived capability of a person to perform a specific action required to achieve a concrete goal .
the concept is competence - based , prospective and action - related . in general , self - efficacy is conceptualized task - specific , for example self - efficacy in managing diabetes self - care tasks like blood sugar testing , keeping to diet and doing physical exercises regularly or pain self - efficacy in ms patients .
patients with higher self - efficacy levels are more likely to start or maintain a specific task even in face of existing barriers .
several self - management programs successfully targeted self - efficacy resulting in improved health outcomes .
the aim of the present study protocol is to examine levels of pain self - efficacy and fatigue in a sample of greek patients suffering from multiple sclerosis .
test - retest reliability of two questionnaires used [ i ) the fatigue assessment scale ( fas ) , ii ) the pain self efficacy questionnaire ( pseq ) ] will be assessed with the same patients rating their situation in a 10 days interval from the first examination in order to examine consistency over time .
forty patients diagnosed with ms will be recruited from hospitals located within the broader area of thessaloniki .
the inclusion criteria are : i ) > 18 years of age ; ii ) ability of communication in greek ; iii ) diagnosed with ms ; iv ) satisfying level of cooperation and perceived ability ; v ) no history of primary psychiatric disease that may interfere with conduct of study ; vi ) clinically stable with no evidence of chronic or acute infections , inflammatory disorders , malignancy .
all subjects will be informed of their rights to refuse or discontinue participation in the study according to the ethical standards of the helsinki declaration .
ethical permission for the study will be obtained from the scientific committees of the participating hospitals .
although these two aspects of fatigue are represented in the questionnaire , the fas has shown to be unidimensional in various populations , as well as in sarcoidosis patients .
the response scale is a 5-point scale ( 1 , never to 5 , always ) .
the cronbach alpha of the fas in a random sample of the dutch working population was 0.90 and in a sarcoidosis population 0.89 .
in addition , the fas had the highest factor loading in a factor analysis incorporating five fatigue questionnaires .
correlations between the fas and the beck depression inventory were 0.59 and between the fas and the ces - d 0.65 .
the pseq is a 10-item questionnaire developed to assess the confidence people with ongoing pain have in performing activities while in pain .
it covers a range of functions , including household chores , socializing , work , as well as coping with pain without medication . the expanded disability status scale ( edss )
is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability over time .
it is widely used in clinical trials and in the assessment of people with ms .
the scale was developed by a neurologist called john kurtzke in 1983 as an advance from his previous 10 step disability status scale ( dss ) .
the edss scale ranges from 0 to 10 in 0.5 unit increments that represent higher levels of disability .
edss steps 1.0 to 4.5 refer to people with ms who are able to walk without any aid and is based on measures of impairment in eight functional systems ( fs ) : i ) pyramidal - weakness or difficulty moving limbs ; ii ) cerebellar - ataxia , loss of coordination or tremor ; iii ) brainstem - problems with speech , swallowing and nystagmus ;
iv ) sensory - numbness or loss of sensations ; v ) bowel and bladder function ; vi ) visual function ; vii ) cerebral ( or mental ) functions ; viii ) other .
each functional system is scored on a scale of 0 ( no disability ) to 5 or 6 ( more severe disability ) .
demographic and clinical characteristics of all patients will be collected as baseline information at the beginning of the study .
a p - value of 0.05 or less will be considered to indicate statistical significance .
all analyses will be performed with the statistical package for the social sciences ( spss 13.0 for windows ) .
forty patients diagnosed with ms will be recruited from hospitals located within the broader area of thessaloniki .
ii ) ability of communication in greek ; iii ) diagnosed with ms ; iv ) satisfying level of cooperation and perceived ability ; v ) no history of primary psychiatric disease that may interfere with conduct of study ; vi ) clinically stable with no evidence of chronic or acute infections , inflammatory disorders , malignancy .
all subjects will be informed of their rights to refuse or discontinue participation in the study according to the ethical standards of the helsinki declaration .
ethical permission for the study will be obtained from the scientific committees of the participating hospitals .
although these two aspects of fatigue are represented in the questionnaire , the fas has shown to be unidimensional in various populations , as well as in sarcoidosis patients .
the response scale is a 5-point scale ( 1 , never to 5 , always ) .
the cronbach alpha of the fas in a random sample of the dutch working population was 0.90 and in a sarcoidosis population 0.89 .
in addition , the fas had the highest factor loading in a factor analysis incorporating five fatigue questionnaires .
correlations between the fas and the beck depression inventory were 0.59 and between the fas and the ces - d 0.65 .
the pseq is a 10-item questionnaire developed to assess the confidence people with ongoing pain have in performing activities while in pain .
it covers a range of functions , including household chores , socializing , work , as well as coping with pain without medication .
the expanded disability status scale ( edss ) is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability over time .
it is widely used in clinical trials and in the assessment of people with ms .
the scale was developed by a neurologist called john kurtzke in 1983 as an advance from his previous 10 step disability status scale ( dss ) .
the edss scale ranges from 0 to 10 in 0.5 unit increments that represent higher levels of disability .
edss steps 1.0 to 4.5 refer to people with ms who are able to walk without any aid and is based on measures of impairment in eight functional systems ( fs ) : i ) pyramidal - weakness or difficulty moving limbs ; ii ) cerebellar - ataxia , loss of coordination or tremor ; iii ) brainstem - problems with speech , swallowing and nystagmus ; iv ) sensory - numbness or loss of sensations ; v ) bowel and bladder function ; vi ) visual function ; vii ) cerebral ( or mental ) functions ; viii ) other .
each functional system is scored on a scale of 0 ( no disability ) to 5 or 6 ( more severe disability ) .
demographic and clinical characteristics of all patients will be collected as baseline information at the beginning of the study .
a p - value of 0.05 or less will be considered to indicate statistical significance .
all analyses will be performed with the statistical package for the social sciences ( spss 13.0 for windows ) .
this study aims to demonstrate fatigue and pain self - efficacy issues in ms patients .
the findings of the present study can be used in the development of health care services and in - patient management .
the role of fatigue and pain self - efficacy in particular may play an important role in the course of illness and treatment outcomes and could therefore be identified as a new area for psychological intervention in people diagnosed with ms .
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the examination of the perception of pain and fatigue in patients with various health problems has received increased research attention in recent years .
the aim of the present study protocol is to examine levels of pain self - efficacy and fatigue in a sample of greek patients suffering from multiple sclerosis .
the association of years and severity of diagnosis with the perception of pain self - efficacy and fatigue will be also investigated .
forty patients from the 3nd neurological department , aristotle university of thessaloniki ( greece ) will participate in this study .
the measurement tools include i ) the fatigue assessment scale , ii ) the pain self efficacy questionnaire and iii ) the expanded disability status scale .
test - retest reliability of the first two questionnaires will be assessed with the same patients rating their situation in a 10 days interval from the first examination in order to examine consistency over time .
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switzerland is a democratic federal state of approximately 7 million inhabitants , in which government responsibilities are divided between three levels : the federal level , the 26 cantons and the municipalities .
the main feature of the swiss healthcare system is its decentralized structure and therefore the relatively high degree of local autonomy .
indeed , switzerland is considered to have 26 slightly different healthcare systems , one for each canton , acting autonomously in the organization of healthcare services in their area . due to subsidiarity ,
universal access to health care is guaranteed since 1996 ( mandatory health insurance ) , and the basic health insurance coverage includes a comprehensive package of health benefits , identical for all insured .
insurers are obliged to accept applicants , theoretically avoiding risk - selection across insurance companies . however , since the risk adjustment system has only poor performance , risk - selection remains a problem .
ambulatory care is provided by physicians working mainly independently in individual private practices . however , a growing part of primary care is provided by small group practices , as well as networks of physicians and health maintenance organizations ( hmos ) acting on the principles of gatekeeping . apart from patients who choose to restrict their choice of doctors in return for lower premiums ( alternative managed care
insurance models allowing patients to receive care from physician networks or hmos ) , patients have direct and unrestricted access to primary care physicians and specialists . while independent private practitioners are paid fee - for - services , physicians working in networks or hmo s may be paid either on a fee - for - service basis or by salary [ 1 , 2 ] .
acute inpatient care is provided by cantonal and publicly subsidized hospitals and to a smaller extend ( 25% ) by private for - profit and not - for - profit hospitals .
inpatient care for patients with basic health insurance is financed by the cantons and the health insurance companies .
the latter cover a maximum of 50% of the costs of the inpatient treatment whereas the cantons meet the remaining costs including investments [ 2 , 3 ] .
spending for health care in switzerland is the third highest , after the usa and france , as expenditure per capita as well as a share of gdp amongst oecd countries .
projections estimate that the percentage of individuals aged 65 years and over will increase from 16% in 2005 to 28% in 2050 .
this ageing of the population is accompanied by a discrete increase in life expectancy , that reached 83.7 years at birth for women ( 78.6 years for men ) in 2004 ( 89.5 years and 85 years estimated , respectively for women and men in 2050 ) , and by an overall decrease in mortality . while life expectancy in switzerland is above eu averages , mortality rates in switzerland
measures of prevalence of risk factors and diseases help estimate the burden of disease and its evolution over time . in switzerland ,
tobacco use places the greatest burden on the swiss population , followed by high blood pressure , overweight , high cholesterol and alcohol consumption . while the prevalence of smoking , between 1992 and 2007 , has been declining both for men and women and most age groups , the prevalence of overweight has been increasing [ 6 , 7 ] .
the prevalence of chronic conditions has augmented markedly . indeed , while 43% of adult swiss residents self - reported at least one chronic condition in 1997 , 51% did so in 2007 .
projections based on epidemiological data estimate that the prevalence of chronic diseases representing the main causes of death , such as diabetes , heart failure , chronic obstructive pulmonary diseases , will increase by at least 50% in 2030 .
integrated care is a polymorphous concept viewed and understood very differently between national systems as well as between the various actors within the health systems . in general , integrated care is a
coherent set of methods and models on the funding , administrative , organisational , service delivery and clinical levels designed to create connectivity , alignment and collaboration within and between the cure and care sectors in order to overcome well - known shortcomings in health care such as the fragmentation of cure and care ( processes ) , the concerns for equal access or the inefficiencies of and within the health systems .
a recent systematic review summarizing the current research literature on health system integration suggested 10 key elements for successful integration : 1 ) comprehensive services across the care continuum , 2 ) patient focus , 3 ) geographic coverage and access , 4 ) standardized care delivery through interprofessional teams , 5 ) performance management , 6 ) information systems , 7 ) organizational culture and leadership , 8) physician integration , 9 ) governance structure and 10 ) financial management .
it is important to note that while there is a wide spectrum of integrated care activities throughout the world , these key elements may represent universal principles and therefore may be helpful to assess specific developments . in switzerland ,
two types of integrated care organizations have been developed since 1990 : physician networks and hmos .
physician networks are defined as organizations which provide healthcare services geared to the requirements of the patients by means of contractually agreed cooperation among themselves , with service providers outside the network and with the insurance companies .
given this definition , swiss physician networks represent hybrid forms or a mixture of integrated medical groups ( img ) and individual practice associations ( ipa ) .
the physicians are employed by the insurance company owning the hmo . for the group model ,
the physicians are the owners of the hmo which is also true for the physician networks .
what all these organizations have in common is the principle of gatekeeping : the insured persons commit to always enter the healthcare system through the same entrance or
this gate may be a physician network , an hmo or a medical call centre reached by telephone .
specialized treatment or in - hospital treatment can be obtained only by presenting a referral from a gatekeeper or care manager . in return , the insured are given a discount on their premiums .
emergencies are exempted from this obligation , and special provisions apply to visits to gynaecologists and paediatricians . according to santesuisse , the association of health insurers in switzerland , the share of insured people choosing an alternative ( managed care ) type of basic health insurance increased continuously since 1990 : in 2010 , only 56.4% had regular health insurance ( down from 75.4% in 2008 ) , 11.2% had managed care health insurance including gp s sharing financial responsibility ( capitation ) ( up from 6.5% in 2008 ) and 32.4% had a managed care type of health insurance without financial responsibility of gp s ( up from 24.6% in 2008 ) [ personal communication axel reichlmeier , santesuisse : data of october 2010 ] .
for the past 10 years the development of physician networks and hmos in switzerland has been evaluated at one- or two - year intervals . in the 2010 survey ,
data has been collected by an online questionnaire from all physician networks and hmos in switzerland under contract to one or several health insurance companies .
all networks and hmos with contracts with one or more health insurers were eligible for the survey . the questionnaire has been developed specifically for this survey and covers the following aspects : ( 1 ) legal form of the network , ( 2 ) number of insured cared for by the network , ( 3 ) number of physicians in the network , ( 4 ) financial co - responsibility of the network , ( 5 ) kind of activities for quality improvement in the network , ( 6 ) information technology used in the network , ( 7 ) disclosure of performance measures by the network , ( 8) kind of contractual cooperation between the network and other care providers , ( 9 ) number and type of preferred provider of the network and ( 10 ) form of management of the network .
prior to the survey , all physician networks and hmos in switzerland received a letter of invitation including a personalized link to the online questionnaire by email .
the representatives of the networks / hmos were asked to complete the questionnaire within six weeks .
data of organizations not completing the questionnaire was gathered by telephone interviews with their representatives .
we got data from all networks and hmos with contracts with one or more health insurers .
the survey was funded by the swiss forum managed care . in 2010 , an average of one out of eight insured person in switzerland , and one out of three in the regions in north - eastern switzerland , opted for the provision of care by general practitioners in physician networks or hmos .
this represented an increase of 34% in comparison with 2008 ( maximum increase , 52% , in the canton of zurich ) ( figure 1 ) .
the geographical distribution of the 86 current networks of physicians , and the number of doctors affiliated with them is shown in figure 2 .
the north - eastern regions of switzerland as well as several other cantons ( e.g. geneva and aargau ) show an above - average percentage of networks .
in contrast , physician networks are ( still ) non - existent in some parts of switzerland especiallyin the french- and italian - speaking regions ( except the canton of geneva ) . in switzerland as a whole , 54% of all basic care providers ( 3488 of total 6418 general practitioners , i.e.
, specialists in general medicine , internists and paediatricians ) and more than 400 other specialists have joined the 86 physician networks . out of 86 networks , 73 ( 84% )
have contracts with healthcare insurance companies with which they agree to assume budgetary co - responsibility , i.e. , to adhere to set cost targets for particular groups of patients .
the members of the physician networks are convinced that the growth and expansion of integrated care will continue .
indeed , 55% of those questioned in the 2010 survey expect the number of insured persons to increase by up to 10% annually , while 26% expect it to increase by up to 20% annually , over the next three years .
the focus here is on quality circles , which 96% of the networks commit to implement .
these circles , which are compulsory for the doctors , are held with a median frequency of eight times a year ( range : 420 times a year ) .
other implemented quality management elements include critical incident reporting ( cirs , 53% ) , use of guidelines ( 41% ) , and contractually agreed disclosure of quality and/or cost data to health insurers ( 55% ) .
more than half of the networks ( 58% ) demonstrate their quality management work by means of a regular report sent to external stakeholders ( 43% ) and/or a quality certificates ( 40% ) .
almost half of the networks ( 43% ) engage in contractually regulated collaboration with other ( external ) service providers in particular , with hospitals ( 36% ) , emergency services ( 33% ) and call centres ( 19% ) . likewise
, about half of the networks ( 51% ) work with and primarily refer their patients topreferred providers ; these are most often specialists ( 45% ) , hospital doctors ( 35% ) , physiotherapists ( 27% ) , radiology institutes ( 27% ) and laboratories ( 23% ) .
preferred providers are selected on the basis of personal experience ( 45% ) and/or of results from the quality circles ( 44% ) ; other factors that play a role in selection are quality data ( 31% ) and/or patient evaluations ( 28% ) .
in 2010 , an average of one out of eight insured person in switzerland , and one out of three in the regions in north - eastern switzerland , opted for the provision of care by general practitioners in physician networks or hmos .
this represented an increase of 34% in comparison with 2008 ( maximum increase , 52% , in the canton of zurich ) ( figure 1 ) .
the geographical distribution of the 86 current networks of physicians , and the number of doctors affiliated with them is shown in figure 2 .
the north - eastern regions of switzerland as well as several other cantons ( e.g. geneva and aargau ) show an above - average percentage of networks .
in contrast , physician networks are ( still ) non - existent in some parts of switzerland especiallyin the french- and italian - speaking regions ( except the canton of geneva ) . in switzerland as a whole , 54% of all basic care providers ( 3488 of total 6418 general practitioners , i.e. , specialists in general medicine
, internists and paediatricians ) and more than 400 other specialists have joined the 86 physician networks . out of 86 networks , 73 ( 84% )
have contracts with healthcare insurance companies with which they agree to assume budgetary co - responsibility , i.e. , to adhere to set cost targets for particular groups of patients .
the members of the physician networks are convinced that the growth and expansion of integrated care will continue .
indeed , 55% of those questioned in the 2010 survey expect the number of insured persons to increase by up to 10% annually , while 26% expect it to increase by up to 20% annually , over the next three years .
the focus here is on quality circles , which 96% of the networks commit to implement .
these circles , which are compulsory for the doctors , are held with a median frequency of eight times a year ( range : 420 times a year ) .
other implemented quality management elements include critical incident reporting ( cirs , 53% ) , use of guidelines ( 41% ) , and contractually agreed disclosure of quality and/or cost data to health insurers ( 55% ) .
more than half of the networks ( 58% ) demonstrate their quality management work by means of a regular report sent to external stakeholders ( 43% ) and/or a quality certificates ( 40% ) .
almost half of the networks ( 43% ) engage in contractually regulated collaboration with other ( external ) service providers in particular , with hospitals ( 36% ) , emergency services ( 33% ) and call centres ( 19% ) .
likewise , about half of the networks ( 51% ) work with and primarily refer their patients topreferred providers ; these are most often specialists ( 45% ) , hospital doctors ( 35% ) , physiotherapists ( 27% ) , radiology institutes ( 27% ) and laboratories ( 23% ) .
preferred providers are selected on the basis of personal experience ( 45% ) and/or of results from the quality circles ( 44% ) ; other factors that play a role in selection are quality data ( 31% ) and/or patient evaluations ( 28% ) .
however , there are two important questions remaining : to what extent do they reduce or stabilize healthcare costs and to what extent do physician networks foster the quality of care ? cost savings of physician networks and hmos in switzerland have been studied repeatedly with mixed results .
early studies evaluating the impact of gatekeeping by physician networks on utilization and financial outcomes indicated transfer of visits from specialists to general practitioners and reduced costs ; the findings were ambiguous due to incomplete case mix adjustment , however [ 16 , 17 ] .
in contrast , a more recent study evaluated the extent to which lower costs in a gatekeeping plan compared with a fee - for - service plan were attributable to more efficient resource management , or explained by risk selection .
the authors showed that the estimated cost savings achieved by replacing fee - for - service based health insurance with gatekeeping in the source population amounted to 15%19% per person not attributable to mere risk selection .
results regarding the impact of physician networks on the quality of care are still sparse . a recent study assessed whether physician networks met a set of 43 self - reported indicators of quality of care ( organization and structure , collaboration , process management , communication , results ) .
the results showed that on average , the 19 participating networks met 69% of the 43 quality indicators .
in another recent study , physicians working in various types of practices ( physician networks , group and single - handed practices ) were asked to assess the extent to which patients with chronic illnesses were receiving care congruent with the chronic care model ( ccm ) , using the assessment of chronic illness care ( acic ) questionnaire .
the authors showed that physician networks practices seemed to implement the different component of the ccm in a greater extent than group and single handed practices . despite these encouraging data , views and opinions on integrated care
indeed , physicians from the french- and italian - speaking part of switzerland , in particular , view the development of physician networks with scepticism . results from a recent survey asking physicians in the ( french - speaking ) canton of geneva to complete a brief questionnaire on integrated care ( i.e. , on physician networks ) showed that a majority ( 56% ) of them was highly critical on such developments .
these physicians believed that physician networks only reduced costs by constraining access to care ( and therefore withholding care ) or by abetting risk selection , and that physicians in networks are distracted of taking enough time to care for their patients .
another recent survey of doctors in the canton of geneva showed that many physicians expressed predominantly negative opinions on the impact of managed care tools such as guidelines , gatekeeping , managed care networks , second opinion requirement , pay - for - performance or utilization review .
although they perceived the impacts of these tools to control healthcare costs as positive , the impacts on professional autonomy were valued predominantly negative .
primary care doctors held more positive opinions than doctors in other specialties , whereas psychiatrists were the most critical .
the fundamental difference between pure gatekeeping systems and integrated care is the dedication of the latter to collaboration over several care levels with upstream and downstream care providers and preferred providers in other words , vertical integration .
vertical integration is the hallmark of integrated delivery systems as it brings together physician practices , hospitals and other service delivery organisations under the roof of shared values , aligned incentives and agreed accountability . regarding the 10 key elements for successful integration described earlier ,
the physician networks in switzerland may be seen as an early form of integrated care organizations .
however , the true importance of integration will become evident in the future in light of the increase in prevalence of chronic diseases and the growing complexity of medical treatment .
it will be seen namely at those locations where numerous different treatments and/or types of care have to be approved and coordinated , i.e. , in patients with complex chronic diseases and in patients undergoing long - term treatment . in this context intensified integration and/or dedicated coordination and control of treatments promise to optimize the quality of medical treatment , secure the provision of medical care , and increase the economy of care . while chronic disease management initiatives have been developed and implemented in several european countries [ 24 , 25 ] , interest towards chronic disease management initiatives is recent in switzerland , and only few programs exist .
nevertheless , during past years , initiatives targeting chronic diseases were developed both within and outside physician networks , at regional and/or cantonal levels . in the french - speaking part of switzerland for example , the implementation of clinical pathways for heart failure and breast cancer patients that do not only include the in - hospital phase of care , chronic disease management programs ( e.g. diabetic and heart failure patients ) at loco - regional levels , and more recently the political decision to implement a diabetes program in the canton of vaud ,
other initiatives in the german - speaking region of switzerland include projects from the zurich university institute of family medicine , based on the chronic care model and its implementation in the general practice [ 2830 ] , as well as other chronic disease management programs developed and proposed by telemedicine support services .
stronger integration and dedicated collaboration are viewed by most stakeholders within healthcare as the most effective and sustainable package of measures suitable for guaranteeing the high quality and economy of healthcare in the future . the reasoning behind this widespread acceptance is the realization , gained from numerous research projects , that healthcare systems are basically fragmented or simply non - integrated because of the contradictory logic espoused by the individual players .
the consequences are all too familiar : patient - care processes of low coherency , duplicate treatment tracks , treatment errors , etc .
however , another realization is even more important : it is not ( only ) self - interest and a clinging to power by the individual players that is responsible for fragmentation ; rather , fragmentation can be considered a normal state of affairs in the healthcare system .
the challenges to be surmounted by any efforts to achieve integration thus become obvious : it is necessary to take account , time and again , of the ( unavoidable ) fact that the individual players all have a mind of their own and to negotiate intelligent compromises .
it is important to note that until now switzerland had no laws and no political blueprints for integrated care , no statutory framework for physician networks , and no public ( start - up ) financing for corresponding projects .
physician networks and hmos were all established solely by initiatives of physicians and health insurance companies .
the sole basis for these activities is the healthcare legislation ( swiss health insurance law , kvg ) which allows for such initiatives and developments . in other words ,
when more than half of all primary care providers in switzerland decided to join physician networks and three out of four of them entered into contracts in which they agree to assume financial co - responsibility , they did so voluntarily .
this is important primarily because contractually stipulated integration and financial co - responsibility are diametrically opposed to the traditional professional understanding of physicians .
we therefore conclude that the initiation and development of physician networks in switzerland may be viewed as a model of
service providers and financing bodies can join forces to develop and implement , on their own initiative , integrated care organisations that frequently extend over large geographical areas .
the future challenges in switzerland are both to develop physician networks and other chronic disease management initiatives towards more comprehensive integrated care .
these developments need to target comprehensive services across the care continuum , standardized care delivery through interprofessional teams , performance management , information systems , governance structure and financial management .
meeting these targets also means modifying practices and attitudes , reorganizing the healthcare system while optimizing and coordinating available resources , and not forgetting to emphasize health promotion and disease prevention .
david perkins , director , centre for remote health research , broken hill , department of rural health , university of sydney ; editor - in - chief of the australian journal of rural health , australia jrgen wasem , professor , institute for health care management and research , university of duisburg / essen , germany volker amelung , dr . , professor , institute for epidemiology , social medicine and health system research , hannover school of medicine , hannover , germany
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introductionthe swiss health care system is characterized by its decentralized structure and high degree of local autonomy .
ambulatory care is provided by physicians working mainly independently in individual private practices .
however , a growing part of primary care is provided by networks of physicians and health maintenance organizations ( hmos ) acting on the principles of gatekeeping.towards integrated care in switzerlandthe share of insured choosing an alternative ( managed care ) type of basic health insurance and therefore restrict their choice of doctors in return for lower premiums increased continuously since 1990 . to date ,
an average of one out of eight insured person in switzerland , and one out of three in the regions in north - eastern switzerland , opted for the provision of care by general practitioners in one of the 86 physician networks or hmos .
about 50% of all general practitioners and more than 400 other specialists have joined a physician networks .
seventy - three of the 86 networks ( 84% ) have contracts with the healthcare insurance companies in which they agree to assume budgetary co - responsibility , i.e. , to adhere to set cost targets for particular groups of patients . within and
outside the physician networks , at regional and/or cantonal levels , several initiatives targeting chronic diseases have been developed , such as clinical pathways for heart failure and breast cancer patients or chronic disease management programs for patients with diabetes.conclusion and implicationsswiss physician networks and hmos were all established solely by initiatives of physicians and health insurance companies on the sole basis of a healthcare legislation ( swiss health insurance law , kvg ) which allows for such initiatives and developments .
the relevance of these developments towards more integration of healthcare as well as their implications for the future are discussed .
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chronic stable angina ( csa ) is a significant and prevalent problem in the united states that can negatively impact quality of life ( qol ) .
data from the centers for disease control and prevention indicate that in 2011 approximately 7.8 million people in the united states aged greater than 20 years experienced angina .
more than 500,000 people aged greater than 45 years are diagnosed with csa each year [ 1 , 2 ] . in a recent study of csa incidence , 29% of patients with csa attending primary care practices experienced at least 1 episode of angina per week .
anginal symptoms can be typical , often described as a burning sensation , pain , pressure , squeezing , or tightness , or atypical , which can include fatigue , indigestion , lightheadedness , nausea , dyspnea , and weakness in addition to pain . moreover , angina can vary from patient to patient and across the sexes and is present in different parts of the body , including the chest , jaw , neck , shoulder , back , and arms .
atypical symptoms can occur with either gender but are more common in females than males [ 57 ] .
the current guidelines from the american college of cardiology foundation and american heart association ( accf / aha ) , published in november 2012 , define the goals of successful treatment in patients with stable heart disease as maximizing health and function and minimizing the likelihood of death .
specific treatment objectives include maintaining and restoring a level of physical activity , functional capacity , and qol that is satisfactory to the patient and the complete or nearly complete elimination of ischemic symptoms .
moreover , to achieve these objectives , the accf / aha guidelines highlight the importance of educating patients about the etiology , clinical manifestations , treatment options , and prognosis of their disease , supporting active patient participation in treatment decisions , and using evidence - based pharmacological treatments that improve the patients ' health status and survival with minimal side effects . pharmacological treatment options for csa include beta - blockers , calcium channel blockers ( ccbs ) , short- and long - acting nitrates , and the late sodium current inhibitor ranolazine .
the first step towards a successful treatment outcome for the patient with csa is an effective evaluation of anginal severity and its impact on patient functional status and qol .
the purpose of this paper is to raise awareness of the prevalence of csa , its impact on qol , and the need for taking an accurate and thorough patient history when assessing patients with probable csa .
in addition , this paper discusses tools that healthcare providers can use to assess the severity of their patients ' angina and the impact on qol , with the aim of providing successful treatment that maximizes survival , eliminates symptoms , and returns the patient to normal functional capacity .
the four - tiered canadian cardiovascular society classification ( ccsc ) system , which measures the limitations to ordinary activity and the timing of angina occurrence , remains the standard method for grading angina in patients with csa .
the classifications range from class i , defined as ordinary physical activity does not cause angina / angina occurs with strenuous , rapid , or prolonged exertion at work or recreation , to class iv , defined as an inability to carry out any physical activity without discomfort / anginal symptoms may be present at rest ( table 1 ) .
chronic stable angina can cause a decrease in the patient 's activity level and their ability to move and participate in normal daily activities and negatively impact their qol . in a comparison of physical activity and health - related qol in patients with and without csa ,
patients with angina curtailed leisure - related physical activity and became more sedentary to avoid an anginal episode , which in turn impaired their overall health and perception of qol ( table 2 ) .
the results of this study found that patients with stable angina have lower health - related qol in multiple domains ( physical function , general health , and vitality ) , impaired exercise performance , and lower levels of physical activity related to leisure compared with patients without stable angina . in the coronary artery disease in general practice ( cadence )
study , which investigated the impact of angina on qol among patients with stable angina attending a primary care practice , approximately 29% of patients experienced at least 1 episode of angina per week , which was associated with worsened qol and greater limitations on physical activity compared with patients with minimal angina ( < 1 episode / week over the preceding 4 weeks ) .
the results of this study demonstrate the important association between symptoms of angina and the health status of patients .
patients with a history of peripheral artery disease , heart failure , and female sex were more likely to have at least 1 episode of angina per week .
these data are important in light of earlier studies showing that a reduction in exercise tolerance is associated with an increase in the risk of mortality in patients with cardiovascular ( cv ) disease [ 11 , 12 ] .
a comparison of the prognostic value of exercise tolerance in patients with csa to risk factors of death ( pack - years of cigarette smoking , hypertension , history of congestive heart failure , or myocardial infarction ) found that peak of exercise capacity was the best predictor of death in both patients with and without cv disease .
an increase in exercise tolerance can improve a patient 's ability to perform daily functions , with every one metabolic equivalent ( met ) increase in exercise capacity correlating to a 12% improvement in survival .
in addition to improving exercise tolerance , the accf / aha guidelines recommend that patients with stable heart disease be educated about the importance of lifestyle modifications , such as smoking cessation , bp control , and weight , lipid , and diabetes management in improving their qol . increased exercise tolerance may also reduce cv risk and enhance qol in patients with csa who have comorbid type 2 diabetes mellitus ( t2 dm ) , and exercise is recommended by the american diabetes association for improving qol and blood glucose control and contributing to weight loss in patients with t2 dm . in patients with t2 dm ,
a structured exercise program has been shown to lower glycosylated hemoglobin by 0.66% and may reduce the risk of diabetic complications .
an important aspect of assessing qol in patients with csa is optimal communication between the patient and their healthcare provider .
patients with csa can vary in their tolerance for symptoms , making it challenging for healthcare providers to provide an accurate assessment of their condition .
for example , in the cadence study , a marked discordance was observed between the physician and patient perspective on the effect of angina on qol . while physicians classified 61% of patients
as having minimal impediment in physical activity ( ccsc class i ) and considered patients to be optimally controlled in 80% of cases regardless of the frequency of their anginal episodes , only 52% of patients reported no angina and 47% reported no diminished qol with increased frequency of angina .
an added challenge for healthcare providers is patients who do not engage in physical activity in order to avoid anginal episodes .
although these patients may become less symptomatic , their increasingly sedentary lifestyle may promote further declines in physical function and qol .
patients with chronic conditions tend to normalize over time and may not realize the extent to which they have become sedentary . as a result , they are unlikely to proactively communicate this information to their healthcare provider .
for example , a female patient who is prescribed a beta - blocker for her csa may report no episodes of chest pain at her 6-month follow - up with her physician and subsequently would be advised to continue with her medication as prescribed until her next follow - up in 6 months . however , in her daily routine she may participate in behaviors that reduce her symptoms , such as choosing the closest parking spot to the entry of her grocery store because she experiences shortness of breath if she walks long distances . as another example , a male patient who underwent percutaneous coronary intervention for csa may report no chest pain or shortness of breath at his 1-year follow - up with his physician .
however , he allows his neighbor to continue mowing his lawn because of his fatigue and lack of energy .
these two cases are great illustrations of how people with csa downregulate their lives without openly reporting it to healthcare providers .
nurse practitioners ( nps ) and physician assistants ( pas ) have an important role in evaluating patients ' condition , as they are likely to be more involved in patient care and may have more opportunity than physicians to spend time with patients and elicit information regarding their experiences . successfully treating csa begins with an accurate assessment of the severity of a patient 's angina , the impact on their functional status , and their risk of cv complications .
generic health status questionnaires , such as the short form 36 , may not focus on symptoms that are specific to coronary artery disease ( cad ) ; therefore , disease - specific tools that measure cad are important when assessing the health status of patients with cad .
several tools have been designed to facilitate assessment of patient - reported functional status and qol of patients with cad in the research and clinical settings .
these include the seattle angina questionnaire ( saq ) , the macnew heart disease health - related qol questionnaire , the ferrans and powers qol index , the duke activity status index ( dasi ) , and the speak from the heart chronic angina checklist .
the saq is a self - administered disease - specific measure for patients with cad that is demonstrably valid , reproducible , and sensitive to clinical change .
it is used extensively in the research trial setting to quantify the symptoms , functional limitations , and qol of patients with stable heart disease in the previous 4 weeks [ 16 , 17 ] .
use of the saq can help healthcare providers identify patients at high risk for morbidity and mortality and can also help identify those patients who may require more aggressive medical therapy or revascularization .
spertus and colleagues evaluated the prognostic utility of the saq among patients with cad and found that a patient 's health status ( symptoms , qol , and physical function ) was a strong predictor of 1-year mortality and hospitalization for acute coronary syndrome ( acs ) .
the macnew heart disease health - related qol questionnaire , also used in patients with heart disease , evaluates the effect of coronary heart treatments on daily activities and physical , emotional , and social functioning [ 18 , 19 ] .
this self - administered questionnaire generates an overall score from 27 questions that govern physical limitations , emotional and social function , and angina symptoms experienced in the previous 2 weeks , and it has been used in multiple clinical studies in patients with heart disease [ 20 , 21 ] . assessing patients ' health status during hospitalization for
angina can be helpful in predicting those patients who may experience a worsened qol 6 months later .
heller and colleagues found that , among 303 patients who were hospitalized with a diagnosis of acute myocardial infarction or angina and completed a follow - up disease - specific qol questionnaire at 6 months , scores were consistently lower in patients with angina .
the ferrans and powers qol index , which can be used in either a self - administered or interview format , measures both the satisfaction with and the importance of various aspects of life .
this qol index produces an overall qol score and four additional scores in the health and functioning , psychological / spiritual , social and economic , and family domains .
this index has been used to evaluate qol in multiple research trial settings in patients with disorders that include cad , chronic fatigue , and migraines [ 2225 ] .
the qol index instrument was used to assess life satisfaction among 47 subjects with cad who completed the instrument 6 to 8 weeks following a coronary event . a strong relationship between psychosocial functioning and life satisfaction was observed .
it is self - administered and uses 12 questions to assess self - reported measures of physical capacity to estimate peak mets , gauge the patient 's functional capacity , and assess aspects of qol .
the dasi was administered to 50 subjects undergoing exercise testing with measurement of peak oxygen uptake and 50 subjects in a control group .
the dasi was found to be a valid measure of functional capacity ; there was a significant correlation between the dasi and peak oxygen uptake .
the speak from the heart chronic angina checklist is a self - administered seven - item questionnaire that allows patients to share with their healthcare provider how angina is affecting their qol , by logging information about each anginal episode ( figure 1(a ) ) .
designed primarily for use in a clinical setting , this tool also provides the patient with an interactive symptom tracker that allows accurate documentation of the occurrence and frequency of angina episodes ( figure 1(b ) ) .
these tools let patients document the frequency of angina , the impact of angina on daily activities , and the impact of angina on qol and share this information with their healthcare provider . although many of the tools discussed are used primarily in the research trial setting , the accf / aha guidelines recommend the formal assessment of a patient 's disease - specific health status and that these tools be used serially in clinical practice to assess and monitor the effectiveness of antianginal medications , revascularization , and qol in patients with stable heart disease .
as physicians and patients can have different impressions of the impact of angina on patient qol , healthcare providers need to be aware of additional signs or atypical symptoms in their patients with csa .
effective communication and clinical assessment of the severity of patient symptoms and functional status are necessary to ensure that patients receive optimal therapy .
in addition to the tools mentioned above , questions that go beyond are you experiencing any chest pain or shortness of breath ?
( table 3 ) may be necessary to determine whether a patient is receiving the treatment necessary to attain the accf / aha treatment objectives of the complete or nearly complete elimination of anginal chest pain and restoration or maintenance of a level of activity , functional capacity , and qol that is satisfactory to the patient . for
the np and pa , asking the appropriate questions and taking an appropriate and thorough history is the key to the assessment of angina .
just because patients are not complaining of chest pain does not mean they are not having angina .
the majority of patients with chronic angina do not present with classic chest discomfort but with atypical symptoms that include but are not limited to fatigue , dyspnea , lightheadedness , weakness , nausea , neck pain , shoulder pain , mid- and lower back pain , and arm pain ( left or right ) .
as angina symptoms typically occur at the end of the ischemic cascade , patients undergo a biochemical alteration and a decrease in relaxation , contraction , and diastolic filling before having symptoms . as such , appropriate identification of symptoms when they do occur is of the utmost importance . asking the questions outlined in table 3 as well as using tools that facilitate interaction with the patient in the clinical setting
moreover , patients are more apt to withhold telling their cardiologist they are having angina for fear of going back to the catheterization laboratory and are much more likely to communicate openly with the np or pa ( if asked ) about any symptoms of typical or atypical angina they may be experiencing , as nps and pas are more apt to maximize medical therapy before sending patients back to the catheterization laboratory .
current pharmacological options for csa include beta - blockers , ccbs , short- and long - acting nitrates , and ranolazine [ 4 , 8 ] .
selection of optimal therapy for csa is critical , as patients have been shown to experience symptoms in the year after being prescribed antianginal therapy .
the antianginal efficacy and safety of beta - blockers and ccbs have been demonstrated in several randomized , controlled clinical trials [ 2944 ] .
beta - blockers and ccbs help reduce symptoms of angina , decrease the frequency of anginal episodes , increase exercise duration , prolong the time to st - segment depression following exercise testing , and improve qol in patients with csa [ 8 , 45 , 46 ] .
although these agents are generally well tolerated , some therapies can also have hemodynamic effects ; for example , ccbs lower blood pressure while beta - blockers and nondihydropyridine ccbs can decrease heart rate ( table 4 ) [ 8 , 47 , 48 ] . therefore ,
beta - blockers should be avoided or used with caution in patients with hypotension and they are contraindicated in patients with significant sinus bradycardia and partial atrioventricular block . therapy with beta - blockers
can also reduce exercise capacity and impair sexual function in some patients [ 49 , 50 ] .
similar to beta - blockers , ccbs also exert their effects by lowering blood pressure and heart rate ; therefore , nondihydropyridine ccbs are contraindicated in patients with left ventricular systolic dysfunction with or without heart failure and in patients with heart block and disorders of the sinus node .
long - acting nitrates are recommended when initial therapy with beta - blockers or ccbs is contraindicated or poorly tolerated .
the antianginal efficacy and safety of long - acting nitrates have been demonstrated in several randomized , controlled clinical trials [ 5156 ] .
long - acting nitrates decrease the frequency of anginal episodes and increase exercise duration in patients with csa but may be ineffective in improving qol [ 57 , 58 ] .
nitrate therapy can also lower blood pressure , with prolonged nitrate therapy leading to the development of tolerance .
a minimum 12-hour nitrate - free interval must be maintained every 24 hours to avoid the development of nitrate tolerance .
in addition , nitrate therapy is absolutely contraindicated in patients prescribed phosphodiesterase-5 ( pde-5 ) inhibitors , as the concomitant use of nitrates and pde-5 inhibitors can potentiate hypotension [ 4 , 57 ] .
ranolazine , a sodium channel inhibitor , is a newer medication approved in 2006 for the treatment of csa .
ranolazine is recommended when therapy with beta - blockers , ccbs , and nitrates is not tolerated or contraindicated or if initial treatment is ineffective .
therapy with ranolazine , alone and in combination with standard doses of beta - blockers , ccbs , and nitrates , can reduce the frequency of anginal episodes and nitroglycerin use and improve exercise duration , the time to onset of angina , st - segment depression , and qol in patients with csa , with no significant effect on blood pressure or heart rate [ 4 , 5963 ] .
the most common adverse effects of ranolazine are constipation , nausea , headache , and dizziness .
unlike beta - blockers and ccbs , ranolazine treats angina without causing significant changes to blood pressure and heart rate and it can be used in combination therapy with other antianginal agents ( beta - blockers , ccbs , and nitrates ) [ 4 , 59 , 60 , 64 ] .
ranolazine is contraindicated for use in patients with corrected qt interval prolongation , clinically significant hepatic impairments , and it should not be used in combination with drugs such as ketoconazole and macrolide antibiotics that are potent inhibitors of the cyp3a4 pathway .
the medical indications for revascularization are the prevention of death and cv complications and the improvement of symptoms and qol .
revascularization procedures such as coronary artery bypass graft or percutaneous coronary intervention can reduce the symptoms of csa and improve morbidity , mortality , and qol in patients with csa [ 6567 ] . however , there are clinical challenges to revascularization , including restenosis / acute coronary occlusion , diffuse disease and/or poor distal target vessels , and other comorbid conditions that increase perioperative complications .
these challenges may preclude revascularization as a treatment option for some patients , and the results of several clinical trials have shown that angina may persist in 20% to 34% of patients even 1 year after revascularization [ 6870 ] .
patients may not be receiving optimal treatment for their chronic angina and may continue to suffer from anginal episodes due in part to ineffective communication with and incomplete assessment by their healthcare provider . as a consequence , patients with chronic angina often experience impaired qol , as they restrict their physical activities and adopt a more sedentary lifestyle in order to minimize anginal episodes .
tools designed to be used in a clinical setting that facilitate proactive and effective communication between a patient and their healthcare provider can be an asset in improving patient - provider communication .
these tools can aid providers in more thoroughly assessing angina severity and the impact on a patient 's qol .
in addition , these tools can help healthcare providers attain the accf / aha treatment objectives of complete or nearly complete elimination of angina chest pain , with the goals of maximizing health and function and minimizing the likelihood of death in patients with csa .
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chronic stable angina ( csa ) is a significant problem in the united states that can negatively impact patient quality of life ( qol ) .
an accurate assessment of the severity of a patient 's angina , the impact on their functional status , and their risk of cardiovascular complications is key to successful treatment of csa .
active communication between the patient and their healthcare provider is necessary to ensure that patients receive optimal therapy .
healthcare providers should be aware of atypical symptoms of csa in their patients , as patients may continue to suffer from angina despite the availability of multiple therapies .
patient questionnaires and symptom checklists can help patients communicate proactively with their healthcare providers .
this paper discusses the prevalence of csa , its impact on qol , and the tools that healthcare providers can use to assess the severity of their patients ' angina and the impact on qol .
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recent advances in equipment and surgical techniques have made minimally invasive surgery ( mis ) a well - tolerated and efficient technique in several fields of surgery .
it has several advantages over standard surgical approaches , including more rapid recovery , lower rate of postoperative infection , decreased pain , better postoperative immune function , and cosmetic results [ 13 ] . in this way ,
robotic - assisted surgery ( ras ) has gained popularity in several surgical specialties and many institutions are now investing in medical robotic technology for applications in general , urological , cardiac , gynecological , and neurological surgery .
this new and exciting technology has been shown to be safe , have better or comparable outcomes , and can be cost effective when compared with conventional surgical approaches [ 13 ] .
this has raised interest in its use in other surgical fields , such as otolaryngology and head and neck surgery .
head and neck and several airway procedures have been associated with a large amount of surgical dissection with associated large surgical incisions .
this can result in major tissue damage , functional impairment , and a decreased quality of life .
however , with minimally invasive approaches , the improved video imaging , endoscopic technology , and instrumentation has provided the surgeon with multiple endoscopic access points .
while the advance of endoscopic technology has increased surgeon capabilities , the technique still has several challenges associated with it .
examples include : ( 1 ) the limited range and degree of motion of instrumentation , ( 2 ) operative field limited to line of sight ( 3 ) lack of three - dimensional imaging of the operative field ( 4 ) amplification of physiologic tremors , ( 5 ) compromised dexterity and ( 6 ) mismatched hand - eye coordination [ 5 , 6 ] . with these challenges in mind , the development of surgical robotics was rooted in the desire to overcome the limitations of current endoscopic technologies and to expand the benefits of mis .
the first robotic surgical system developed was the puma 560 , which was used in 1985 to perform neurosurgical biopsies with increased precision . since this time , a series of robots have been developed .
however , the only fda approved and actively marketed system ( 2009 , for transoral robotic surgery tors ) for head and neck surgery is the da vinci surgical robot ( intuitive surgical inc . , sunnyvale , ca , usa ) .
this system has its roots in the national aeronautics and space administration 's ( nasa ) desire to develop a method to provide surgical care to orbiting astronauts via telepresence surgery .
[ 7 , 9 ] interest in this technology came from both the stanford research institute and the us army , which saw promise in bringing the technology to the battlefield to provide surgical care to a wounded soldier as soon as possible even with the surgeon operating remotely .
thereafter , in 1995 , the intuitive surgical corporation was set up to produce telerobotic systems for commercial public use , where it was first used in general surgery .
reported the first two cases of robot - assisted fundoplication in 1999 , and weber et al .
the first robotic surgery performed transorally in the head and neck was carried out in 2005 by macleod and melder whereby a vallecular cyst was excised . in 2006 , three patients with tongue base tumors underwent tors as part of prospective clinical trial by o'malley jr .
at its core , the intuitive surgical corporation system is a comprehensive master - slave arrangement , with the surgical robotic cart containing multiple manipulation arms that are operated remotely from a console .
the robot contains video - assisted visualization and computer enhancement and is composed of three components : the surgical cart , the vision cart , and the surgeon 's console ( figure 1 ) . the surgical cart ( or slave unit ) is equipped with four arms ; one arm holds a 0 or 30 12 mm stereoscopic camera ( with 2 optical channels , each 5 mm ) , and the other three arms hold 5 mm ( pediatric size ) or 8 mm ( conventional ) endowrist instruments ( intuitive surgical inc . ) , that are easily interchangeable by surgical staff according to the surgeon 's desire and procedure requirement .
the vision cart is equipped with two light sources , an insufflator , and hardware that generates the three - dimensional image .
the cart usually holds another monitor for the assistant surgeon . the surgeon 's console ( or master unit ) displays two images , one for each eye .
in addition , the console is the interface for the surgeon to control the instrument , by controlling the hand manipulators .
the surgeon 's console is equipped with pedals to control the camera and instrument arm clutching ( disengagement of the hand controllers from the surgical arms ) camera controller , focus adjustment , and electrocautery .
the endowrist instruments are controlled by the surgeon at the master console and provide multiple degrees of freedom , including pitch , yaw , and roll plus two additional degrees of freedom in the wrist and two others for tool actuation a total of seven degrees of freedom in all .
the 3-dimensional visualization and tenfold magnification of the operative field enhance the depth of the field and the clarity of the tissue planes during dissection . this can be especially helpful during head and neck surgery and pediatric surgery , because of the small size of the surgical field and the inability to maneuver the instruments and the camera within it .
in addition , movements can be scaled , whereby large hand movements can be translated into micromovements inside the operative field , allowing the surgeon more precision .
endowrist instruments have 7 degrees of freedom , which improves dexterity , allowing maneuverability that approaches that of open surgery . during the robotic portion of the surgery the surgeon is sitting with his / her forearms resting comfortably on a pad and the head resting against the console , therefore improving ergonomics .
this results in reduced body fatigue . with the surgeon sitting at a remote workstation ,
it eliminates the need to physically twist and turn in awkward positions to move instruments within the operative field while simultaneously visualizing a monitor .
in addition , hand muscle fatigue is reduced , which when considered together with improved visualization , makes tasks such as suturing substantially easier .
studies suggest ( berguer and smith ) that robotic surgery is less stressful for the surgeon .
the robotic system eliminates the fulcrum effect of endoscopic surgery and makes instrument and camera manipulation more intuitive , emulating another property of open surgery .
since the inception of robotic surgery , the wish to overcome geographical constraints and the availability of specialists was an important goal .
marescaux and collaborators described the feasibility and safety of a robot - assisted laparoscopic cholecystectomy at distance using high - speed connection between the surgical unit at strasbourg , france , and the surgical console in new york .
telesurgery allows for these barriers to be overcome as well as offering new teaching and tutoring possibilities .
an experienced surgeon can use another console next to the trainee , which can be activated to command the main arms or auxiliary arms .
the vinci skills simulator ( intuitive surgical inc . ) can be attached to the console , allowing a virtual training environment to be creating while maintaining the same robotic interface . however , there are currently no standardized residency curriculums that formally support the teaching of robotic surgical skills .
the surgeon is unable to feel tissue resistance or how tight a knot is being tied .
this can be a significant problem early on , although the improvement in visualization is such that the surgeon rapidly learns visual clues to compensate for his lack of feedback . despite this
increased physical space requirements in the operating room are needed to accommodate the large and heavy equipment .
additional time and personnel are needed to set it up , along with specialized training for or staff .
initial installation cost ranges from 1.5 to 2.5 million dollars ( us ) depending on the model , along with an approximately 100,000 dollars annual maintenance fee and 2000 dollars per instrument ( each instrument has a ten use lifespan ) ; the da vinci robotic system is one of the most expensive operating tools available , making it impractical for many institutions .
stronger studies are needed to assess the real cost - benefit of this technology compared to other techniques .
the description below applies to the tors procedures , although not all procedures in the head and neck region use this approach .
transoral robotic surgery ( tors ) is defined as surgery performed via the oral cavity that uses a minimum of three robotic arms and allows bimanual manipulation of tissues .
it was first developed by weinstein and o'malley , who have assessed the feasibility of this technique using the da vinci robotic system [ 13 , 2227 ] . to minimize obstruction and maximize the communication between the surgeon and his / her assistants in tors surgery
, the surgeon 's cart should be located at the end of the operating room , allowing free space to maneuver the surgical cart that is placed on the right side of the patient , opposite to the surgeon .
the support staff and instrument carts are located on the side of the patient , opposite the surgeon as well .
the anesthesia machine and anesthesiologist are at the patient 's foot ( figure 1 ) .
anesthesia induction is usually done without moving the patient ; this technique is described in detail by chi et al . .
this method of organization slightly complicates the induction , but vastly simplifies setup for procedure , saving 1520 minutes per case . performing the induction across from the anesthesia unit
does not require the disconnection / reconnection of iv lines , monitor devices , or the anesthesia circuit , avoiding entanglement with the robotic equipment .
next , with the patient in supine position , the airway is secured via standard endotracheal intubation and the tube is appropriately secured .
safety goggles and a molded dental guard are used to protect the patient . following induction ,
the robotic cart is brought in to the right of the patient , and the endoscopy tower and scrub table are brought in to the left .
the surgeon then places a retractor in the patient 's mouth to gain surgical exposure , and the 3 sterilely draped robotic arms are placed in surgical position ( figures 2 and 3 ) .
o'malley jr . et al . initiated the tors studies in canine and cadaveric models [ 13 , 2227 ] and applied the technique to clinical practice . in 2006 ,
three patients underwent robot - assisted transoral tongue base resection in a prospective clinical trial . in this study , the robot enabled the surgeons to easily identify the glossopharyngeal , hypoglossal and lingual nerves , as well as the lingual artery .
one t1 and one t2 squamous cell ( ajcc cancer staging ): two instances of squamous cell carcinoma ( one t1 and one t2 ) were adequately resected with negative margins , good hemostasis , and no postoperative complications .
the different retractor types were assessed first during the cadaveric part of the study , and then at the beginning of each procedure performed in patients .
the fk retractor achieved the best ( versus crowe davis and dingman retractors ) tissue exposure and retraction . the same group published another study in which robot - assisted tonsillectomy was performed on 27 patients with squamous cell carcinoma .
25 of the 27 patients had negative cancer margins and 26 of the 27 patients were able to swallow postoperatively . in 2007 , solares and strome described transoral carbon dioxide ( co2 ) laser robotic - assisted supraglottic laryngectomy in a 74-year - old woman with a large supraglottic tumor .
the use of the carbon dioxide laser linked to the surgical robotic system allows more maneuverability of the instrument 's tips and improves beyond sight of beam limitations .
in addition to tumor resection , robotic surgery can be used in the reconstruction of postresection defects .
reported two cases of robotic - assisted free flap reconstruction in the oral cavity and oropharynx .
these studies highlight the improved visualization provided by ras , avoiding the need to perform a mandibulotomy for access , thereby reducing morbidity and operative time .
after preliminary studies assessing the feasibility of tors for oncologic resection , a series of studies were performed to examine the functional outcomes of these procedures [ 8 , 15 , 3239 ] .
most studies primarily report on oropharyngeal and oral cavity cancer , however , there are also case series on hypopharyngeal and laryngeal malignancy treated with tors .
failure due to suboptimal access has been reported . in the study performed by weinstein et al . in 2010
, only 3 of 47 patients were converted to open surgery after attempts failed to reach adequate exposure for resection .
, who reported 3 of 29 , and iseli et al . found 5 of 54 .
a comprehensive panendoscopy prior to scheduling patients for tors can identify unsuitable patients and thus reduce surgical risk .
also report a successful swallowing rate of 97.6% at 12-month followup , while boudreaux et al . found 79% at last follow up ( 3 months ) , and iseli 's study found 83% ( 12 months of followup ) .
report that all patients returned to normal swallowing ( followup time ranged 3 months to 2 years ) .
predictive factors of poor swallowing following robotic resection included : higher tnm stage , preoperative nasogastric feeding requirement , tumor site ( oropharyngeal or laryngeal ) , and recurrent or second primary tumor resection .
regarding the overall procedure time , we observed a trend to faster procedure times as more cases were being performed .
lawson et al . assessed the robotic learning curve for procedures in the head and neck and found that both set - up and operative times showed a reduction in time as more procedures were performed .
for the operative segment , time was reduced from 88 53 to 47 29 minutes . for the overall procedure , time
was reduced from 117 64 to 66 33 minutes . however
although there are no studies assessing recurrence rates at 5 years , preliminary outcomes have been encouraging . in weinstein 's report of advanced oropharyngeal carcinoma ,
regional control was obtained in 96% and distant control in 91% of cases at 18 months follow up . accordingly with machtay et al .
the robot can thus provide an excellent approach to cancer , improving the ability to interpret the adequacy of the resection margins an important factor in determining whether adjuvant therapy is indicated .
further studies are needed to assess the short- and long - term outcomes of tors when compared to other more established techniques table 1 .
the first published clinical application of tors , performed by macleod and melder , was marsupialization of a vallecular cyst .
assessed the effectiveness of robot - assisted surgery in obstructive sleep apnea - hypopnea syndrome ( osahs ) [ 44 , 45 ] . in these studies ,
20 patients underwent a tongue base resection , with some patients also having a supraglottoplasty and uvulopalatoplasty performed .
overall patient satisfaction , assessed by a visual analogue scale ( vas , 0 to 100% ) was 94% .
a reduction in the epworth score ( mean ess improvement was 5.9 + 4.4 sd ) and apnoea - hypopnoea index was seen ( mean ahi improvement was 24.6 + 22.2 sd ) .
all patients were decannulated between day 4 and 13 after surgery and regained a satisfactory ability to swallow within 2 weeks .
this study showed the feasibility and safety of robotic tongue base resection techniques . another cadaveric study conducted at the university of pennsylvania in 2010 by lee et al . showed the feasibility of transoral approach for decompression of the craniocervical junction , demonstrating the possible use of the robot in the future for conditions such as compression for basilar invagination , congenital skull base malformations , extradural lesions , and skull - base tumors .
the transaxillary robotic technique was first described in 2005 by lobe et al . , where a hemithyroidectomy was successfully performed in a pediatric patient . in 2008 ,
the same group reported a bilateral axillary approach for total thyroidectomy in two pediatric patients . in adults , the largest experience in robot - assisted thyroidectomy by kang et al . who developed the gasless transaxillary technique [ 49 , 50 ] and reported a series of 338 patients .
in 2009 , a case control study of 41 robotic cases and 43 conventional thyroid surgery patients was reported [ 51 , 52 ] . unlike the transoral technique described previously
, this procedure dissects a tunnel on the anterior surface of the pectoralis major muscle and clavicle by electrocautery under direct vision , before the robotic portion of the surgery . with the patient - placed supine under general anesthesia ,
the neck is slightly extended , and the ipsilateral arm is abducted at the shoulder to minimize the distance between the axilla and neck .
a second incision is made on the medial side of the anterior chest wall to insert the 4th robot arm that will be used for thyroid retraction , and it is connected to a continuous suction system .
the dissection is approached through the avascular space of the sternocleidomastoid muscle branches and beneath the strap muscle until the contralateral lobe of the thyroid is exposed .
two 8 mm instruments are introduced through the breast incision , and the 3rd arm carries the 12 mm endoscope .
robotic thyroidectomy using a transaxillary approach leaves a scar in the axilla that is covered by the patient 's arm .
this is important when we consider that thyroid disease is more common in women , and the incidence is increasing in young women , raising concerns about cosmetic results .
robotic - assisted thyroidectomy has been associated with a lower degree of postoperative discomfort , a higher degree of patient cosmetic satisfaction , and subjective improvements in swallowing discomfort , when compared to the conventional surgery [ 5153 ] .
a few cases of recurrent laryngeal nerve injury have been reported . in 2011 , lee et al .
published a multicenter retrospective study of 1,043 cases of low - risk differentiated thyroid carcinoma and compared the results of robotic - assisted thyroidectomy to laparoscopic and open thyroidectomy surgical series .
this study supports the statement that robotic use is safe , feasible , and provides the similar outcomes to other techniques , while also overcoming their limitations .
in addition , it seems that the indication for robotic thyroidectomy can be expanded to include advanced thyroid cancer , because lymph node resection can be performed with great dexterity , removing a similar number of lymph nodes as in open surgery .
inserted the 4th arm trocar through an ipsilateral periareolar nipple incision , while lee et al . used a bilateral transaxillary approach with co2 insufflation . in any case , these techniques were shown to be feasible and have comparable results to open surgery , although co2 insufflation has been associated with increased probability of pneumomediastinum and air embolism table 2 . technically similar to the surgery performed for thyroidectomy ,
this technique involves a 5-to-6 cm vertical skin incision in the axilla with a subcutaneous skin flap created from the axilla to the anterior neck area over the pectoralis major muscle and clavicle under direct vision .
a second 0.8 cm skin incision is made on the anterior chest . with these 2 incisions ,
4 robotic arms can be inserted3 in the axilla and 1 in the anterior chest wall . following this study , other publications detailed further robot - assisted parathyroidectomy [ 6369 ] .
the most recent and largest study tolley et al . included 11 patients with hyperparathyroidism .
this study showed that the robot - assisted surgery allowed adequate visualization of important anantomicanatomic structures in this region , good resection , and a hospital length of stay comparable to nonrobotic minimally invasive surgeries [ 7177 ] .
only one case needed to be converted to open surgery due to the patient 's large body habitus a factor shown to be a predictor of longer operative times . validated questionnaires regarding quality of life and cosmetic appearance showed good subjective results for this new approach .
the fundamental studies that established the technical feasibility of tors to gain access to many regions , such as the oral cavity , oropharynx , hypopharynx , and larynx , raised the question about whether the robot can reach more difficult places .
they also reported the first human case a patient that underwent resection of parapharyngeal cystic neoplasm extending into the infratemporal fossa .
concern regarding identification of important structures , such as the carotid artery , jugular vein and cranial nerves was raised , and was solved by appropriate demonstration of surgical technique and hemostasis . in 2010 , another study performed by o'malley jr .
and weinstein assessed the outcomes of 10 patients undergoing parapharyngeal space resection using the tors approach .
the surgery was performed in 9 of the 10 patients , with acceptable operative time and blood loss , and no significant complications such as hemorrhage , infection , trismus or tumor spillage .
one patient was converted to an open transcervical approach due to difficulties found during resection and to avoid the risk of tumor spillage . in 7 patients that had resection of a parapharyngeal space pleomorphic adenoma
, local control was obtained in all 7 patients , although tumor spillage was reported in one patient .
the tors approach was found to offer reduced complication rates when compared to the transcervical approach [ 79 , 80 ] .
, in which 6 complete and 2 partial resections were performed using a suprahyoid port , while the other arms were placed transorally . in another report , hanna et al .
obtained excellent access to the anterior and central skull base in cadavers , including the cribriform plate , fovea ethmoidalis , medial orbits , planum sphenoidale , nasopharynx , pterygopalatine fossa , and clivus .
in addition , sella turcica and suprasellar and parasellar access was achieved using the robotic arms .
however , there is a continuing need for further development of appropriate instruments , in terms of size , flexibility , and function .
although there are studies of robotic surgery thyroidectomy in children [ 47 , 48 ] , which we have discussed previously , studies of robotic surgery in the pediatric population are sparse .
to date , the only pediatric case series is that described by rahbar et al . in 2007 at children 's hospital boston . in this study ,
4 pediatric cadaver larynxes were used to assess precision and tissue handling using a robotic - system .
equipment size was the main limiting factor for these procedures , resulting in limited transoral access in 3 of 5 the patients .
the other 2 patients , who had type 1 and type 2 laryngeal clefts , had successful surgical repairs using the robotic system .
the trend towards the use of minimally invasive surgery has had an impact on the way new technology is thought of , developed , and incorporated into clinical practice .
it is improving the outcomes , such as reducing hospital stays and infection rates , and allowing for better cosmetic results .
however , surgical robots were developed to perform procedures in spacious cavities , such as the abdomen , and thus , the instruments are over sized to perform many of the otolaryngology and head and neck procedures .
the da vinci robot system is starting to be adopted to carry out a number of otolaryngology procedures , and it has done so with excellent results so far
. other limitations of robotic surgery are like the large size of the robotic system , which necessitates additional manpower to set it up and creates new challenges for the anesthesia team and surgical assistants .
unfortunately , the high cost of the robotic equipment forbids its routine presence and use in most operating rooms across the globe .
this calls for the development of smaller , less expensive and easy to operate robotic platforms , which are portable and flexible to use , as well as specific instruments for tasks in head and neck surgery . besides the evidence of robotic feasibility and safety in head and neck surgery , postoperative outcomes regarding airway management and oropharyngeal function are comparable or better to traditional surgical approaches .
although we did not explore the details concerning oncologic results , robot - assisted surgery showed a trend towards favorable cure and recurrence rates .
this can be attributed to its capability to resect tumour en - bloc a feature that is provided by the increased dexterity and 3d visualization of the robotic system .
we believe that future studies comparing robotic techniques to transoral laser microsurgery ( tlm ) , open surgery and chemoradiotherapy are required to support these assertions .
reported studies are supportive of the feasibility and safety of robotic surgery in head and neck procedures and encourage its continuing use and exploration .
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recent advancements in robotics technology have allowed more complex surgical procedures to be performed using minimally invasive approaches . in this article
, we reviewed the role of robotic assistance in otolaryngology and head and neck surgery .
we highlight the advantages of robot - assisted surgery and its clinical application in this field .
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the satisfaction of basic needs constitutes quality of life ( qol ) which is related to health .
these are : physiological needs , safety needs , need of love and affection , need to belong , need for esteem and need for self actualization .
each of these needs in the hierarchy has to be satisfied before the higher need can be met .
understanding these needs is important because the greater the acquisition of knowledge of people 's natural way of being , the less difficult it becomes to guide people about how to fulfill their greatest potential , how to respect the self , how to love and be productive , and how to be good and happy .
this transcendence occurs because these people are guided by internal values and rules that foster a self - governing character , detachment and independence .
scores on belief in an internal locus of control and neuroticism were predicted by maslow 's need for satisfaction .
maslow argues that people have the potential for growth and innate goodness , and are able to strive when faced with adversity .
women associated a comfortable life , pleasure , values and happiness with health , unlike men who associated health with national security and family .
the values of women satisfied their fundamental needs , while those of men satisfied their higher order needs .
this difference suggests that men can be motivated to engage in healthy behavior after they have fulfilled their more fundamental needs , compared to women who may strive for health before they are motivated by other needs . however , there is no gender difference in self - actualization scores , but women score lower on perceived self - presentation , confidence , physical self - efficacy and perceived physical ability .
therefore , caring for these patients requires that their self - actualization and self - esteem needs are met , and not just their physiological health .
in addition , the unmet physiological and safety needs of patients who suffer from chronic vestibular dysfunction means that these patients can not progress to higher order needs .
maslow 's hierarchy of needs is also important for health education because the status of people 's basic needs influences their health - promoting self care behavior .
some 64% of the variance in health - promoting self - care behavior was influenced by the physical : love , belonging , need , satisfaction and self - actualization .
unhealthy behavior and health disparities based on race and class can be reduced through health promotion programmes that respond to the basic needs of people , which will allow them to achieve self-actualization.this self - actualization influences the quality of life .
the hierarchy of needs was applied to the development of the quality of life in 88 countries between 1964 and 1994 .
there is a significant association between the predictions of maslow 's theory and the quality of life , including part of the s - shaped course and the sequence of needs achievement which influences health .
published evidence on the health status and quality of life of jamaicans is lacking , and not much research has been done in this area in the english - speaking caribbean .
this study examined how jamaicans conceptualize health and quality of life , and investigated any possible relationship between the two variables .
the current study utilized two different cross - sectional probability surveys which were conducted in 2007 to examine the health status and quality of life of jamaicans .
the studies were conducted by ( 1 ) the centre for leadership and governance ( clg ) , department of government , the university of the west indies ( uwi ) , mona , and ( 2 ) the planning institute of jamaica ( pioj ) and the statistical institute of jamaica ( statin )
the sample for the current study was 8,120 participants : 1,338 from the clg and 6,782 from the jslc .
each survey was independently collected by the organization , and both the clg and the jslc collected data at the same time . during the months of july and august 2007 , clg conducted a stratified probability sample of 1,338 respondents .
face - to - face interviews were used to collect the data on an instrument which took about 90 minutes .
the instrument consisted of questions about abraham maslow 's hierarchy of needs ( physiological needs , safety needs , social needs , self - esteem and self - actualization ) which were used to determine the participant 's quality of life .
it was vetted by senior scholars , researchers , and interviewers from statin and the social development commission ( sdc ) .
after the vetting phase , the questionnaire was pre - tested in a number of communities across the 14 parishes of jamaica , as well as among uwi faculty members and the student population .
modifications were made at a training symposium , based on the comments of the different interviewers and the remarks of trained researchers .
all the interviewers employed by the clg 's team were data collectors from either statin or sdc .
the interviewers who are trained data collectors underwent further training with the clg team for a 3-day period .
the project manager of clg travelled across the country to verify the data collection process .
a data template was created before the data was entered and data entry clerks were trained to work with the instrument .
three different groups independently entered the data , which was cross - referenced and reviewed for accuracy by two members of the research team , who also validated the data entry process and cleaned the data .
the jslc was commissioned by pioj and statin in 1988 , and these organizations have been collecting data since 1989 .
the jslc is done through the administering of questionnaires modeled on the world bank 's living standards measurement study ( lsms ) household survey .
the jslc questionnaire consists of variables dealing with demographics , health , the immunization of children aged 0 - 59 months , education , daily expenses , non - food consumption expenditure , housing conditions , inventory of durable goods and social assistance .
quality of life was defined as the overall self - reported life satisfaction of an individual .
it was measured as the mean summation of the five - item needs from abraham maslow 's hierarchy .
these items were physiological needs , safety needs , social needs , self - esteem and self - actualization .
using cronbach alpha for the five - item scale , reliability was 0.841 ( or = 84% ) .
qoli = 1/5*ni where i is each need ( i.e. i = 1 , 2 , 3 , 4 , 5 ) , and where the qol index is : 0qoli10 . cohen and holliday stated that correlation can be very low / weak ( 0.0 - 0.19 ) ; weak ( 0.2 - 0.39 ) ; moderate ( 0.4 - 0.69 ) , strong ( 0.7 - 0.89 ) and very strong ( 0.9 - 1.0 ) . cohen and holliday 's interpretation will be applied to categorizing qol i into five groups : very poor ( values range from 0 to 1.9 ) ; poor ( values from 0.2 to 3.9 ) ; moderate ( values from 4.0 to 6.9 ) , good ( values ranging from 7.0 to 8.9 ) and very good ( values ranging from 9 to 10 ) .
health status was measured by the question generally , how do you feel about your health ? answers to this question were on a likert scale ranging from excellent to poor .
quality of life was defined as the overall self - reported life satisfaction of an individual .
it was measured as the mean summation of the five - item needs from abraham maslow 's hierarchy .
these items were physiological needs , safety needs , social needs , self - esteem and self - actualization .
using cronbach alpha for the five - item scale , reliability was 0.841 ( or = 84% ) .
qoli = 1/5*ni where i is each need ( i.e. i = 1 , 2 , 3 , 4 , 5 ) , and where the qol index is : 0qoli10 .
cohen and holliday stated that correlation can be very low / weak ( 0.0 - 0.19 ) ; weak ( 0.2 - 0.39 ) ; moderate ( 0.4 - 0.69 ) , strong ( 0.7 - 0.89 ) and very strong ( 0.9 - 1.0 ) .
cohen and holliday 's interpretation will be applied to categorizing qol i into five groups : very poor ( values range from 0 to 1.9 ) ; poor ( values from 0.2 to 3.9 ) ; moderate ( values from 4.0 to 6.9 ) , good ( values ranging from 7.0 to 8.9 ) and very good ( values ranging from 9 to 10 ) .
health status was measured by the question generally , how do you feel about your health ? answers to this question were on a likert scale ranging from excellent to poor .
quality of life was defined as the overall self - reported life satisfaction of an individual .
it was measured as the mean summation of the five - item needs from abraham maslow 's hierarchy .
these items were physiological needs , safety needs , social needs , self - esteem and self - actualization .
using cronbach alpha for the five - item scale , reliability was 0.841 ( or = 84% ) .
qoli = 1/5*ni where i is each need ( i.e. i = 1 , 2 , 3 , 4 , 5 ) , and where the qol index is : 0qoli10 .
cohen and holliday stated that correlation can be very low / weak ( 0.0 - 0.19 ) ; weak ( 0.2 - 0.39 ) ; moderate ( 0.4 - 0.69 ) , strong ( 0.7 - 0.89 ) and very strong ( 0.9 - 1.0 ) .
cohen and holliday 's interpretation will be applied to categorizing qol i into five groups : very poor ( values range from 0 to 1.9 ) ; poor ( values from 0.2 to 3.9 ) ; moderate ( values from 4.0 to 6.9 ) , good ( values ranging from 7.0 to 8.9 ) and very good ( values ranging from 9 to 10 ) .
health status was measured by the question generally , how do you feel about your health ? answers to this question were on a likert scale ranging from excellent to poor .
in examining the demographic characteristics of the sample as well as qol and health status , forty three percent of the clg 's respondents ( n = 1338 ) were males compared to 49% for the jslc ( n = 6,782 ; table 1 ) .
fifty - four percent of clg 's respondents indicated at least good qol ( of which 10.3% claimed very good ) compared to 82.2% of those in the jslc who indicated at least good health status ( of which 37% mentioned very good ) .
demographic characteristics of sample for clg and jslc , 2007 a statistical relationship was found between qol and gender [ qol ( df = 4 ) = 11.9 , p < 0.018 ] , and health status and gender [ jslc ( df = 4 ) = 46.5 , p < 0.0001 ; table 2 ] .
a cross - tabulation between qol and area of residence revealed no significant statistical relationship [ qol ( df = 4 ) = 6.98 , p < 0.137 ; table 3 ] .
however there was a significant relationship between health status and area of residence ( jslc ( df = 4 ) = 27.51 , p < 0.0001 ] .
quality of life and health status by gender of respondents , clg and jslc quality of life and health status by area of residence , clg and jslc using the standardized health status and qol a significant statistical association was found between the two variables [ ( df = 4 ) = 388.9 , p < 0.0001 ; table 4 ] .
in addition , a statistical relationship was found between the two variables [ ( df = 16 ) = 85.477 , p < 0.0001 ; table 5 ] .
quality of life , health status and standardized health status qol by economic situation of individual and family , clg using data from jslc 's survey , a statistical relationship was found between the health status and self - reported illness of respondents variables [ ( df = 4 ) = 1323.470 , p < 0.0001 ] .
the statistical association was moderate , as given by the contingency coefficient with a value of 0.450 . of those who indicated that they had an illness ( n = 976 )
, 3.0% claimed very poor ; 17.4% said poor ; 36.8% indicated moderate ; 31.3% mentioned good and 11.6% reported very good health status .
in the same way , of those who indicated that they had not experienced an illness in the last 4-week period ( n = 5569 ) , 0.4% reported very poor health status ; 1.8% said poor ; 8.7% moderate health status ; 47.7% claimed good and 41.4% reported very good health status .
the majority of the respondents in the clg and jslc surveys stated that they had good health status .
the jslc survey had the greater majority with 28.2% more of the respondents stating that they had good health status than their counterparts in the clg survey .
for both surveys there was no significant gender difference in terms of qol as there was a weak statistical relationship between gender and qol .
this latter finding suggests that men and women view their quality of life or basic needs similarly , despite the patriarchal nature of jamaican society and the attendant gender inequality .
there was also a weak statistical relationship between the economic situation of the respondents and their families , and qol .
this finding suggests that the respondents in their self - reports did not view their economic status as influencing their qol .
therefore , in the respondents understanding of their basic needs there are other explanatory factors that will have to be explored in future research .
there was a significant difference in the health status of the respondents in rural and urban areas because there was a strong statistical relationship between area of residence and health status , unlike the relationship between area of residence and qol .
these findings suggest that the respondents , in their self - reporting , view their health status and their qol dichotomously , which is different from the results obtained in previous studies .
moreover , in the current study a significant relationship was found between qol and health status .
this finding suggests that although qol and health status are related , they are viewed by the respondents as dichotomous domains in their lives .
the dichotomous conceptualization of qol and health status may be explained by the finding that a significant relationship was found between health status and self - reported illness .
the respondents in this study viewed their health status based on the absence or presence of an illness , and did not include qol .
the respondents exclusion of basic needs in their health status suggests that their conceptualization of health as the presence or absence of illness is culturally determined , because this is different from the findings of previous studies .
therefore , within the jamaican culture qol is multi - dimensional and health status is one - dimensional , so these conceptualizations are antonymous .
the preponderance of illness accounting for most of health is not atypical to jamaica , as a study conducted by hambleton et al .
's work found that 88% of the variability in health status was accounted for by current illness . while this study can not allude to the generalizability of this to the caribbean , clearly in both of the aforementioned nations , health still carries a narrow definition .
this narrow definition of health was the justification of the world health organization 's ( who ) concept of health in 1948 .
the who postulated a definition which states that health is more than the absence of disease , as it includes social , psychological and physical wellbeing .
this is a negative approach to the image and study of health , and does not encompass wellbeing or the positive side to health .
both the who in the preamble to its constitution in 1948 , and engel[2426 ] , have sought to conceptualize and provide a rationale for the image and study of health that extends beyond illness or the antithesis of disease . despite the contributions of social scholars as well as the who and engel to the discourse of health , in contemporary jamaica the image of health is still the antithesis of illnesses .
bok opined that the who 's conceptualization of health is too broad , and therefore poses a problem to operationalize in research .
embedded in bok 's claim is the difficulty in quantifying social and psychological conditions in health , and explaining the use of diagnosed illnesses , mortality and life expectancy instead of wellbeing . like other scholars[2830 ] , bok sees health as an objective phenomenon which explains the use of life expectancy , diagnosed illness and mortality .
life expectancy relies on mortality data , and while it is an objective measure of the health of people or a society , it is similar to the use of the antithesis of illness and not wellbeing .
it is this narrow approach to the use of life expectancy that justifies the world health organization 's ( who ) introduction of healthy life expectancy . recognizing the limitations of life expectancy , the who discounted life expectancy for disability .
disability adjusted life expectancy ( dale ) summarizes the expected number of years of life of an individual , which might be termed the equivalent of full health .
to calculate dale , the years of ill health are weighted according to severity , and subtracted from the expected overall life expectancy , to give the equivalent years of healthy life . this study has contributed to our understanding of health status by highlighting the culture - bound conceptualization of health status in jamaica , which is different from how it is conceptualized in the literature which includes qol .
another contribution is the generalization of the findings , with the combination of the findings from two large - scale random national surveys .
. we did not measure the factors influencing how jamaicans conceptualize illness which would inform interventions . also , the clg and jslc surveys relied on self - reports so there was the possibility of social desirability bias , where the respondents might have told the interviewers what they wanted to hear to get their approval .
qol is concerned with how people assess their lives which includes a wide range of issues from health , life satisfaction , momentary moods , economic wellbeing , happiness to needs satisfaction and a global assessment of all aspects of life[3234 ] .
health status , however , is synonymous with physical health ( illness ) which means that collecting data on illness and self - rated health status is one of the same and therefore adds nothing new to understanding general health as defined by the who . in keeping with the broad definition of health forwarded by the who , qol should be used in addition to illness or self - rated health status , as self - reported illness and self - rated health status
jamaicans view their health status and their qol as distinct domains in their lives . this surprising distinction is culturally determined because the difference has not been empirically observed elsewhere except barbados .
the exclusion of qol or basic needs from their conceptualization of health status should be noted by medical practitioners and researchers when they assess the health of jamaicans .
the aforementioned findings highlight that collecting data on health status and illness in jamaica is one and the same , and therefore other subjective indices such as qol , life satisfaction and happiness would yield more information than health status and/or illness .
if health is multi - faceted , then health status would not be a good measure of this broad conceptualization .
further research is needed to uncover the reasons for the one - dimensional view jamaicans have of their health status , and how this conceptualization affects their health .
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background : health is defined as the presence or absence of illness . this conceptualization of health status is dominant in health treatment and in fashioning the health care system .
however , very little research has been done on how jamaicans view health status and quality of life ( qol).aims : this article seeks to understand how jamaicans conceptualize health status and qol because definitional content has implications for their health.material and methods : the current study utilized two national cross - sectional probability surveys from the centre for leadership and governance ( clg ) which looked at qol among other variables and the jamaican survey of living conditions ( jslc ) which measured living standards including health status .
the sample in both surveys was 8,120 participants.results:the majority of the respondents in the clg ( 54% ) and the jslc ( 82.2% ) surveys reported good health status .
there was a strong statistical relationship between area of residence and health status ( p < 0.0001 ) unlike the relationship between area of residence and quality of life ( p < 0.137 ) .
the respondents dichotomized health status and qol and a significant relationship was found between both variables ( p < 0.0001 ) .
the respondents dichotomization of health status and qol is explained by the significant relationship between health status and self reported illness ( p < 0.0001 ) where respondents view health status as the absence or presence of illness , excluding qol.conclusion:health status means the presence or absence of illness and excludes qol which is not in keeping with previous findings .
this distinction is culturally determined .
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hepatocellular carcinoma ( hcc ) is the third leading cause of death from cancer worldwide and is the ninth leading cause of cancer - related deaths in the united states .
hepatogastric fistula ( hgf ) development is a rare complication of transcatheter arterial chemoembolization ( tace ) of the hepatic artery for hcc .
tace is performed as a palliative measure for unresectable malignant tumors to reduce pain and slow growth of the mass by producing vascular compromise leading to necrosis of the mass .
such tissue hypoxia can be destructive to normal tissue and can increase the tumor burden leading to metastatic disease .
an unfortunate complication of tace is an abscess development that occurs in less than 1% of the cases .
it is hypothesized that the ischemic necrosis that follows the tace procedure may partially be responsible for the occurrence of hgf .
the complications from this aberrant communication of visceral organs lead to rapid status decline and to patient opting for hospice care .
we present a case and literature review of hgf following tace , which was performed to provide palliative care for an unresectable hepatitis b virus - related hcc tumor .
a 51-year - old caucasian male with chronic hepatitis b virus - associated cirrhosis and biopsy - proven hcc was treated with tace for an unresectable malignant liver mass . at his initial presentation ,
the patient complained of right upper quadrant pain , a 20-lb unintentional weight loss ( initial weight : 180 lbs , weight at presentation : 160 lbs ) , and an epigastric mass .
the laboratory results were significant for anemia of chronic disease , elevated liver enzymes with a total bilirubin of 2.2 mg / dl , marked thrombocytopenia , and an elevated international normalized ratio of 1.72 .
the patient was child class b and had a model for end - stage liver disease ( meld ) score of 21 .
an abdominal computed tomography ( ct ) scan showed a heterogeneous , hypervascular mass arising in the lateral segment of the lower left lobe of the liver ( segments ii and iii ) measuring 11.5 9.5 5.3 cm in size suggestive of hcc .
the patient subsequently underwent tace of the hepatic mass , which later was complicated by a liver abscess . out of concern for infectious etiology ,
approximately 6 months following the first tace , the patient presented for his second tace . at that time , he reported a recent history of melena and coffee ground emesis , which were suggestive of an upper gastrointestinal hemorrhage . unfortunately , the patient could not accurately correlate the symptoms in relation to the first tace .
a gastroenterology consultation was requested , and the team suspected a fistulous connection between the hcc mass and the stomach , which was seen on the abdominal ct ( fig .
the patient underwent esophagogastroduodenoscopy that revealed a 2-cm ulcer to the lesser curvature of the stomach communicating with the liver , forming a hgf ( fig .
surgical consultation again recommended supportive care after considering the patient 's advanced disease status and poor candidacy for surgical intervention .
gastroenterology recommended the use of proton pump inhibitors and the avoidance of nasogastric and orogastric tubes to prevent further complications while hospitalized .
ct and esophagogastroduodenoscopy results along with poor prognosis secondary to the advanced disease were discussed with the patient , following which , the patient opted for hospice care .
direct invasion of hcc to adjacent organs is one of the most common routes of metastasis along with hematogenous and lymphatic spread .
the most common site of direct tumor invasion is the stomach followed by the duodenum and colon .
most metastases to the gastrointestinal tract ( git ) are found incidentally secondary to their asymptomatic presentation , but bleeding from the upper git is the most common initial presentation [ 6 , 7 ] .
risk factors for the development of hgf include liver cirrhosis , local or regional radiation , previous tace of the liver or stomach , large lesions from hcc which are close to the git , and hepatic abscess .
indications for tace include : confirmed diagnosis of hcc , absence of extrahepatic metastases , targeted tumor involving 50% or more of the liver parenchyma , disease recurrence after potentially curative treatment , and if ablation is unfeasible [ 8 , 9 ] . a further indication is given in a potential transplant recipient requiring bridge therapy to minimize the drop - off rate from the transplant list .
contraindications to tace include : portal vein thrombosis , portal flow reversal , and child class c cirrhosis [ 10 , 11 ] .
contraindications to the chemotherapeutic agent and anaphylactic reactions to the contrast media also remain to be considered . when considering tace as a therapy option , the metabolic status of the candidate must be evaluated .
serum creatinine must be 2 mg / dl , platelet count 50,000/mm , and prothrombin activity 50% .
few case reports of hgf following tace exist , and the mechanism is not clearly understood , with even less literature available concerning treatment options .
while there is no widely accepted pathophysiologic mechanism for hgf development , a review of the literature suggests that a direct invasion of hcc to the stomach is more likely to develop in patients with a history of loco - regional treatment such as tace or intra - arterial chemotherapy [ 13 , 14 ] .
as such , we can hypothesize that the direct tumor invasion might result from the adhesion of hcc after loco - regional treatment .
tace for hcc might induce an inflammatory reaction along with local tissue hypoxia in the proximity , which causes the serosal side of the stomach to become adherent to a tumor capsule [ 13 , 15 ] .
park et al . reported that the main factors for the direct invasion were growth pattern , tumor size , and location rather than a previous history of loco - regional treatment . in the case
documented above , the patient was treated with repeated tace , and there was a fistulous connection noted between the left lobe of the liver and the stomach following the procedure .
all the factors discussed above and the abscess formation might have contributed to a direct gastric invasion by hcc and resulted in the formation of hgf . regardless of the etiology , this inappropriate communication between the stomach and the liver results in hepatogastric irritation and inflammation that leads to symptoms such as pain , hemorrhage , melena , and hematemesis .
closer surveillance is warranted in patients undergoing successive tace or radiotherapy for hcc close to the git , as complications including fistula , abscesses , hemorrhage , and perforation can develop leading to devastating consequences .
more studies are needed to indicate the potential benefits of routine surveillance with ultrasound or ct scan for an early detection of asymptomatic fistulas that represent disease metastasis .
quality of life and surgical risks should also be taken into consideration before undertaking additional procedures . in conclusion ,
hgf secondary to an invasion by hcc is rare and should be included in the differential diagnosis of patients presenting with upper git bleed who have undergone tace for advanced hcc .
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hepatogastric fistula ( hgf ) formation following transcatheter arterial chemoembolization ( tace ) leads to increased morbidity and mortality .
a 51-year - old caucasian male with chronic hepatitis b virus - associated cirrhosis and unresectable hepatocellular carcinoma ( hcc ) presented to the interventional radiology unit for tace to achieve tumor necrosis .
following the procedure , the patient was admitted with symptoms of fever , epigastric and right upper quadrant pain secondary to the development of an abscess .
the abscess was drained ; however , an exceedingly rare hgf resulted that was favored to represent a direct invasion of hcc .
hgf , the rare complication following tace , leads to grave consequences and vigilant monitoring , for the development of this entity is recommended to reduce patient mortality .
we present a case and literature review of hgf development following tace for hcc .
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epstein - barr virus ( ebv ) is associated with a variety of lymphoproliferative disorders ( lpds ) and other malignancies [ 15 ] , including nasopharyngeal carcinoma , hodgkin disease , and burkitt lymphoma [ 69 ] .
ebv - driven b - cell lpds can be age - related or can occur in patients who are immunosuppressed due to primary immune deficiency , hiv infection , organ transplantation , and treatment with methotrexate or tumor necrosis factor- antagonist for rheumatoid arthritis [ 10 , 11 ] .
the major ebv oncogene , latent membrane protein-1 ( lmp-1 ) , activates signaling pathways such as those involving nuclear factor - kappa - light - chain - enhancer of activated b - cells ( nf-b ) , which enhances b - cell survival and is essential for ebv - induced transformation [ 1216 ] .
lmp-1 is a 63 kda integral membrane protein with three domains and contains two distinct functional regions within its c - terminus , designated c - terminal activating regions 1 and 2 ( ctar1 and ctar2 ) .
the protein also protects cancer cells from apoptosis , by inducing antiapoptotic proteins , including bcl-2 , mcl-1 , a20 , early growth response transcription factor-1 ( egr-1 ) , and snark [ 1719 ] .
recent studies have shown that ebv - infected cells undergo hypermutation or switching of recombination in vivo via upregulation of activation - induced cytidine deaminase ( aid ) and also that ebv - induced aid is associated with oncogene mutations , which contribute to lymphomagenesis .
the relationship between lmp-1 and cancer has been relatively well established , while the molecular mechanisms underlying aid induction remain to be fully clarified .
aid is normally expressed in germinal center ( gc ) b - cells , where it plays a central role in both somatic hypermutation and class switch recombination in humans and mice [ 23 , 24 ] .
aid converts single - stranded genomic cytidine into uracil , with pronounced activity in the immunoglobulin variable and switch regions [ 2528 ] .
aberrant expression of aid and abnormal targeting of aid activation in both b- and non - b - cells cause dna double - strand breaks ( dsbs ) and dna point mutations in both ig and non - ig genes , inducing tumorigenesis . aid is required for chromosomal dsbs at the c - myc and igh loci , which lead to reciprocal c - myc / igh translocations , resulting in the development of b - cell lymphomas , such as burkitt lymphoma in humans and plasmacytoma in mice .
aid protein is localized more in the cytoplasm than in the nucleus in normal and neoplastic b - cells , and cytoplasmic aid protein relocates to the nucleus when pathological change occurs in b - cells [ 31 , 32 ] . a recent in vitro study by kim et al .
has shown that lmp-1 increases genomic instability through egr-1-mediated upregulation of aid in b - cell lymphoma cell lines . however , to our knowledge , no clinicopathological case study has examined the expression of lmp-1 , aid , and egr-1 , including the distribution and density of positive cells , on lpds .
it is therefore important to clarify the expression pathway and distribution of positive cells in lesions of human tissues .
we considered that aid positive cells would be more numerous in the ebv - driven lpds than in dlbcls showing a monotonous growth pattern .
therefore , we conducted an immunohistochemical study to investigate the relationship between lmp-1 , aid , and egr-1 expression in lpds ( mtx-/age - related ebv - associated ) , including dlbcls .
a total of 29 biopsy specimens were retrieved from the three hospitals to which the authors have contributed pathological diagnosis and were presented for investigation .
tissue samples from 17 cases of mtx - ebv - lpd , 2 cases of age - ebv - lpd , and 10 cases of dlbcl were used ( table 1 ) .
sporadic - burkitt lymphoma ( sbl ) , mtx - lpd , age - lpd , and oral squamous cell carcinoma ( oscc ) were used as an overexpressing positive control for aid , lmp-1 , ebv - encoded small rna ( eber ) , and egr-1 .
ten samples of cervical lymph nodes ( lns ) showing reactive lymphoid hyperplasia ( rlh ) were used as normal positive controls for aid , lmp-1 , eber , and egr-1 .
each section was prepared for immunohistochemical analysis ( ihc ) and in situ hybridization ( ish ) .
the case study protocol was reviewed and approved by the research ethics committee of meikai university school of dentistry ( a0832 , a1321 ) .
deparaffinized sections were immersed for 15 min at room temperature in absolute methanol containing 0.3% h2o2 to block endogenous peroxidase activity and then treated with 2% bovine serum albumin for 15 min to block nonspecific reactions .
after washing , they were incubated with an appropriately diluted mouse monoclonal antibody against human lmp-1 and rabbit polyclonal antibodies against aid and egr-1 ( table 2 ) .
after washing , the sections were incubated with a prediluted anti - mouse or rabbit igg antibody conjugated with peroxidase ( nichirei , tokyo , japan ) for 30 min at room temperature .
they were then immersed for 8 min in 0.05% 3,3-diaminobenzidine tetrahydrochloride ( dab ) in 0.05 m tris - hcl buffer ( ph 8.5 ) containing 0.01% h2o2 and counterstained with mayer 's haematoxylin for 90 s. ish for eber oligonucleotides was performed to detect the presence of ebv small rna in formalin - fixed paraffin - embedded sections using a hybridization kit ( dako , a / s , denmark ) in accordance with the manufacturer 's instructions .
reactivity for each of the antigens and eber was evaluated semiquantitatively using a light microscope ( model bh2 , olympus corp . ) .
the distribution of the staining was categorized semiquantitatively according to the ratio of the positive area as follows : diffuse ( + + + ) 75% ; focal ( + + ) < 75% to 25% ; partial ( + ) < 25% to 5% ; few ( / ) negative/<5% or nonspecific .
the intensity of the staining was categorized semiquantitatively as strong ( s ) , moderate ( m ) , weak ( w ) , or negative ( n ) relative to each control specimen .
aid intensity was compared with that in the sbl case sample used as a positive control and expressed as strong ( s ) when higher or of the same intensity as that in sbl , moderate ( m ) when lower than that in sbl or of the same intensity as that in rlh , weak ( w ) when lower than that in rlh , and negative ( n ) in case of no staining or nonspecific staining .
lmp-1 and eber intensity were compared with those in mtx - lpd and age - ebv - lpd used as a positive control [ 35 , 36 ] and evaluated in the same manner as those for aid .
egr-1 intensity was compared with that in oscc used as a positive control and evaluated in the same manner as that for aid .
the significance of differences between the mean values was determined by using the mann - whitney u test or exact binominal test for comparing two categories .
strong aid , lmp-1 , eber , and egr-1 reactivity were observed in overexpressing positive control specimens in sbl , mtx - lpd , age - lpd , and oscc by ihc and ish ( figures 1(a)1(d ) ) .
moderate aid , lmp-1 , eber , and egr-1 reactivity were observed in normal positive control specimens rlm by ihc and ish ( figures 1(e)1(h ) ) .
aid expression was diffuse and strongly positive in mtx-/age - ebv - lpds ( figures 2(a ) , 2(b ) , 2(d ) , and 2(e ) ) and was few and moderately positive in dlbcls ( figures 2(c ) and 2(f ) ) .
although lmp-1 expression was diffuse and strongly positive in mtx-/age - ebv - lpds ( figures 3(a ) , 3(b ) , 3(d ) , and 3(e ) ) , the expression was few and weakly positive in dlbcls ( figures 3(c ) and 3(f ) ) .
eber expression was sporadic diffuse and strongly positive in mtx-/age - ebv - lpds ( figures 4(a ) , 4(b ) , 4(d ) , and 4(e ) ) , while eber reactivity was negative in dlbcls ( figures 4(c ) and 4(f ) ) .
expression of aid , lmp-1 , and eber was higher in mtx-/age - ebv - lpds than in dlbcls . staining patterns , aid , lmp-1 , and eber ,
the distribution of aid ( p < 0.000005 ) , lmp-1 ( p < 0.05 ) , and eber ( p < 0.00001 ) expression was significantly more extensive in the mtx-/age - ebv - lpds than in the dlbcls ( figures 5(a)5(c ) ) .
in addition , aid expression was significantly more intense in mtx-/age - ebv - lpds than in dlbcls ( p < 0.000005 ) ( figures 6(a ) and 6(b ) ) , and expression of lmp-1 ( p < 0.0005 ) ( figures 6(c ) and 6(d ) ) and eber ( p <
0.0001 ) was more intense in mtx-/age - ebv - lpds than in dlbcls ( figures 6(e ) and 6(f ) ) .
the high intensity ( strong and moderate ) of aid , lmp-1 , and eber expression was greater in mtx / age - ebv - lpds ( 89.5% , 68.4% , and 94.7% ) than in dlbcls ( 20.0% , 10.0% , and 20.0% ) ( figures 6 and 7 ) .
conversely , the low intensity ( weak and negative ) of aid , lmp-1 , and eber expression was greater in dlbcls ( 80.0% , 90.0% , and 80.0% ) than in mtx / age - ebv - lpds ( 10.5% , 31.6% , and 5.3% ) ( figures 6 and 7 ) . in mtx-/age - ebv - lpds ,
the intensity of aid , lmp-1 , and eber expression was stronger than in dlbcls ( figure 7 ) .
egr-1 expression was diffuse and strongly positive in mtx-/age - ebv - lpds ( figures 8(a)8(c ) ) and was a positive variety in the dlbcls ( figures 8(d)8(f ) ) .
distribution of egr-1 expression was significantly more extensive in mtx-/age - ebv - lpds than in the dlbcls ( p < 0.001 ) ( figure 9(a ) ) .
the intensity of egr-1 was significantly different between mtx-/age - ebv - lpds and dlbcls ( p < 0.01 ) ( figures 9(b ) and 9(c ) ) .
although the high intensity of egr-1 expression was comparable rate in both mtx-/age - ebv - lpds ( 94.4% ) and dlbcls ( 80.0% ) , strong intensity was higher in mtx-/age - ebv - lpds ( 83.3% ) ( figure 9(b ) ) than in dlbcls ( 30.0% ) ( figure 9(c ) ) .
immunohistochemical analysis in this study revealed that the expression of aid , lmp-1 , and egr-1 had a much more diffuse distribution and was stronger in intensity in lpd than in dlbcl cases .
furthermore , lpd cases showed a more diffuse distribution and stronger intensity of eber - ish than dlbcl cases .
ebv is associated with a variety of lpds and malignant lymphomas [ 1 , 35 ] .
ebv - driven b - cell lpds occur in patients who are immunosuppressed due to primary immune deficiency , hiv infection , or organ transplantation or patients who have received other treatments including methotrexate and tumor necrosis factor- antagonists [ 10 , 11 ] .
primary ebv infection is usually asymptomatic and leads to latent infection in memory b - cells , which do not permit viral replication .
although newly infected naive b - cells have the phenotypes of transformed cells , they are controlled by both ebv - specific cytotoxic t lymphocytes and natural killer cells unless immunity is suppressed [ 37 , 38 ] . in immunocompromised hosts ,
transformed cells become proliferating blasts that can result in symptomatic disease , such as immunodeficiency - associated lpd [ 1 , 10 , 37 , 38 ] .
lpd is characterized pathologically by focal or diffuse proliferation of atypical large b - cells including reed - sternberg - like cells with reactive components , which pose a diagnostic problem for pathologists .
the spectrum of ebv - lpd is broad , ranging from benign polyclonal reactivation lesions to monoclonal ebv - dlbcl .
the major ebv - encoded lmp-1 is an integral membrane protein , which activates signaling pathways such as that involving nf-b , which increases b - cell survival and induces transformation [ 1216 ] by inducing antiapoptotic protein [ 1719 ] .
an in vitro study has reported that ebv - infected cells undergo hypermutation or switching of recombination via aid upregulation , and ebv - induced aid is also associated with oncogene mutations , which contribute to lymphomagenesis . in a mouse bone marrow transplantation model , aid overexpression was reported to promote b - cell lymphomagenesis .
although the relationship between lmp-1 and lymphomagenesis has been relatively well established , the molecular mechanisms underlying aid induction remain to be fully clarified .
recently , kim et al . have reported that lmp-1 increases genomic instability through egr-1-mediated upregulation of aid in b - cell lymphoma . the egr-1 gene ( also named zif268 , ngfi - a , or krox24 ) encodes an 80 kda dna - binding transcription factor .
egr-1 is an exceptionally multifunctional transcription factor . in response to growth factors and cytokine signaling , egr-1 regulates cell growth , differentiation , and apoptosis .
egr-1 has been associated with ebv infection , a human gamma herpes virus closely associated with several lymphoid and epithelial malignancies .
first , egr-1 is upregulated when ebv interacts with b lymphocytes at the initial infection stage , and constitutive expression of egr-1 correlates with certain types of ebv latency in b - lymphoid cell lines .
ebv reactivation is associated with upregulation of egr-1 , and egr-1 can be induced as an ebv lytic transactivator . however , there are no reports of any clinicopathological studies on lmp-1 , aid , and egr-1 in samples of human tissue . therefore , we examined the density and distribution of aid , lmp-1 , eber , and egr-1 in 19 cases of lpd and 10 cases of dlbcl .
the distribution of aid , lmp-1 , and eber expression was more extensive in patients with lpd than in patients with dlbcl .
the intensity of aid , lmp-1 , and eber expression was higher in lpd ( 89.5% , 68.4% , and 94.7% ) than in dlbcl ( 20.0% , 10.0% , and 20.0% ) patients ( figure 7 ) .
although a higher intensity of expression was seen in lpd ( 94.4% ) and dlbcl ( 80% ) , the intensity of egr-1 expression was stronger in the former ( 83.3% ) than in the latter ( 30.0% ) ( figures 9(b ) and 9(c ) ) .
these in vivo results partly supported the previous in vitro study by kim et al . and suggest that overexpression of aid in lpds may be one process in the course of tumorigenic transformation .
the factor responsible for the lack of lymphoid tissue involvement in oral areas in patients with primary lymphoma / lpd is unclear , but there may be some association with bacteria in and around the teeth , together with chronic inflammation such as apical and marginal periodontitis .
the copy number of ebv - dna in subgingival plaque is associated with the presence of some periodontal bacteria .
a recent study has shown that periodontal disease could act as a risk factor for hiv reactivation and similarly induce ebv reactivation .
thus , there may be a relationship between aid , lmp-1 , and egr-1 expression in ebv - infected b - cells .
further studies , including the head and neck , will be needed to confirm the causal link between oral bacteria and ebv - positive lymphoma / lpds of the oral cavity .
these results suggest that increased aid expression in lpds may be part of the process of lymphomagenesis , thereby further increasing the survival of genetically destabilized b - cells .
the reason why aid , lmp-1 , and eber were expressed more in the ebv related lpds compared to dlbcl could be either the ebv infection or immunosuppression that is predominant in age - related lymphoma or in autoimmune diseases of patients taking methotrexate .
the intensity and distribution of aid expression may be an indicator for differentiating ebv - driven lpds from dlbcls .
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recent research has shown that activation - induced cytidine deaminase ( aid ) triggers somatic hypermutation and recombination , in turn contributing to lymphomagenesis .
such aberrant aid expression is seen in b - cell leukemia / lymphomas , including burkitt lymphoma which is associated with c - myc translocation .
moreover , epstein - barr virus ( ebv ) latent membrane protein-1 ( lmp-1 ) increases genomic instability through early growth transcription response-1 ( egr-1 ) mediated upregulation of aid in b - cell lymphoma .
however , few clinicopathological studies have focused on aid expression in lymphoproliferative disorders ( lpds ) .
therefore , we conducted an immunohistochemical study to investigate the relationship between aid and lmp-1 expression in lpds ( mtx-/age - related ebv - associated ) , including diffuse large b - cell lymphomas ( dlbcls ) .
more intense aid expression was detected in lpds ( 89.5% ) than in dlbcls ( 20.0% ) , and the expression of lmp-1 and eber was more intense in lpds ( 68.4% and 94.7% ) than in dlbcls ( 10.0% and 20.0% ) .
furthermore , stronger egr-1 expression was found in mtx / age - ebv - lpds ( 83.3% ) than in dlbcls ( 30.0% ) .
aid expression was significantly constitutively overexpressed in lpds as compared with dlbcls .
these results suggest that increased aid expression in lpds may be one of the processes involved in lymphomagenesis , thereby further increasing the survival of genetically destabilized b - cells .
aid expression may be a useful indicator for differentiation between lpds and dlbcls .
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impaired wound healing is a substantial problem in both type 1 and type 2 diabetes .
the wound is often resistant to conventional wound management and may ultimately threaten limb viability .
analysis of the diabetic wound microenvironment has revealed that healing impairment is characterized by a number of local cytokine and cellular abnormalities such as reduced angiogenesis , decreased collagen synthesis and wound breaking strengths .
growth factors such as platelet - derived growth factor ( pdgf ) and vascular endothelial growth factor ( vegf ) have been found to be diminished in the diabetic wound , whereas levels of matrix metalloproteinase and superoxide are elevated in diabetic wound fluid [ 3 , 4 ] .
for example , reduced wound nitric oxide ( no ) concentration has been shown in experimental diabetic animal models of acute cutaneous wound healing .
diabetic wounds have also evident reduction in cutaneous blood flow , and abnormal angiogenesis supports the theory of impaired endothelial function and consequently delayed wound repair [ 6 , 7 ] .
so , restoration of endothelial function may be a good therapeutic approach in the diabetic wound healing process .
angiotensin - converting enzyme inhibitors ( aceis ) have known restorative effects on endothelial cell functions .
aceis enhanced endothelial cell survival through activation of prosurvival signals such as akt phosphorylation and endothelial no synthase ( enos ) expression . however , aceis block the production of angiotensin ii ( ang ii ) which has many proangiogenic activities [ 8 , 9 ] .
aceis are recommended for the prevention of chronic kidney disease in diabetic patients , while the effect of aceis on diabetic wound healing is still unclear .
we aimed to investigate whether oral administration of captopril modulate wound healing and no and vegf concentration in wound fluid in an acute incisional wound model in diabetic rats .
48 male sprague - dawley rats , weighing 180 to 220 g , from razi institute of iran , were housed one per cage , maintained under controlled environmental conditions ( 12-hour light / dark cycle , temperature approximately 23c ) , and provided with standard laboratory food and water ad libitum .
the study protocol was approved by institutional animal ethics committee according to principles of laboratory animal care .
diabetes was induced by a single 65 mg / kg intravenous injection of streptozotocin ( stz , sigma , usa ) in saline - sodium citrate buffer ( sigma , inc . , st .
blood glucose levels were measured using a glucometer ( gr-102 , terumo co. , tokyo , japan ) .
two weeks after stz injection , animals with blood glucose levels above 300 mg / dl were defined as diabetic and used in the study .
the animals of the first diabetic group received captopril ( 25 mg / kg / day ) ( dm - cap25 ) .
the animals of the second diabetic group received captopril ( 50 mg / kg / day ) ( dm - cap50 ) , while the animals of the third diabetic group received only distilled water ( dm - control ) for 11 days through gavage .
the fourth group , consisting of nondiabetic rats , were given distilled water , by gavage for 11 days ( non - dm - control ) .
the doses were selected based on work by other studies [ 11 , 12 ] . in the first day of the study , after general anesthesia with sodium pentobarbital (
80 mg / kg , ip ) , hair on the back was shaved and the skin was washed with a povidone - iodine solution and then wiped with sterile water .
full - thickness wounds ( 1 cm ) were made on the dorsum of the rats . by the end of the study
, the fasting plasma glucose ( fpg ) was measured again in all animals by enzymatic method ( boehringer mannheim , germany ) .
two samples of wound fluid were collected using sterile nitrate - free absorbent paper strips placed on the edges of the wound for 10 min , in order to measure vegf in the 5th day and no levels in 5th , 7th , and 11th days of the study .
this method for the measurement has been validated for other sample types , particularly for tears [ 13 , 14 ] . for vegf measurement , protein elution from the shirmer strips
was performed by stirring the strips in 0.5 ml of buffer ( 50 mm tris , 50 mm nacl , 0.05% brij 35 , ph 7.6 ) for at least 2 h at + 4c . for wound fluid nox determinations ,
the amount of vegf in wound fluid was measured using enzyme - linked immunosorbent assay using available reagents and recombinant standards ( r&d systems , minneapolis , mn , usa ) according to manufactures instruction only in 5th day samples .
the total nitrite level of wound fluid was measured using the griess assay after conversion of no3 to no2 with the no3 reductase enzyme as described previously .
all wound tissue specimens were fixed in 10% neutral - buffered formalin for at least 24 h at room temperature .
after fixation , vertical sections to the anterior - posterior axis of the wound were dehydrated in graded ethanol , cleared in xylene , and embedded in paraffin . four - micron - thick sections were mounted on glass slides , dewaxed , rehydrated to distilled water , and stained with hematoxylin and eosin .
for histological evaluation , all slides were examined by two pathologists , without knowledge of the prior treatment , under a microscope from 20 to 100 magnification .
the histological score adopted in this study was performed according to the previous study concerning wound healing in experimental models .
all data are expressed as the mean the standard deviation ( mean sd ) .
a statistical software package , spss ( version 14 ) , was used to perform statistical analysis .
data were analyzed by analysis of variance ( anova ) , followed by a post hoc multiple comparison .
the mean of fpg levels has been illustrated in table 2 . by the end of the study
, there was no significant difference between diabetic groups , while all of the diabetic groups had significantly more fpg than non - dm control group ( p < 0.001 ) .
total nitrite / nitrate concentrations ( nox ) in biological fluids can be used as an index of nos activity . as it has been illustrated in figure 1 ,
at the 5th day dm - cap25 group had significantly more wound fluid nox level than other groups ( p < 0.001 ) .
dm - cap50 group had significantly more wound fluid nox level than dm - control group ( p < 0.001 ) , though there was no significant difference between dm - cap50 group compared to non - dm - control group .
the nox level of wound fluid in non - dm - control group was significantly more than dm - control group ( p < 0.001 ) .
at the 7th day , the dm - cap25 group had significantly more wound fluid nox level than dm - control ( p < 0.001 ) and non - dm - control groups ( p < 0.003 ) , while the difference of wound fluid nox level between dm - cap25 group and dm - cap50 group was not significant .
the nox level of wond fluid in dm - cap50 group was significantly more than dm - control group ( p < 0.001 ) and non - dm - control group ( p < 0.05 ) .
non - dm - control group had significantly more wound fluid nox level than dm - control group ( p < 0.05 ) ( figure 2 ) . at the 11th day ,
the wound fluid nox level of dm - cap25 group was more than other groups but the difference only between dm - cap25 group and dm - control group was significant ( p < 0.002 ) .
the nox level of wound fluid in dm - cap50 group was significantly more than dm - control group ( p < 0.007 ) .
non - dm - control group had significantly more wound fluid nox level than dm - control group ( p < 0.03 ) ( figure 3 ) .
the vegf levels of wound fluid were only measured in the 5th day of the study .
dm - cap25 group vegf concentration was significantly higher than other groups ( p < 0.02 ) .
vegf concentration dm - cap50 group was significantly more than dm - control group ( p < 0.05 ) .
non - dm - control group had more vegf concentration than dm - cap50 and dm - control groups , but the difference was significantly between non - dm - control and dm - control groups ( p < 0.002 ) ( figure 4 ) .
there was no significant difference in wound healing score between captopril - treated groups and non - dm - control group .
the wound healing score in captopril - treated groups and non - dm - control group was significantly more than dm - control group ( p < 0.05 ) ( figure 5 ) .
the wound healing process involves a complex interplay of cells , mediators , growth factors , and cytokines .
therefore , targeting of endothelial dysfunction may represent a promising focus for treatment of the diabetic wound .
so , in this study , we tested whether captopril as a known effective drug on the endothelial dysfunction as well as frequently used drug in diabetic patients influences diabetic wound healing . in our study , there was a significant decreased level of no in wound fluid of diabetic rats . decreased level of no parallel to impaired healing has been shown in experimental diabetic animal models of wound healing
furthermore , abnormalities in endothelial and inducible nos expression have been demonstrated in diabetic animal wound tissue when compared to nondiabetic counterparts [ 20 , 21 ] .
it has been shown that treatment of diabetic patients with aceis can improve nos - dependent responses of large peripheral vessels [ 23 , 24 ] .
the significant different levels of wound fluid no between the two doses of the ace inhibitor in 5th day of the study may show a different dose - dependent effect of aceis on the no .
it may be explained by an inverse relationship between the expression / activity of ace and enos via feedback regulation .
it has been shown that the regulation of ace is correlated with changes of the expression and activity of enos .
conversely , a decreased ace expression / activity is observed when enos expression / activity is increased .
thus , it can be postulated that more ace inhibition may be associated with more no production .
furthermore , more ace inhibition leads to further increase of endothelial - derived kinins and consequently more no production . in agreement with our results ,
vegf concentration was significantly higher in both captopril - treated groups compared to both dm and non - dm control groups .
it has been shown that impaired angiogenesis in diabetes is related to vegf signaling and in inflammation - related pathway .
the results of several studies suggest that the effect of aceis on angiogenesis is related to acei - induced potentiation of endogenous bradykinin .
li et al . showed that bradykinin receptor1 ( bk - b1r ) antagonist severely suppressed the imidapril - induced angiogenesis in angiotensin receptor 1a knockout ( at1ako ) mice , while nos inhibitor moderately attenuated the imidapril - mediated angiogenesis .
furthermore , it has been shown that proangiogenic effects of aceis are blunted in bk - b2r receptor - deficient diabetic mice .
similar to our results , gao and yu have shown that acei treatment resulted in increased level of vegf mrna and protein expression by 1.44- and 1.30-fold compared with untreated diabetic rats in hind limb ischemic model .
though , the combination of a bk - b1r antagonist to acei - treated group made the expression of vegf decreased in their study .
in addition , no can induce vegf synthesis through an hif-1-mediated pathway , and vegf enhances no production by enos [ 32 , 33 ] .
furthermore , it has been shown that proangiogenic actions of ang ii could be mediated via the angiotensin type 1 ( at1 ) receptor , in part , through activation of vegf - related pathway .
several studies suggest that ang ii promotes cell proliferation and blood vessel growth [ 35 , 36 ] . in line of these evidences
, it can be speculated that low dose of aceis would be more effective in wound healing .
it has been shown that very low dose of perindopril induces an early and sustained effect on the revascularization in ischemic tissue through a vegf - dependent pathway . in conclusion
, it seems that low dose of captopril is safe and effective in diabetic wound healing .
. it would be better to study further doses of captopril and have a captopril - treated nondiabetic control group .
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we aimed to investigate whether oral administration of captopril modulate wound healing , nitric oxide ( no ) , and vascular endothelial growth factor ( vegf ) concentration in wound fluid of diabetic rats .
48 male sprague - dawley rats were divided in four groups ( n = 12 ) .
the 36 rats were rendered diabetic by streptozotocin .
the animals of the first and second groups received 25 and 50 mg / kg / day captopril , respectively , ( dm - cap25 and dm - cap50 ) .
the animals of the third group were treated by distilled water ( dm - control ) .
control rats had no intervention .
the wound fluid level of no and vegf were measured . wound specimens were investigated histopathologically . at the 5th day , there was significantly more nox in wound fluid of dm - cap25 compared to other groups . at the 7th day , both captopril - treated groups had more nox in wound fluid compared to other groups . at the 11th day ,
both captopril - treated groups had more nox in wound fluid compared to dm - control group .
vegf concentration was significantly higher in both captopril - treated groups versus dm - control group ( p < .05 ) .
there were significant higher wound healing scores in captopril - treated groups compared with dm - control group ( p < .05 ) .
these results suggest that captopril might be useful in diabetic wound healing .
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xanthogranulomatous pyelonephritis ( xgp ) is an inflammatory condition of chronically obstructed and infected kidneys that is typically refractory to medical management .
successful laparoscopic nephrectomies have been reported , albeit with high conversion and complication rates . in this report
, we describe successful laparoscopic heminephrectomy for a horseshoe kidney moiety that was subsequently shown to have pathologic features of xgp .
we report the case of a woman who underwent laparoscopic heminephrectomy for xgp of a horseshoe kidney .
however , this is a challenging procedure that should be the domain of advanced laparoscopic surgeons .
horseshoe kidney ( hsk ) is a common congenital renal abnormality with an incidence of 1 in 400 .
horseshoe kidneys may be associated with urologic abnormalities including obstruction , reflux , and calculi .
hsks present unique challenges to the laparoscopic surgeon owing to their position , the frequency of vascular anomalies , and the isthmus of renal parenchyma connecting the 2 sides .
. it does not respond to medical therapy and has typically been managed via surgical removal .
however , due to its inflammatory nature , it poses additional challenges to a laparoscopic approach , causing debate as to the most prudent technique for nephrectomy .
we present here a case of xgp that developed in a horseshoe kidney and was surgically removed via laparoscopic heminephrectomy .
this case highlights some of the difficulties encountered in the minimally invasive management of both xgp and hsk .
a 48-year - old woman with a known hsk presented to the urology service after having multiple hospital admissions for febrile urinary tract infections .
ct angiography of the abdomen and pelvis ( figure 1 ) revealed features of the left renal moiety that were interpreted as compatible with xgp as well as a branch calculus .
tc - mag3 scanning revealed 12% differential function in the left renal moiety . the patient elected to undergo laparoscopic left heminephrectomy .
two 12-mm step ports were placed along the lateral border of the rectus muscle , and two 5-mm step ports were placed , one between the 12-mm ports and one off the tip of the left 12th rib .
dense adhesions emanated from the upper pole of the left kidney that invested the spleen and the descending colon .
the colonic adhesions were sharply divided with an ensuing 5-mm serosal tear that was intracorporeally oversewn .
the hilar dissection was also challenging , owing to intense local inflammation ; the vessels and ureters were divided with gia staplers using an endovascular load .
the isthmus was noted to be fairly thin , and a plane was created with relative ease between the isthmus and the aorta .
two loads of staples from the 45-mm endo - gia stapler cartridge divided the isthmus with excellent hemostasis .
the specimen was collected in an endo - catch bag and retrieved through a lower midline incision .
issues raised by this case report include those pertinent to laparoscopic management of xgp and those related to minimally invasive surgery for horseshoe kidneys .
laparoscopic extirpative surgery for xgp has been controversial since its initial description by bercowsky et al who noted a high complication rate and no advantage over open surgery with regards to pain or length of hospitalization .
open surgery has been regarded as the reference standard for management of xgp ; in the 41-patient series reported by korkes et al , laparoscopic nephrectomy was attempted twice and completed only once .
tobias - machado et al reported 11 successful retroperitoneoscopic and 6 attempted transperitoneal laparoscopic nephrectomies for inflammatory renal disease , 2 of which were converted to open surgery .
they found that hand assistance was very helpful , using it in all 6 transperitoneal nephrectomies .
kapoor et al performed 10 transperitoneal laparoscopic nephrectomies for pathologically confirmed xgp , noting a significant reduction in hospital stay and requiring conversion to open surgery in 20% of cases .
although it is still technically challenging , laparoscopic nephrectomy for xgp is evolving as an option for select patients and surgeons .
laparoscopic management of hsk is also an evolving field , with technical challenges arising from the aberrant location and vasculature of these kidneys .
both transperitoneal and retroperitoneal approaches have been used , and hand assistance is frequently used .
yohannes et al note that while the retroperitoneoscopic approach may provide better exposure of the renal hila , inadvertent entry into the peritoneum may be difficult to avoid given the intimate relation between the anterior aspects of the kidneys and the posterior peritoneum .
bhayani and andriole performed a transperitoneal pure laparoscopic heminephrectomy for a horseshoe kidney with a 6-cm renal cell carcinoma ( rcc ) .
transperitoneal laparoscopic partial nephrectomy has been used for tumors in hsks as reported by tsivian and colleagues , who recommend the transperitoneal approach for anterior and isthmic lesions .
molina and gill reported a partial nephrectomy for a tumor in an hsk treated using a retroperitoneal laparoscopic approach .
strategies for dividing the renal isthmus have included staplers , bipolar cautery , the harmonic scalpel , and cold shears followed by argon beam fulguration .
the vascular supply of horseshoe kidneys is variable and has been described in great detail by glodny and colleagues .
accordingly , preoperative studies are useful in understanding the vascular anatomy of a given patient .
glodny et al compared ct to conventional angiography and mri and found that when ct was used as the gold standard , conventional angiography had a sensitivity of 0.2 for identifying renal arteries , while that of mri was 0.22 .
this case represents a combination of some of the unique challenges in the laparoscopic management of both xgp and hsk . in our hands , the transperitoneal approach was successful with minimal morbidity and short convalescence .
we did not encounter any difficulty in identifying or controlling major vascular structures , and patient dissection was sufficient to free the left renal moiety from its adhesions .
we found that the endo - gia stapler divided the isthmus effectively and with minimal additional blood loss .
the operative time was quite long at 307 minutes ; this was the result of significant inflammatory adhesions involving the spleen and colon . in this case
, we felt that persistence with the laparoscopic technique was worthwhile given that progress was steadily being made , despite the tedious nature of the dissection .
moreover , the patient 's convalescence might not have been as rapid if an open technique had been used .
xgp and hsk are difficult to manage laparoscopically . however , as technique evolves , experienced laparoscopic surgeons should regard both of these entities as amenable to minimally invasive management .
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background and objectives : horseshoe kidney is a relatively common renal fusion anomaly .
there are increasing reports of successful laparoscopic management of surgical disease in this entity .
xanthogranulomatous pyelonephritis ( xgp ) is an inflammatory condition of chronically obstructed and infected kidneys that is typically refractory to medical management .
xgp has been regarded as a contraindication to laparoscopic surgery .
successful laparoscopic nephrectomies have been reported , albeit with high conversion and complication rates . in this report
, we describe successful laparoscopic heminephrectomy for a horseshoe kidney moiety that was subsequently shown to have pathologic features of xgp.methods:we report the case of a woman who underwent laparoscopic heminephrectomy for xgp of a horseshoe kidney .
we discuss surgical technique and patient outcome .
we also performed and report on a focused review of the relevant literature.results:pure laparoscopic heminephrectomy was completed by a transperitoneal approach .
the patient was discharged the day after surgery.conclusions:laparoscopy is a viable approach for the management of xgp in horseshoe kidneys . however
, this is a challenging procedure that should be the domain of advanced laparoscopic surgeons .
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evaluation of the circulatory system is essential during examination of any hospitalized patient . as a supplement to the initial clinical evaluation ,
point - of - care ( poc ) echocardiography is performed with increasing frequency , in particular among physicians in intensive care , emergency medicine and anesthesia settings .
physicians are able to evaluate the patients at the bedside and commence appropriate initiatives without further delay . however , while some pathologic findings are dichotomous and most often very apparent ( e.g. pericardial exudates or gross chamber enlargement ) others can be a challenge ( e.g. moderately impaired systolic function or discrete valvular disease ) .
in particular , quantification of systolic function is relevant in most situations ; although ejection fraction ( ef ) is probably not the most accurate and descriptive parameter of left ventricular systolic function it is widely used clinically and it remains a key criterion for different pharmacological and invasive treatment strategies .
three - dimensional echocardiography , cardiac magnetic resonance and cardiac computed tomography provide non - user dependent and reproducible estimates of ef [ 8 , 9 ] . however , none of the methods are feasible as poc examinations . in consequence ,
assessment of ef at the bedside has to rely on imprecise surrogate markers , visual estimates or manual tracing of the endocardial border .
previous attempts have been made to circumvent the issues with user - dependent assessment of ef by means of semi - automated endocardial tracking .
however , difficulties with gain - dependence and suboptimal tracing throughout the cardiac cycle have limited the applicability . a relatively new semi - automated method for estimation of ef
the method is named autoef ( ge healthcare , horten , norway ) and is based on angle - independent speckle tracking for detection of the myocardium throughout the cardiac cycle [ 13 , 14 ] . however , no studies have so far assessed the feasibility and diagnostic performance of autoef measurements at the bedside .
the aim of this study was to compare estimation of ef at the bedside by autoef compared with conventional methods and to assess feasibility and time consumption .
the study was performed in accordance with the helsinki declaration and informed consent was obtained from all patients .
the study was reviewed by the central denmark region committees on biomedical research ethics and due to the design of the study it was exempt from further ethical approval .
patients undergoing a standard echocardiographic examination at the department of cardiology , aarhus university hospital were eligible for inclusion .
the selection process was performed by an independent nurse and physician affiliated with the study . during data collection , patients were screened and included consecutively to avoid selection bias .
all eligible patients were assessed on all study days ; details about the selection process are described elsewhere .
the clinical presentation of enrolled patients was characterized by hemodynamic stability and no severe distress symptoms .
all patients were admitted at the department of cardiology or the department of cardiothoracic and vascular surgery , aarhus university hospital .
equipment and data acquisition . a vivid s6 ( ge healthcare , horten , norway ) ultrasound system equipped with a m4s phased array transducer ( 1.5 - 4.5 mhz ) with second harmonic imaging was used to obtain data .
all patients underwent poc echocardiography at the bedside performed by a novice examiner ( limited experience in echocardiography and certification equal to level i ) [ 16 , 17 ] .
the examination was initially performed with patients placed in the supine position ; if the condition of the patient allowed it , image acquisition was also performed in the left lateral position .
the poc echocardiography included the following views : subcostal 4-chamber view , apical 4-chamber view , parasternal long- and short - axis views .
raw data were digitally stored in cineloop format defined by the r - wave in the corresponding electrocardiogram for off - line analyses .
were done as post - examination analyses using echopac software ( ge healthcare , horten , norway ) .
three assessments of ef were made : expert eyeballing : a single specialist in cardiology and expert ( experience equivalent to level iii ) [ 18 , 19 ] in echocardiography visually estimated all the poc echocardiography recordings blinded to any previous assessments .
all the obtained images were taken into account when the assessments were performed and the ef was reported as a percentage from 5%-75% and when in doubt as a range of 5%.manual planimetry : when feasible in relation to the image quality , all apical 4-chamber views were analysed by an experienced examiner blinded to previous assessments ( experience equivalent to level ii ) .
manual endocardial border tracing from end - diastole to end - systole was applied and ef was calculated according to the simpson s method of discs .autoef :
when feasible in relation to the image quality , all apical 4-chamber views were analysed by a novice examiner blinded to previous assessments and with limited experience in echocardiography and certification equal to level i. this examiner was not experienced enough to perform eyeballing estimates of ef .
expert eyeballing : a single specialist in cardiology and expert ( experience equivalent to level iii ) [ 18 , 19 ] in echocardiography visually estimated all the poc echocardiography recordings blinded to any previous assessments .
all the obtained images were taken into account when the assessments were performed and the ef was reported as a percentage from 5%-75% and when in doubt as a range of 5% .
manual planimetry : when feasible in relation to the image quality , all apical 4-chamber views were analysed by an experienced examiner blinded to previous assessments ( experience equivalent to level ii ) .
manual endocardial border tracing from end - diastole to end - systole was applied and ef was calculated according to the simpson s method of discs .
autoef : when feasible in relation to the image quality , all apical 4-chamber views were analysed by a novice examiner blinded to previous assessments and with limited experience in echocardiography and certification equal to level i. this examiner was not experienced enough to perform eyeballing estimates of ef .
the autoef software relies on 2d speckle tracking , utilizing natural acoustic markers in the tissue ( figure 1 ) .
the endocardial border is traced throughout the cardiac cycle by means of speckle tracking and the software automatically locates end - systolic and end - diastolic frames .
the software requires the examiner to define three regions of interest in the left ventricle , the endocardial border is then traced throughout the cardiac cycle and the software automatically locates the end - systolic and end - diastolic frames .
end - diastolic and end - systolic volumes are calculated based on the tracings and serves as the basis for the ef calculation . during manual planimetry and autoef assessments , the time spent from the beginning of the procedure to the determination of the ef was recorded .
in addition , all quantifications of ef by manual planimetry and autoef were performed twice in order to assess intra - observer variability .
comparisons of methods were performed as proposed by bland and altman and by means of the intraclass correlation coefficients .
tests and calculations were performed using stata 11.0 software ( statacorp lp , texas , usa ) .
the mean age of patients was 63.2 years ( 16.4 ) and 31% were females .
the mean ef in the expert eyeballing group was 45% ( 17 ) , in the manual planimetry group 49% ( 16 ) and in the autoef group 45% ( 14 ) .
there difference between the mean ef in the expert eyeballing group and the autoef group was 0.08% ( -2.0 - 2.2 ) and not statistically significant ( p=0.94 ) .
however , there was a significant difference in the mean ef between the manual planimetry group and the autoef group of 3.8% ( 1.8 - 5.8 ) ( p=0.0003 ) as well as between the expert eyeballing group and the manual planimetry group of 3.6% ( 1.4 - 5.6 ) ( p=0.0016 ) .
the relationship between expert eyeballing and autoef is shown in figure 2 ( r=0.82 , p < 0.001 ) , corresponding bland - altman analysis revealed 95% limits of agreement ranging from -19% to 19% , bias was 0% .
scatter plots with line of identity on the left side and bland - altman plots illustrating agreement in ef measurements on the right side .
central horizontal line in the bland - altman plot represents mean bias or systematic difference , upper and lower dashed horizontal lines represent 95% confidence intervals of differences ( limits of agreement ) .
the bias between expert eyeballing and manual planimetry was -3.6% and 95% limits of agreement ranged from -24% to 17% .
the correlation for this comparison is shown in figure 3 ( r=0.80 , p < 0.001 ) .
scatter plots with line of identity on the left side and bland - altman plots illustrating agreement in ef measurements on the right side .
central horizontal line in the bland - altman plot represents mean bias or systematic difference ; upper and lower dashed horizontal lines represent 95% confidence intervals of differences ( limits of agreement ) .
figure 4 shows the relationship between manual planimetry and autoef ( r=0.82 , p < 0.001 ) . in this case
figure 5 shows intra - observer data from manual planimetry assessments ( r=0.87 , p < 0.001 ) and autoef assessments ( 0.94 , p < 0.001 ) .
intra - observer variability of manual planimetry measurements on the top and autoef on the bottom .
scatter plots with line of identity on the left side and bland - altman plots illustrating agreement in ef measurements on the right side .
central horizontal line in the bland - altman plot represents mean bias or systematic difference , upper and lower dashed horizontal lines represent 95% confidence intervals of differences ( limits of agreement ) .
the 95% limits of agreement for manual planimetry are -12% to 15% and in the case of autoef -8% to 11% . when excluding patients with atrial fibrillation from the analysis , the correlation between expert eyeballing and autoef was 0.81 ; between expert eyeballing and manual planimetry it was 0.80 and between autoef and manual planimetry 0.82 . the mean time spent for manual planimetry was 98 ( 90 - 106 ) seconds ; the corresponding mean time spent for autoef was 41 ( 36 - 46 ) seconds , which was significantly less ( p < 0.001 ) .
this is the first study to evaluate semi - automated estimation of ef based on speckle tracking technology .
in contrast to previous automated border - detection methods suffering from gain dependency , autoef analyses standard 2d gray scales images in a rapid and semi - automated manner . however , autoef depends on the same parameters as speckle tracking strain analyses , thus frame rates need to be high ( 60 - 80 ) and images free from myocardial dropouts .
these issues explain why autoef was only possible in 83% of cases , when manual planimetry was possible in 89% of cases .
the agreement between our chosen reference ( expert eyeballing ) and autoef was moderate to good and in fact very similar to the agreement between expert eyeballing and manual planimetry .
this is in spite of the fact that the manual planimetryexaminer had experience level ii and the autoef examiner had level i experience .
in addition , intra - observer variability and time consumption was considerably less for autoef compared to manual planimetry .
two previous studies have evaluated an automated method for assessment of ef , based on artificial intelligence [ 22 , 23 ] .
however , one of the studies showed poor correlation ( r=0.64 ) between visual reading and automated reading and the other showed good correlation ( r=0.98 ) .
however , the autoef assessments were performed by a level i examiner , and it is possible that the agreement would improve with a more experienced examiner .
atrial fibrillation is a challenge when estimating ef and guidelines suggest multiple measurements because of the cycle variability .
however , only 5.9% of the patients included in this study had atrial fibrillation and they were thus part of the analyses to show robustness of the method .
however , we still recommend multiple measurements of ef for clinical practice as described in the chamber quantification guidelines . clinical implications .
although ef is probably not the best measure of systolic function in the left ventricle it still plays a major role in the clinical management of many patients . in everyday clinical practice , visual assessment of ef
however , visual assessments are highly dependent on the examiner s experience ; this is probably why many less experienced examiners rely on manual planimetry . for the sake of less time consumption and intra - observer variability
, autoef seems to be a valid alternative to manual planimetry . in particular , emergency situations where time is of the essence
however , caution is advised for novice examiners , because speckle tracking based methods are dependent on frame rates and myocardial presentation and in some cases requiring manual editing .
no true gold standard method like magnetic resonance imaging or 3-dimensional echocardiography was used for reference purposes in the current study . however , as expert eyeballing is the everyday clinical gold standard in many clinical settings it seems to be a valid choice for the current study .
manual planimetry and advanced methods also report diastolic and systolic volumes which have prognostic value and these parameters should be included in future studies of the autoef method . since 34.3% of the research cohort had ischemic heart disease or acute coronary syndrome , some of these patients may have regional wall motion abnormalities . in these patients
the lack of information about regional dyskinesia obviously represents a minor limitation to the use of autoef . in accordance with guideline recommendations , manual planimetry needs to be a biplane assessment
however , due to the specification in the poc protocol used to acquire images for this study , no 2-chamber view was available .
this has possibly contributed to the ( somewhat ) less accurate agreement of autoef compared to previous studies . however , in spite of this limitation , we still obtained better agreement than the method based on artificial intelligence without manual editing .
as no hemodynamically unstable patients were included is this study , the results need to be further validated in the acute setting with patients in supine- or semirecumbent position as well as intubated / ventilated patients .
autoef seems to be a valid supplement to the clinical assessment of ef in the hands of less experienced examiners , yielding results similar to manual planimetry when compared to expert eyeballing with less time consumption and less intra - observer variability .
however , manual editing may be required and this is why some training is recommended before autoef is applicable for use by novices .
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introductionto compare estimation of ejection fraction at the bedside by autoef compared with conventional methods and to assess feasibility and time consumption.methodsa total of 102 relatively hemodynanically stable mixed medical and surgical patients were included .
all patients underwent ultrasonography of the heart at the bedside performed by a novice examiner .
three assessments of ejection fraction were made : 1 ) expert eyeballing by a single specialist in cardiology and expert in echocardiography ; 2 ) manual planimetry by an experienced examiner ; 3 ) autoef by a novice examiner with limited experience in echocardiography.resultsexpert eyeballing of ejection fraction was performed in 100% of cases .
manual planimetry was possible in 89% of cases and autoef was possible in 83% of cases .
the correlation between expert eyeballing and autoef was r=0.82 , p < 0.001 , for manual planimetry and for autoef it was r=0.82 , p < 0.001 ; for expert eyeballing and manual planimetry it was r=0.80 , p < 0.001 .
the mean time consumption for manual planimetry was 98 ( 90 - 106 ) seconds ; correspondingly the mean time spent for autoef was 41 ( 36 - 46 ) seconds , which was significantly less ( p < 0.001).conclusionsautoef seems to be a valid supplement to the clinical assessment of ejection fraction in the hands of less experienced examiners , yielding result similar to manual planimetry with less time consumption and less intra - observer variability .
however , manual editing may be required and training is thus recommended before autoef is applicable for use by novices .
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phenylthiocarbamide ( ptc ) , a bitter chemical synthesized by fox has been widely used for genetic and anthropological studies .
the inability to taste ptc is a simple mendelian recessive trait,[37 ] wherein the individuals with two recessive alleles ( tt ) are nontasters for ptc and individuals with one dominant allele ( tt ) or two dominant alleles ( tt ) are tasters for ptc .
there are many reports , which contradict this model , some of them are : incomplete dominance , two locus model[912 ] and polygenic model .
kim et al have identified a small region on chromosome 7q by genome - wide linkage analysis , which shows strong linkage disequilibrium between single - nucleotide polymorphism markers and ptc taste sensitivity in unrelated subjects .
this region harbors a gene that encodes a member of the tas2r bitter taste receptor family . a major locus on 7q35-q36 and a secondary locus on chromosome 16p
bufe et al have demonstrated that alleles of htas2r38 codes for functionally different receptor types that directly affect bitterness perception containing compounds .
they suggested that these polymorphisms might be due to evolutionary pressures that foster variability at the receptor to enhance bitter detection and rejection of these compounds . in the present study
, we report that the tasters are more frequent than nontasters in mysore , south india .
about 3282 subjects were randomly selected from various parts of the city irrespective of age , sex , religion , food habits and socio - economic status .
subjects were classified into four different categories viz , unbiased category (= individuals screened randomly in public places without looking into religion , region , age and sex ) , hindu , christian and muslim religious groups . in order to assess the pattern of inheritance and the frequency of the ptc tasting in different religious groups , a total of 180 families , which included christian ( 50 ) , hindu ( 61 ) and muslim ( 69 ) were screened from different localities of the city .
harris and kalmus method was used to assess the ptc taster and nontaster phenotype . with their consent ,
the subjects and their family members were asked to taste the ptc solution and the results were recorded in the proforma used to collect the family history .
information about any possible migration in or out of these families or any associated diseases in the family was also recorded .
chi square test was applied to justify the deviations from the expected ratio of taster and nontaster alleles .
one of the limitations of this study was , the number of offspring born in these families were less .
however , this can be justified by the fact that in humans each conception is an independent event ; the probability of having another child in these families who could be a taster can not be ruled out .
another limitation is that the availability of families over three to four generations is very rare and even some of these families were not given consent for this study .
ptc taster trait was analyzed in four different categories namely unbiased , hindu , muslim and christian groups .
it was found that tasters were significantly more frequent than nontasters in all the four categories [ table 1 ] .
the incidence of tasters was more in the unbiased category ( 85% ) and less in the muslim category ( 58% ) .
based on the taster phenotype of the progeny and the parents analyzed in 180 families , five different genotypic groups were classified .
they are ( a ) tt tt , ( b ) tt tt , ( c ) tt(t ) tt , ( d ) tt tt(t ) and ( e ) tt tt . pedigrees were constructed for all the five different genotypic groups [ figure 1 ] . of
the 180 families studied , parents of 55 families were in homozygous dominant for taster trait and parents of 12 families were in heterozygous condition [ table 2 ] .
taster parents ( 67 families ) had produced both taster and nontaster children , of which 89% were taster children , whereas nontaster parents ( 27 families ) , had produced only nontaster children ( 100% ) .
figure 2 illustrates the above frequency of distribution of taster gene in 180 families studied .
distribution of tasters and nontasters for phenylthiocarbamide in mysore , south india pedigree of homozygous dominant ( a ) , heterozygous dominant ( b ) , nontaster taster ( c ) , taster nontaster ( d ) and homozygous recessive ( e ) .
inheritance pattern of taster gene in 180 families frequency of distribution of taster gene in 180 families in four different genotypic categories - a = tt ( t ) tt ( t ) , b = tt(t ) tt , c = tt tt(t ) , d = tt tt when one of the parents was a nontaster , the frequency of tasters and nontasters in the progeny varies . by analyzing the taster phenotype of the progeny in the above 86 families , 15 and 12 families had homozygous taster father and mother respectively [ table 3 ] .
the remaining 59 families , that is , heterozygous father or mother for taster gene with nontaster partners ( test cross ) had taster and nontaster progenies in the ratio 1.0 : 1.54 .
this indicates the deviation in the segregation pattern with increased variable penetrance of nontaster allele [ figure 3 ] in these families .
segregation of homozygous and heterozygous taster gene in 86 families based on the progeny data presence of homozygous and heterozygous taster gene in 86 families studied with the genotypic categories - a = tt tt and b = tt tt . in the category
a all the individuals were tasters and in category b tasters and nontasters were in the frequency of 53% and 82% respectively .
substantial variation in taste sensitivity exists along with great degree of sequence diversity and variation in bitter taste receptor genes .
wooding et al have examined patterns of dna sequence variation in the ptc gene of different populations , which accounts for up to 85% of phenotypic variance in the taster trait .
they found excess of intermediate - frequency variants than neutral variants and suggested that this variation is due to the balance natural selection , which has acted to maintain
however , the frequency of tasters in different populations as available in the literature indicates that papago indians in arizona shows the highest incidence of tasters ( 98.6% ) , while kondhs population in orissa , india has shown only 35% of tasters .
studies in other regions showed the frequency of tasters ranged in between the two extreme cases [ table 4 ] indicating the wide range of variations found in the frequency of taster and nontaster genotypes . the ability to taste ptc shows complex inheritance in humans . using quantitative trait locus mapping techniques ,
kim et al have found a gene that accounts for 55 - 85% of the variance in ptc taste sensitivity .
frequency of tasters in world populations in the present study , the frequency of ptc tasters is greater than nontasters .
however , based on the segregation analysis of heterozygous families , nontaster alleles are increased in their progeny . since variable penetrance is more common in autosomal dominant condition and deviation in segregation pattern may be due to variable penetrance of taster allele , as heterozygotes can not be identified .
the other possibility that some of the tt tt marriages may be infact tt tt , can not be ruled out , which may be the reason for deviation in the segregation of taster and nontaster progeny .
another possibility of the change in the genetic ratio of the ptc taster allele in heterozygotes could be due to inbreeding .
the religious groups in india particularly in south india exhibit high level of second degree consanguinity .
the inbreeding within these religious groups for several generations and selection of preferentially segregated nontaster allele to the subsequent generations can not be ruled out . on the other hand , although ptc itself has not been found in nature , the ability to taste ptc is correlated strongly with the ability to taste other naturally occurring bitter substances , many of which are toxic . therefore ,
understanding the nature of the variation in bitter taste and its relationship to diet and other behaviors may have important implications on human health .
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background : the ability to taste phenylthiocarbamide ( ptc ) , a bitter chemical has long been known to be a heritable trait , which is being widely used for both genetic and anthropological studies .
the frequency of taster and non - taster allele is found to vary in different populations.aims and objective : to investigate the frequency of taster trait in mysore , south india.materials and methods : the present investigation was conducted in mysore , south india during 2002 - 2003 .
about 3282 subjects irrespective of age , sex , religion , food habits , socio - economic status were randomly selected from various parts of the city and a total of 180 families , which included christian ( 50 ) , hindu ( 61 ) and muslim ( 69 ) were screened from different localities of the city .
harris and kalmus method was used to assess the ptc taster and nontaster phenotype.results:it was found that tasters were significantly more frequent than nontasters in all the four categories .
the incidence of tasters was more in unbiased category ( 85% ) and less in muslim category ( 58% ) .
investigations on ptc tasting in the families of three different religious groups revealed that the tasters were significantly more frequent than nontasters .
it was also found that heterozygous father or mother for the taster genes with nontaster partner had taster and nontaster progenies in the ratio 1.0 : 1.54 indicating the deviation in the segregation pattern of test cross.conclusion:in mysore , tasters are more frequent than nontasters .
variation in the frequency of nontaster allele in the religious groups could be due to inbreeding .
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operatory microscope has generally brought a new dimension in dentistry and particularly in endodontic surgery [ figure 1a ] . ( a )
pecora , rubinstein , kim the new dimension of teaching ( b ) mirror and micromirrors ( c and d ) we can treat only what we can see light and magnification have determined a new higher standard of quality in the profession .
microscopy is a young specialty in a great and continuous evolution , not only in the field of application , but also in the development of new instruments and techniques .
concepts based on evolution , advanced technology applications , characteristics of perfectioned materials have brought major improvment on long - term results and an excellent predictability in the surgical techniques .
in particular , the extensive use of the microscope in conventional endo has enlarged the indications for the positive treatment of failures and restricted the indications for surgical treatment .
the concept of apical surgery has been expanded to periradicular surgery and today we speak in terms of micro - endo surgery . throughout the history of endodontics , never before there have been as many changes as in the last 25 years .
new dimension has been created working in different directions , which we can summarize in :
new operative protocolguided regeneration principlesmaxillary sinus management in endo surgerylimits of conservative therapyimplants as an alternative .
new operative protocol guided regeneration principles maxillary sinus management in endo surgery limits of conservative therapy implants as an alternative .
one of the most important advantages in using the microscope is the evaluation of our surgical technique .
more light can be shed on the rationality of the treatment to perform procedures in certain ways .
microsurgery is defined as a surgical procedure on exceptionally small and complex structures with an operating microscope .
this instrument enables the surgeon to assess pathological changes more precisely and to treat pathological lesions with the greatest precision , thus minimizing tissue damage during surgery .
endodontic microsurgery combines magnification and illumination , provided by the microscope , with the proper use of new microinstruments [ figure 1b ] .
if the access is limited for traditional surgery , it will also be limited when the microscope is placed between the surgeon and the surgical field .
however , the microscope creates a much better view of the surgical field by appropriate magnification and highly focused illumination .
since vision is greatly enhanced , cases can be better treated with higher precision and accuracy .
there is a maxim : to see better is to do better. we might add : to do it more easily. the microscope allows an exceptional evidentiation of the anatomy with the consequent possibility of a more accurate diagnosis and a more incisive operative capacity .
furthermore , the better vision leads to a less invasive approach respecting the tissues and , therefore , a lesser post - operative discomfort and faster healing .
the advanced technologies , applied to medical surgical specialties , have brought to exceptional progresses , achievement of unthinkable results and have also contributed to simplify technical steps , making them accessible to a larger number of specialists and more predictable in the results .
just think to the use of air drills and ultrasounds to the application of magnetic resonance and laser .
think to limbs reattachment , to otosclerosis surgery , to eye surgery , and you will understand that advanced technology and technical operative improvements today allow the opening of new frontiers . in the 80 's we had a raising interest for microscope in dentistry .
sometimes , endodontics has a limited visual field , with little and very little anatomic structures , and has particularly difficult vision of the inside of the tooth and the canals .
therefore , it has got major benefits from the possibility to see better through light and magnification . under microscopic control ,
the evolution towards suitable and practical modification , allows a better precision and quality standards .
we can treat only what we can see. the introduction of the microscope in endodontic surgery will give clinicians the necessary means to treat difficult cases with a higher degree of confidence and clinical success .
once we reached the goal of an hermetic sealing of the root canal system ( rcs ) exits and , in particular , the sealing of the new apex , we expanded our horizon to endo perio surgery .
we begun our treatments applying guided tissue regeneration ( gtr ) and guided bone regeneration ( gbr ) principles to the borderline cases obtaining encouraging results .
the use of the microscope allowed us to gain access to the maxillary sinus in a very conservative way .
the diagnostic potential has grown as well as the therapeutic options and this allowed us to successfully intervene on the oro - antral pathologies .
on one hand , the microscope enhanced the orthograde treatment as well as the retreatment , reducing the indications for surgery ; on the other hand , we started to deal with
in which the extraction of the tooth was the only alternative , we claimed for the endodontist the right to insert implants simoultaneously to the extraction with immediate loading . the challenge for the future will be predictable for thesuccessfull management of the endo perio lesions .
the step from apical surgery to periradicular micro surgery has involved new concepts , new techniques , new materials , new instruments but , particularly , the use of light and magnification through the surgical microscope [ figures 1c and d ] .
all these phases of the surgical protocol have taken advantage from the use of this instrument and there have been tremendous changes in the approach and the execution of surgery .
many comparative clinical studies have been carried out to evidentiate the differences between traditional and micro surgery and the advantages in the use of the microscope [ figures 2a - f ] .
( a ) failure of endo - treatment ( b ) apicoectomy without retrofilling ( c ) recurrency at 3 years ( d ) retrofilling 22x ( e ) rx post - op control ( f ) healing at 6 months rubinstein and kim have given scientific evidence that the increase of long - term success with micro - endo surgery on molars premolars and single - rooted teeth has to be attributed to the utilization of the microscope . with its use
, we can consider the following as the main advantages in our practice :
easy identification of the apical thirdsmaller osteotomiesefficient evaluation of cut surface with minimal resection angleincreased diagnostic powereasier and safer management of anatomic structuresmininvasive approach to pathological tissuespotential of creating an hermetic seal in the neo - apex .
easy identification of the apical third efficient evaluation of cut surface with minimal resection angle increased diagnostic power easier and safer management of anatomic structures mininvasive approach to pathological tissues potential of creating an hermetic seal in the neo - apex .
the combination of microscope , ultrasonic tips and microintruments allows conservative , coaxial , deep root - end preparation and retrofilling .
this satisfies the requirements for rcs sealing and the realization of the triad : cleaning , shaping , filling of the rcs itself . when we begin a surgical treatment .
the primary concern is to create conditions that favor healing of periradicular tissues either for regeneration or for repair .
these conditions include :
removal of necrotic tissuesremoval of disintegration tissuesdecontamination of root surfaceentomb bacteria present in rcsremoval of the apical part with the most accessory canalscreation of the best neo - apical seal .
removal of necrotic tissues removal of disintegration tissues decontamination of root surface entomb bacteria present in rcs removal of the apical part with the most accessory canals creation of the best neo - apical seal .
in the last decade , the operative protocol has undergone major evolution , achieving better results . from the common experience of different operators and from literature investigation ,
we have stated technical determinant factors in the following steps :
diagnosisflap design and elevationosteotomyapicoectomy : courettagebiopsyemostasiscut surfaceretrofillinggtr evaluationsuture .
flap design and elevation apicoectomy : courettage hermetic seal of the neo - apex and exits of rcs is the target of surgical treatment [ figures 3a and b ] .
( a ) cutting surface evaluation , methylene blue 2% staining ( b ) retro - filling control 22x to perform the operative phases at best , we need :
a correct diagnosis and adequate treatment planningthe possibility to evaluate and choose the most favorable solution in case of controversial situationsa complete identification and visualization of the anatomy of the surgical areathe best quality level of each technical moment in the surgerycorrect evaluation of operatory risksminimal dissection and damage of healthy tissues .
a correct diagnosis and adequate treatment planning the possibility to evaluate and choose the most favorable solution in case of controversial situations a complete identification and visualization of the anatomy of the surgical area the best quality level of each technical moment in the surgery correct evaluation of operatory risks minimal dissection and damage of healthy tissues .
critical size defect has its total application in endodontic micro - surgery . in some clinical situations ,
there are many misunderstandings not only for the indication to guide the healing toward regeneration , but also among researchers on the techniques and the grafting materials .
a small defect undergoes spontaneous healing while a large defect has a different and non difficult healing .
the ultimate goal of micro - endo surgery is the predictability of the regeneration of the peri - apical tissues .
regeneration is a biological process to replace the destroyed tissues with new tissue that has same cells , same architecture , same function and capability of reaction against the pathologic stimulations .
repair is a biologic process where the lost tissue is substituted by a new tissue with different cells , different architecture , different capability of reaction against the pathologic stimulations .
this is an acceptable type of healing depending on which part of the root it occurs . according to pecora 's anatomo - pathologic classification
, we have : type 1 ( above the apex ) ; type 2 ( middle root area ) ; type 3 ( endo - perio communications ) [ figures 4a - c ] .
( a ) lesion above the apex type i ( b ) middle root lesion type ii ( c ) endo - marginal lesion when a defect can spontaneously heal , bone fillers are ineffective or inappropriate .
however , there are many clinical situations requiring the use of regenerative materials and techniques :
large lesions ( more than 5 mm)t & t lesionsendo - perio lesionssinus membrane perforationroot perforations with large lesions .
large lesions ( more than 5 mm ) endo - perio lesions sinus membrane perforation root perforations with large lesions . the prognostic limit of the regenerative techniques and therapeutic possibilities can be evaluated with pecora 's classification , where class e lesions represent the borderline and the evaluation criteria are based on the distance of bony walls and their thickness .
large defects , endo - perio communications and through - and - through defects need a guide for healing .
the healing of the bone defect depends on the presence of these cells in the surrounding bone and tissue as well as on their capability to colonize the area and differentiate into osteoblasts . in borderline cases
, a lesion of endodontic origin is complicated by the loss of the marginal attachment .
the effectiveness of endo - surgery may be diminuished , if epithelial cells are allowed to populate the root surface .
membrane barriers inhibit this epithelial proliferation and , thereby , promote regeneration of funcional attachment .
today , the controversial use of regenerative therapies , even if limited in the indications and cases,- allows the clinicians to treat extreme cases and to have an optimal and predictable treatment of those lesions considered as
( a ) large lesion with endo - perio communication ( b ) apicoectomy with calcium sulfate graft ( c ) rx at 2 months ( d ) rx at 6 months anatomical relations between apices of postero - superior teeth and maxillary sinus , imply the risk of endo - perio pathologies transmission , bacteria , debris , instruments penetration and resected apices violation during endo - micro surgery .
in the last years , the utilization of the microscope has determined new horizons in the prevention and therapeutic options in maxillary sinus ( ms ) involvement during endo - surgery or consequent to endo - perio lesions .
the endodontist has to be very careful when dealing with the following ms involvements :
endo - periocysteas ( endo antral syndrome)oac ( oro antral communication)oaf ( oro antral fistula ) .
eas ( endo antral syndrome ) oac ( oro antral communication ) oaf ( oro antral fistula ) .
the schneider 's membrane of the sinus may present :
thickeningperforation .
with or without symptoms .
the must is to prevent foreign bodies , bacteria , debris penetration into the sinus , and if a perforationexists , the correct choice is its immediate closure . if we have to perform an endo - surgery and if a thin layer of bone separates the apex from the sinus or if a communication pre or intra op occurs , the correct therapy is to close the perforation and increase the amount of bone above the neo - apex to prevent future recurrencies . in case of perforation ,
particular attention has to be given to the type of healing and how the event may influence the healing process .
a study from matisko et al . , evidentiated that the use of gtr ( collagene membranes ) in ms perforation brings to bone formation when compared to spontaneous healing with fibrous tissue in the control sites , in rabbits .
microsurgical procedures have implemented our skills to treat successfully most of the failures in the endo treatments .
heroic cases towards the conservative approach . which is the limit between conservative treatment and overtreatment ?
it is too simple to consider the lack of symptoms or clinical signs or a decreasing of the radiolucency a success .
many teeth , also if they can not be included into the success criteria , remain asymptomatic and in - function for years .
the critical point is when the progression of infection is out of control and the situation may lead to massive bone loss , jeopardizing the future correct implant insertion .
necessarily we have to respect a logic protocol , which foresees :
endo - treatmenteventual retreatmentindication for surgerydiagnostic flappossibility of infection controlevaluation of healing and regenerative potential .
indication for surgery possibility of infection control evaluation of healing and regenerative potential . in the final decision
, we have to consider , beyond periodontal and restorative considerations , factors as : esthetic , proprioceptive sensibility , postural balance , prosthetic relevance of the tooth , anatomic limitations , economical aspects and patient 's will .
to the traditional goals of therapy :
decreasing or disappearing of radiolucencycontrol of symptomsrestoring of function .
decreasing or disappearing of radiolucency restoring of function .
therapeutic option , correct diagnosis , predictability of the results have to be validated with a diagnostic flap .
the microscope and the methylene blue as biological indicators , make it easier to chose between the different options .
once the decision to extract the tooth is made , clinicians have to face two therapeutic options :
immediate post - extraction regenerationfresh socket implant with immediate loading .
immediate post - extraction regeneration fresh socket implant with immediate loading .
there are no objective criteria of evaluation , but only clinical experience and studies focusing single aspects of the problem exists
the most favorable compromise when we combine experience of the operator , logical approach and a few fundamental points [ figures 6a and b ] .
( a ) untreatable horizontal fracture ( tooth 2.1 ) ( b ) fresh socket implant with immediate loading quality of bone and primary stability are the main factors .
it is essential for the surgeon to decide how and where to place the implant and whether an implant can be immediately loaded .
the leading concept is the evaluation of the post - extractive defect :
presence of bony wall and thickness;endo - perio condition of the closer teeth;esthetic aspect ( anterior or posterior teeth);acute infection or suppuration .
presence of bony wall and thickness ; endo - perio condition of the closer teeth ; esthetic aspect ( anterior or posterior teeth ) ; acute infection or suppuration . in a clinical study , novaes and
novaes concluded that the chronically infected sites do not constitute a contraindication for immediate implant placement if some pre - and post - operative clinical cares are followed :
antimicrobical therapy pre- and post - op;meticolous cleansing and debridement of the alveoli before implant placement .
antimicrobical therapy pre- and post - op ; meticolous cleansing and debridement of the alveoli before implant placement . in presence of bone dehischence or missing bone wall ,
the choice is first to regenerate the bone and then place the implant , because the predictability of gtr techniques is questionable .
immediate loading can be applied in some clinical situations where the control of micromotion is possible and if there are some important advantages :
decreased healing timereduced resorption of alveolar boneachievement of optimal esthetic results .
decreased healing time reduced resorption of alveolar bone achievement of optimal esthetic results . in case of extraction sites in esthetic areas
, there is a restorative challange and particular steps have to be followed in order to preserve site morphology and to support the existing hard and soft tissues :
atraumatic tooth extraction with mininvasive operative techniques that minimize the treatment of the tissues;appropriate placement , orientation and stabilization of the implant .
atraumatic tooth extraction with mininvasive operative techniques that minimize the treatment of the tissues ; appropriate placement , orientation and stabilization of the implant .
flapless surgery has been suggested as a favorable option to enhance implant esthetic , with several advantages :
reduction of pain and swellingminimal intra operative bleedingreduction of surgical timeno need of suturemaintenance of blood spplypreservation of hard and soft tissues .
reduction of pain and swelling minimal intra operative bleeding reduction of surgical time maintenance of blood spply preservation of hard and soft tissues .
the only limitation is that this process requires more operative experience and pre surgical plannig for the inhability to visualize anatomic landmarks and vital structures . a clinical study of pecora et al .
in 1996 , pointed out that the endodontist now has an additional choice of treatment in cases where tooth extraction is inevitable. in conclusion , the placement of implants immediately after extraction , seems to be a successful procedure .
even if we have plenty of papers in literature that evidentiate the good results of the immediate implants , why they speak in terms of implant survival , remains an interrogative .
survival is characterized by the maintainance of a certain function for a certain period of time without the respect of fixed parameters and without a long - term control .
success is qualified and codificated in compliance with fixed parameters declared in the protocol and respected in the world 's documented long - term results .
the optimal treatment plan incorporates the best available evidence together with specific case factors and the patient 's desires and needs .
dental implants provide a useful alternative to replace teeth that can not otherwise be treated with a good prognosis .
it is impossible to give a proper evaluation to the success rate of the various treatments since the same criteria of evaluation of the results are inconsistent themselves .
realistically , we can not say that implants have a higher percentage of survival if compared with real teeth treated and reconstructed in a proper way .
the availability of osteointegrated implants does not justify the extraction of natural teeth that can still offer a stable function .
a compromised tooth should be managed with a multidisciplinary approach and dental implants should be reserved for patients with truly end - stage tooth failure .
the step from apical surgery to periradicular micro surgery has involved new concepts , new techniques , new materials , new instruments but , particularly , the use of light and magnification through the surgical microscope [ figures 1c and d ] .
all these phases of the surgical protocol have taken advantage from the use of this instrument and there have been tremendous changes in the approach and the execution of surgery .
many comparative clinical studies have been carried out to evidentiate the differences between traditional and micro surgery and the advantages in the use of the microscope [ figures 2a - f ] .
( a ) failure of endo - treatment ( b ) apicoectomy without retrofilling ( c ) recurrency at 3 years ( d ) retrofilling 22x ( e ) rx post - op control ( f ) healing at 6 months rubinstein and kim have given scientific evidence that the increase of long - term success with micro - endo surgery on molars premolars and single - rooted teeth has to be attributed to the utilization of the microscope .
with its use , we can consider the following as the main advantages in our practice :
easy identification of the apical thirdsmaller osteotomiesefficient evaluation of cut surface with minimal resection angleincreased diagnostic powereasier and safer management of anatomic structuresmininvasive approach to pathological tissuespotential of creating an hermetic seal in the neo - apex .
easy identification of the apical third efficient evaluation of cut surface with minimal resection angle increased diagnostic power easier and safer management of anatomic structures mininvasive approach to pathological tissues potential of creating an hermetic seal in the neo - apex .
the combination of microscope , ultrasonic tips and microintruments allows conservative , coaxial , deep root - end preparation and retrofilling .
this satisfies the requirements for rcs sealing and the realization of the triad : cleaning , shaping , filling of the rcs itself . when we begin a surgical treatment .
the primary concern is to create conditions that favor healing of periradicular tissues either for regeneration or for repair .
these conditions include :
removal of necrotic tissuesremoval of disintegration tissuesdecontamination of root surfaceentomb bacteria present in rcsremoval of the apical part with the most accessory canalscreation of the best neo - apical seal .
removal of necrotic tissues removal of disintegration tissues decontamination of root surface entomb bacteria present in rcs removal of the apical part with the most accessory canals creation of the best neo - apical seal . in the last decade , the operative protocol has undergone major evolution , achieving better results . from the common experience of different operators and from literature investigation ,
we have stated technical determinant factors in the following steps :
diagnosisflap design and elevationosteotomyapicoectomy : courettagebiopsyemostasiscut surfaceretrofillinggtr evaluationsuture .
flap design and elevation apicoectomy : courettage hermetic seal of the neo - apex and exits of rcs is the target of surgical treatment [ figures 3a and b ] . ( a ) cutting surface evaluation , methylene blue 2% staining ( b ) retro - filling control 22x to perform the operative phases at best ,
we need :
a correct diagnosis and adequate treatment planningthe possibility to evaluate and choose the most favorable solution in case of controversial situationsa complete identification and visualization of the anatomy of the surgical areathe best quality level of each technical moment in the surgerycorrect evaluation of operatory risksminimal dissection and damage of healthy tissues .
a correct diagnosis and adequate treatment planning the possibility to evaluate and choose the most favorable solution in case of controversial situations a complete identification and visualization of the anatomy of the surgical area the best quality level of each technical moment in the surgery correct evaluation of operatory risks minimal dissection and damage of healthy tissues .
critical size defect has its total application in endodontic micro - surgery . in some clinical situations ,
there are many misunderstandings not only for the indication to guide the healing toward regeneration , but also among researchers on the techniques and the grafting materials .
a small defect undergoes spontaneous healing while a large defect has a different and non difficult healing .
the ultimate goal of micro - endo surgery is the predictability of the regeneration of the peri - apical tissues .
regeneration is a biological process to replace the destroyed tissues with new tissue that has same cells , same architecture , same function and capability of reaction against the pathologic stimulations .
repair is a biologic process where the lost tissue is substituted by a new tissue with different cells , different architecture , different capability of reaction against the pathologic stimulations .
this is an acceptable type of healing depending on which part of the root it occurs . according to pecora 's anatomo - pathologic classification
, we have : type 1 ( above the apex ) ; type 2 ( middle root area ) ; type 3 ( endo - perio communications ) [ figures 4a - c ] .
( a ) lesion above the apex type i ( b ) middle root lesion type ii ( c ) endo - marginal lesion when a defect can spontaneously heal , bone fillers are ineffective or inappropriate .
however , there are many clinical situations requiring the use of regenerative materials and techniques :
large lesions ( more than 5 mm)t & t lesionsendo - perio lesionssinus membrane perforationroot perforations with large lesions .
large lesions ( more than 5 mm ) endo - perio lesions sinus membrane perforation root perforations with large lesions . the prognostic limit of the regenerative techniques and therapeutic possibilities can be evaluated with pecora 's classification , where class e lesions represent the borderline and the evaluation criteria are based on the distance of bony walls and their thickness .
large defects , endo - perio communications and through - and - through defects need a guide for healing .
the healing of the bone defect depends on the presence of these cells in the surrounding bone and tissue as well as on their capability to colonize the area and differentiate into osteoblasts .
in borderline cases , a lesion of endodontic origin is complicated by the loss of the marginal attachment .
the effectiveness of endo - surgery may be diminuished , if epithelial cells are allowed to populate the root surface .
membrane barriers inhibit this epithelial proliferation and , thereby , promote regeneration of funcional attachment .
today , the controversial use of regenerative therapies , even if limited in the indications and cases,- allows the clinicians to treat extreme cases and to have an optimal and predictable treatment of those lesions considered as
( a ) large lesion with endo - perio communication ( b ) apicoectomy with calcium sulfate graft ( c ) rx at 2 months ( d ) rx at 6 months
anatomical relations between apices of postero - superior teeth and maxillary sinus , imply the risk of endo - perio pathologies transmission , bacteria , debris , instruments penetration and resected apices violation during endo - micro surgery .
all these potential clinical situations determine therapeutic needs , diagnostic and prognostic problems . in the last years
, the utilization of the microscope has determined new horizons in the prevention and therapeutic options in maxillary sinus ( ms ) involvement during endo - surgery or consequent to endo - perio lesions .
the endodontist has to be very careful when dealing with the following ms involvements :
endo - periocysteas ( endo antral syndrome)oac ( oro antral communication)oaf ( oro antral fistula ) .
eas ( endo antral syndrome ) oac ( oro antral communication ) oaf ( oro antral fistula ) .
the schneider 's membrane of the sinus may present :
thickeningperforation .
with or without symptoms .
the must is to prevent foreign bodies , bacteria , debris penetration into the sinus , and if a perforationexists , the correct choice is its immediate closure .
if we have to perform an endo - surgery and if a thin layer of bone separates the apex from the sinus or if a communication pre or intra op occurs , the correct therapy is to close the perforation and increase the amount of bone above the neo - apex to prevent future recurrencies . in case of perforation ,
particular attention has to be given to the type of healing and how the event may influence the healing process .
a study from matisko et al . , evidentiated that the use of gtr ( collagene membranes ) in ms perforation brings to bone formation when compared to spontaneous healing with fibrous tissue in the control sites , in rabbits .
microsurgical procedures have implemented our skills to treat successfully most of the failures in the endo treatments .
the application of gtr principles has moved many heroic cases towards the conservative approach . which is the limit between conservative treatment and overtreatment ?
it is too simple to consider the lack of symptoms or clinical signs or a decreasing of the radiolucency a success .
many teeth , also if they can not be included into the success criteria , remain asymptomatic and in - function for years .
the critical point is when the progression of infection is out of control and the situation may lead to massive bone loss , jeopardizing the future correct implant insertion .
necessarily we have to respect a logic protocol , which foresees :
endo - treatmenteventual retreatmentindication for surgerydiagnostic flappossibility of infection controlevaluation of healing and regenerative potential .
indication for surgery possibility of infection control evaluation of healing and regenerative potential . in the final decision
, we have to consider , beyond periodontal and restorative considerations , factors as : esthetic , proprioceptive sensibility , postural balance , prosthetic relevance of the tooth , anatomic limitations , economical aspects and patient 's will .
to the traditional goals of therapy :
decreasing or disappearing of radiolucencycontrol of symptomsrestoring of function .
decreasing or disappearing of radiolucency restoring of function .
therapeutic option , correct diagnosis , predictability of the results have to be validated with a diagnostic flap .
the microscope and the methylene blue as biological indicators , make it easier to chose between the different options .
once the decision to extract the tooth is made , clinicians have to face two therapeutic options :
immediate post - extraction regenerationfresh socket implant with immediate loading .
immediate post - extraction regeneration fresh socket implant with immediate loading .
there are no objective criteria of evaluation , but only clinical experience and studies focusing single aspects of the problem exists
the most favorable compromise when we combine experience of the operator , logical approach and a few fundamental points [ figures 6a and b ] .
( a ) untreatable horizontal fracture ( tooth 2.1 ) ( b ) fresh socket implant with immediate loading quality of bone and primary stability are the main factors .
it is essential for the surgeon to decide how and where to place the implant and whether an implant can be immediately loaded .
the leading concept is the evaluation of the post - extractive defect :
presence of bony wall and thickness;endo - perio condition of the closer teeth;esthetic aspect ( anterior or posterior teeth);acute infection or suppuration .
presence of bony wall and thickness ; endo - perio condition of the closer teeth ; esthetic aspect ( anterior or posterior teeth ) ; acute infection or suppuration . in a clinical study , novaes and
novaes concluded that the chronically infected sites do not constitute a contraindication for immediate implant placement if some pre - and post - operative clinical cares are followed :
antimicrobical therapy pre- and post - op;meticolous cleansing and debridement of the alveoli before implant placement .
antimicrobical therapy pre- and post - op ; meticolous cleansing and debridement of the alveoli before implant placement . in presence of bone dehischence or missing bone wall ,
the choice is first to regenerate the bone and then place the implant , because the predictability of gtr techniques is questionable .
immediate loading can be applied in some clinical situations where the control of micromotion is possible and if there are some important advantages :
decreased healing timereduced resorption of alveolar boneachievement of optimal esthetic results .
decreased healing time reduced resorption of alveolar bone achievement of optimal esthetic results . in case of extraction sites in esthetic areas
, there is a restorative challange and particular steps have to be followed in order to preserve site morphology and to support the existing hard and soft tissues :
atraumatic tooth extraction with mininvasive operative techniques that minimize the treatment of the tissues;appropriate placement , orientation and stabilization of the implant .
atraumatic tooth extraction with mininvasive operative techniques that minimize the treatment of the tissues ; appropriate placement , orientation and stabilization of the implant .
flapless surgery has been suggested as a favorable option to enhance implant esthetic , with several advantages :
reduction of pain and swellingminimal intra operative bleedingreduction of surgical timeno need of suturemaintenance of blood spplypreservation of hard and soft tissues .
reduction of pain and swelling minimal intra operative bleeding reduction of surgical time maintenance of blood spply preservation of hard and soft tissues .
the only limitation is that this process requires more operative experience and pre surgical plannig for the inhability to visualize anatomic landmarks and vital structures . a clinical study of pecora et al .
in 1996 , pointed out that the endodontist now has an additional choice of treatment in cases where tooth extraction is inevitable. in conclusion , the placement of implants immediately after extraction , seems to be a successful procedure .
even if we have plenty of papers in literature that evidentiate the good results of the immediate implants , why they speak in terms of implant survival , remains an interrogative .
survival is characterized by the maintainance of a certain function for a certain period of time without the respect of fixed parameters and without a long - term control .
success is qualified and codificated in compliance with fixed parameters declared in the protocol and respected in the world 's documented long - term results .
the optimal treatment plan incorporates the best available evidence together with specific case factors and the patient 's desires and needs .
dental implants provide a useful alternative to replace teeth that can not otherwise be treated with a good prognosis .
it is impossible to give a proper evaluation to the success rate of the various treatments since the same criteria of evaluation of the results are inconsistent themselves .
realistically , we can not say that implants have a higher percentage of survival if compared with real teeth treated and reconstructed in a proper way .
the availability of osteointegrated implants does not justify the extraction of natural teeth that can still offer a stable function .
a compromised tooth should be managed with a multidisciplinary approach and dental implants should be reserved for patients with truly end - stage tooth failure .
the dramatic evolution from endo - surgery to micro - endo surgery has enlarged the horizon of therapeutic options . illumination and magnification through the microscope
the clinical experience has evidentiated the following benefits :
optimal hermetic seal of the neo - apexgtr application increases the long - term results in the treatment of borderline casescorrect and favorable management of endo - sinus pathologiesexpanded diagnostic power in difficult clinical situationsexcellent evaluation of clinical data to accelerate the correct choice in the determination between tooth or implant .
optimal hermetic seal of the neo - apex gtr application increases the long - term results in the treatment of borderline cases correct and favorable management of endo - sinus pathologies expanded diagnostic power in difficult clinical situations excellent evaluation of clinical data to accelerate the correct choice in the determination between tooth or implant .
do better. finally , we would like to stress the concept that fresh socket implants with endodontic indications have to be considered an important part of the endodontists daily practice .
the diagnostic flap helped us to clarify that the post - extractive therapeutic options have to follow the decision to extract the tooth since the immediate loading has become a trustable and predictable solution .
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there is an immense difference between tradizional endodontic surgery and micro - endo surgery.microsurgical techniques made possible and accessible results , that were unimaginable before . under microscopic control ,
the operative techniques reached continous changes , allowing a better precision and quality standards.the dramatic evolution from endo surgery to micro - endo surgery has enlarged the horizon of therapeutic options.illumination and magnification through the microscope has fundamentally and radically changed the way endo surgery can be performed .
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while macroautophagy , herein simply termed autophagy , plays an indispensible role in the intracellular degradation of proteins and organelles , there is growing evidence that also implicates this process in protein secretion .
this link includes a role for autophagy in regulating unconventional secretion of acb1 in yeast and interleukin-1 ( il-1 ) in mammalian cells .
for instance , the constitutive secretion of il-6 and il-8 from senescent cells appears to involve the formation of a specialized cellular domain termed the tor - autophagy spatial coupling compartment ( tascc ) .
this compartment is directly adjacent to the trans golgi network and is enriched for both signaling proteins such as mtor , as well as for autophagic vacuoles . finally , there are a growing number of studies suggesting a connection between components of the autophagy machinery and the regulated secretion of intracellular lysosomes or granules . for instance ,
deficiency in specific essential autophagy proteins appear to impair regulated secretion from the intestinal paneth cell , bone marrow derived mast cells , pancreatic -cells , melanocytes , osteoclasts and vestibular epithelial cells .
exocytosis from endothelial cells represents one of the first lines of defense following vascular injury .
specific secretory granules within the endothelial cell known as weibel - palade bodies ( wpbs ) contain numerous biologically active molecules , although von willebrand factor ( vwf ) is by far the most abundant .
indeed , vwf is required for formation of wpbs , and heterologous expression of vwf in non - endothelial cells can induce the formation of wpb - like structures .
once secreted , vwf multimers can assemble into long strings that can become tethered to the underlying connective tissue while simultaneously ensnaring circulating platelets .
impaired production , processing or secretion of vwf results in the clinical syndrome known as von willebrand disease , recognized as the most common inherited bleeding disorder .
the production of wpbs is a complex process involving dimerization and disulfide bond formation in the endoplasmic reticulum followed by furin - dependent cleavage in the golgi complex .
the vwf multimers that form can then be folded , in a ph - sensitive fashion , into tubular structures . while structural mutations in vwf can lead to defects in multimer formation or tubulation , relatively little
is known about what other cellular processes regulate the number , ph or secretory potential of wpbs . here
, we demonstrate that autophagy plays a critical role in the biology of wpbs and regulates the in vitro and in vivo release of vwf .
while there is a growing understanding of the molecular and biological role of autophagy , the role this process plays within the vasculature is poorly understood . in an effort to better understand how autophagy might contribute to vessel homeostasis , we analyzed electron micrographs of primary human umbilical vein endothelial cells ( huvecs ) .
both wpbs and autophagosomes have distinctive appearance by electron microscopy , with the former exhibiting a classical cigar shaped appearance and the latter being an intracellular organelle often filled with diverse cellular contents surrounded by a unique double membrane structure .
surprisingly , we noted that within endothelial cells , wpbs and autophagosomes were often found in close proximity .
moreover , we noted multiple instances in which a wpb appeared to be very near and actively fusing with an autophagosome ( fig . 1a and supplemental fig .
examination of the contents of autophagosomes also revealed the presence of what appeared to be wholly or partially intact wpbs , clearly identifiable by their unique shape and internal striations running parallel to their long axis ( fig .
1b ) . to further pursue these observations we performed additional immunogold labeling coupled with electron microscopy analysis to assess the distribution of vwf within endothelial cells .
these studies revealed that as expected , vwf was present at high abundance within mature wpbs ( fig
this immunogold labeled vwf signal appeared to be composed of both free protein , as well as , what appeared to be wholly or partially intact wpbs ( fig . 1d and supplemental fig .
1c - e ) . analyzing 30 random section containing several hundred autophagosomes revealed that 17 % of the autophagosomes had no detectable vwf signal ( 48/279 ) , 42% ( 117/279 ) of the autophagosomes had a moderate amount of vwf ( 1 - 10 immunogold particles ) and 41% ( 114/279 ) of the autophagosomes had a robust signal for vwf ( > 10 immunogold particles per autophagosome ) .
the electron microscopic observations suggested that autophagosomes , and by extension autophagy , might somehow modulate vwf levels or secretion .
autophagosome formation requires that the cytosolic form of lc3 ( lc3-i ) be conjugated in an atg5 and atg7 dependent fashion to form the lipidated lc3-ii species .
based on these past observations , we next sought to analyze the distribution of vwf and lc3 ( atg8 ) using super resolution structured illumination microscopy .
these images revealed the expected cigar shaped wpb morphology and the classical spherical punctae of lipidated lc3-ii ( fig .
1e ) . within endothelial cells , super resolution microscopy revealed that endogenous vwf and lc3 were clearly separable .
nonetheless , at any given time , a substantial fraction of wpbs were closely associated with a lc3 signal ( 37/224 wpbs or 16.7% ) with a smaller percentage of wpbs ( 7/224 or 3.2% ) appearing to be surrounded by a structure consistent with a lc3-coated autophagosome ( fig . 1e and supplemental movie 1 ) .
a separate analysis using confocal microscopy came to a similar conclusion , namely that a substantial fraction ( approximately 30% ) of intracellular lc3 and vwf spots had a centroid to centroid distance of less than 1.5 m ( supplemental fig .
finally , using two color confocal live cell imaging , we could capture in real time , the apparent direct interaction of a lc3-gfp autophagosome with a vwf - mcherry positive wpb ( fig .
nonetheless , additional dual - color ( lc3-mcherry and vwf - gfp ) or single color ( lc3-gfp ) tirf imaging suggested these interactions were rare and that under normal circumstances , autophagosomes do not appear to directly fuse with the plasma membrane ( supplemental movie 2&3 ) . to begin to address whether autophagy modulates vwf biology
, we performed lentiviral shrna mediated knockdown of atg7 in human endothelial cells . in atg7 knockdown cells , western blot analysis confirmed a significant reduction in atg7 expression ( fig .
assessment of autophagic flux often relies on the simultaneous measurements of the level of expression of the multidomain , signaling adaptor protein p62/a170/sqstm1 , hereafter referred to simply as p62 , along with the ratio of lc3-ii to lc3-i .
a rise in p62 levels and a fall in lc3-ii are consistent with impaired autophagic flux .
as expected , knockdown of the essential autophagy gene atg7 had the expected effects on these well characterized markers of autophagic flux ( fig .
we then asked whether knockdown of atg7 altered the stimulated release of vwf . using an elisa - based method of the culture medium , we detected an increase in vwf secretion following stimulation with histamine or vegf ( fig .
we saw similar effects on vwf secretion using an alternative and independent method for knockdown of atg7 that employed sirna ( supplemental fig .
the reduction in the stimulated release of vwf was further confirmed by direct western blot analysis of the endothelial cell supernatant ( fig .
2c , d ) . a recent report has suggested that disruption of autophagy might alter intracellular calcium signaling .
given the central role of calcium in stimulated exocytosis from the endothelium , we wondered whether such a mechanism might explain our observations . using a calcium sensitive fluorometric probe
, we could not however detect significant differences in ligand - induced calcium transients between control and atg7 knockdown cells ( fig .
similarly , other proximal signaling events such as extracellular regulated kinase ( erk ) activation appeared to be unaffected by atg7 knockdown ( supplemental fig .
4 ) . we next sought to establish whether other genetic or pharmacological inhibitors of autophagy had similar effects .
knockdown of the essential autophagy gene atg5 alone or in combination with atg7 also inhibited vwf release ( fig .
following histamine stimulation , the combined knockdown of atg5 and atg7 appeared slightly more effective than single knockdown of either atg7 or atg5 in blocking vwf secretion ( fig .
incubation of human endothelial cells with chloroquine or bafilomycin , two known inhibitors of autophagic flux , also resulted in a marked inhibition of ligand - stimulated vwf secretion ( fig .
in contrast , knockdown of atg5 or atg7 did not appear to alter ligand - stimulated release of endothelin-1 from endothelial cells ( supplemental fig .
5b ) . in addition , while knockdown of atg6 ( beclin 1 ) was quite effective in reducing atg6 expression ( fig 2i ) , it had only a very modest effect on markers of autophagic flux ( supplemental fig .
we next sought to attempt to get a better understanding of why knockdown of an essential autophagy gene such as atg7 might inhibit the secretion of vwf . following synthesis , vwf undergoes a series of processing events in the er and golgi that eventually lead to maturation of the protein and incorporation into wpbs . using a quantitative elisa - based method we determined that the total intracellular level of vwf protein was not significantly different between control and atg7 knockdown endothelial cells ( fig .
similar results were observed following knockdown of atg5 or combined atg5 and atg7 knockdown ( supplemental fig .
we next sought to analyze whether differences might exist in the processing , maturation and secretion of vwf .
the processing of vwf involves a furin - dependent proteoltyic cleavage within the trans - golgi network with the pro - vwf precursor distinguishable from mature vwf based on electrophoretic mobility .
we observed that the steady state ratio of pro - vwf to mature vwf was significantly altered in atg7 or atg5 knockdown cell ( fig .
consistent with a defect in vwf processing , we also noted a reduction in the number of mature wpbs observed in atg7 knockdown cells ( fig .
we saw similar reduction in wpbs in endothelial cells with either atg5 knockdown or following pharmacological inhibition of autophagy using chloroquine or bafilomycin ( supplemental fig .
in contrast , knockdown of atg6 did not alter the number of mature wpbs ( supplemental fig .
these results suggest that one explanation for our observation with atg5 or atg7 knockdown is that these strategies , while not altering overall vwf expression , appear to inhibit the processing and maturation of vwf into mature wpbs .
the formation of wpbs is believed to occur at the trans - golgi network ( tgn ) and autophagy is known to be critical for the homeostatic maintenance of both the er and golgi . furthermore , the ph of wpbs is known to be acidic and this low ph is essential for the shape of the wpbs , as well as vwf tubulation and function . using previously described methods , we were able to establish that wpbs within atg7 knockdown cells exhibited a higher ph than wpbs found in control cells ( fig . 4a and supplemental fig .
consistent with previous results , when assessed by confocal microscopy , this higher ph in atg7 knockdown cells resulted in rounder and less rod - shaped wpbs ( fig .
this also translated into a statistically significant reduction in the length of wpbs found in atg7 knockdown cells ( fig .
similar observations were evident by electron microscopy where we observed that knockdown of atg7 resulted in wpbs that were shorter , rounder and had less distinct tubulation ( fig .
whether in addition to a processing defect , there was also an effect of atg7 on the secretion of mature wpbs , we employed a previously described system using live cell imaging of the secretion of a vwf - gfp chimeric fusion protein .
expression of this tagged vwf results in assembly of wpbs containing the co - expressed fluorescently labeled vwf protein ( supplemental fig .
consistent with the alterations in processing described above , the number of gfp - positive wpbs was reduced in cells where atg7 was knockdown ( supplemental fig .
9b ) and the wpbs that formed , had an altered , more rounded morphology ( supplemental movies 4 , 5 ) .
using this system , in conjunction with total internal reflection fluorescence ( tirf ) microscopy , we could monitor individual secretion events of gfp - positive wpbs that localized near the plasma membrane ( fig .
our analysis demonstrated that knockdown of atg7 resulted in reduced wpb secretion after histamine stimulation ( fig .
4 g ) . in an effort to extend these observations to an in vivo situation
, we conditionally deleted atg7 within endothelial cells by crossing mice harboring atg7 floxed alleles with transgenic mice expressing the previously described ve - cadherin cre recombinase .
parental mice both of which bore the genotype atg7 ; ve - cadherin cre were crossed to generate offspring that were either atg7 ; ve - cadherin cre ( hereafter denoted as atg7 ) or atg7 ; ve - cadherin cre ( hereafter denoted as control ) .
given the relatively small contribution of endothelial cells to overall organ mass , we did not observe a significant reduction in atg7 expression in the tissues and organs isolated from atg7 mice ( supplemental fig .
primary cultures of mouse endothelial cells isolated from atg7 mice exhibited the expected reduction in atg7 expression ( fig .
the residual expression of atg7 in these cells may represent a combination of incomplete deletion with ve - cadherin cre , as well as potentially some degree of contamination of non - endothelial cells in these primary cultures .
nonetheless , this level of atg7 expression was sufficient to induce a reduction in autophagic flux as evidenced by the lc3-i / ii ratio and p62 levels ( fig .
similar to our observations employing knockdown of atg7 in human endothelial cells , we did however observe a reduction in the number of mature wpbs in endothelial cells derived from atg7 mice ( fig 5b ) . while conditional deletion of essential autophagy genes in tissues such as brain led to marked alterations in tissue homeostasis and early mortality , atg7 mice exhibited no obvious vascular phenotypes .
similarly , atg7 mice exhibited no increase in mortality over the first year of life ( unpublished observations ) .
assessment of vascular density demonstrated that atg7 mice were indistinguishable from control animals ( fig .
5c ) . in addition , while autophagy has been implicated in angiogenesis , we saw no consistent or reproducible abnormalities in atg7 mice when post - natal retinal angiogenesis was assessed ( fig .
furthermore , the endothelium and underlying architecture of large vessels were similar in atg7 and control mice ( fig .
we also observed no reduction in the level of vwf expression in atg7 mice either by immunohistochemical ( fig .
we next asked whether atg7 mice exhibited defects in vwf secretion . in control mice ,
6a , fold increase : 1.5 + / 0.04 , n=27 mice , p<0.001 pre versus post ) .
this increase in vwf in response to subcutaneous epinephrine injection is similar in magnitude to previous reports .
in contrast , atg7 mice failed to respond in a significant fashion to an epinephrine challenge ( fig .
6a , fold increase : 1.17 + / 0.05 , n=13 mice , p = ns pre versus post ) .
this lack of stimulated epinephrine release was also evident in mice where atg5 had been conditionally deleted again using ve - cadherin cre .
similar to atg7 mice , atg5 mice also failed to respond to epinephrine ( fig .
6a , fold increase : 1.13 + / 0.07 , n=9 mice , p = ns pre versus post ) .
neither atg5 mice nor atg7 mice had a significant reduction in basal vwf ( fig .
similarly , basal levels of circulating p - selectin levels were not altered between control and atg7 mice ( supplemental fig .
12 ) . to therefore further assess the role of autophagy in regulating basal vwf levels , we analyzed mice having a complete , whole body knockout of atg7 .
previous studies demonstrated that these total body atg7 knockout mice rarely survive for more than 48 hours after birth .
when we analyzed atg7 mice shortly after delivery , we did note a reduction in plasma vwf when compared to wild type littermates , perhaps reflective of a more complete knockout in this model then can be achieved using conditional approaches ( fig .
we next sought to further characterize the physiological importance of atg7 in vwf processing in vivo .
consistent with the observed in vitro deficit in vwf processing , we noted that on non - reducing gels , plasma vwf analyzed from atg7 mice contained diminished levels of the biologically active , higher molecular weight multimers ( fig .
consistent with this reduction in high molecular weight vwf , coupled with the decrement in stimulated vwf release ( fig .
6a ) , we noted a significant increase in measured bleeding time when control mice were compared to atg7 mice ( fig .
such differences occurred in the absence of any measurable differences in various hemostatic parameters ( supplemental fig .
furthermore , we did not see any obvious abnormalities in platelet morphology or platelet vwf levels that could have potentially contributed to the observed alterations in the measured bleeding time ( supplemental fig .
14 ) . given that one of the many effects for chloroquine is inhibition of autophagy and since this agent is approved for human usage , we next randomized mice to saline or chloroquine ( cq ) injections .
after 9 days , basal plasma vwf levels in cq treated and vehicle treated mice were similar ( supplemental fig .
nonetheless , direct assessment of hemostasis revealed that similar to atg7 mice , pharmacological inhibition of autophagic flux resulted in a significant prolongation of bleeding time ( fig . 6e and supplemental fig .
while there is a growing understanding of the molecular and biological role of autophagy , the role this process plays within the vasculature is poorly understood . in an effort to better understand how autophagy might contribute to vessel homeostasis , we analyzed electron micrographs of primary human umbilical vein endothelial cells ( huvecs ) .
both wpbs and autophagosomes have distinctive appearance by electron microscopy , with the former exhibiting a classical cigar shaped appearance and the latter being an intracellular organelle often filled with diverse cellular contents surrounded by a unique double membrane structure .
surprisingly , we noted that within endothelial cells , wpbs and autophagosomes were often found in close proximity .
moreover , we noted multiple instances in which a wpb appeared to be very near and actively fusing with an autophagosome ( fig . 1a and supplemental fig .
examination of the contents of autophagosomes also revealed the presence of what appeared to be wholly or partially intact wpbs , clearly identifiable by their unique shape and internal striations running parallel to their long axis ( fig .
1b ) . to further pursue these observations we performed additional immunogold labeling coupled with electron microscopy analysis to assess the distribution of vwf within endothelial cells .
these studies revealed that as expected , vwf was present at high abundance within mature wpbs ( fig
this immunogold labeled vwf signal appeared to be composed of both free protein , as well as , what appeared to be wholly or partially intact wpbs ( fig . 1d and supplemental fig .
1c - e ) . analyzing 30 random section containing several hundred autophagosomes revealed that 17 % of the autophagosomes had no detectable vwf signal ( 48/279 ) , 42% ( 117/279 ) of the autophagosomes had a moderate amount of vwf ( 1 - 10 immunogold particles ) and 41% ( 114/279 ) of the autophagosomes had a robust signal for vwf ( > 10 immunogold particles per autophagosome ) .
the electron microscopic observations suggested that autophagosomes , and by extension autophagy , might somehow modulate vwf levels or secretion .
autophagosome formation requires that the cytosolic form of lc3 ( lc3-i ) be conjugated in an atg5 and atg7 dependent fashion to form the lipidated lc3-ii species .
based on these past observations , we next sought to analyze the distribution of vwf and lc3 ( atg8 ) using super resolution structured illumination microscopy .
these images revealed the expected cigar shaped wpb morphology and the classical spherical punctae of lipidated lc3-ii ( fig .
1e ) . within endothelial cells , super resolution microscopy revealed that endogenous vwf and lc3 were clearly separable .
nonetheless , at any given time , a substantial fraction of wpbs were closely associated with a lc3 signal ( 37/224 wpbs or 16.7% ) with a smaller percentage of wpbs ( 7/224 or 3.2% ) appearing to be surrounded by a structure consistent with a lc3-coated autophagosome ( fig . 1e and supplemental movie 1 ) .
a separate analysis using confocal microscopy came to a similar conclusion , namely that a substantial fraction ( approximately 30% ) of intracellular lc3 and vwf spots had a centroid to centroid distance of less than 1.5 m ( supplemental fig .
finally , using two color confocal live cell imaging , we could capture in real time , the apparent direct interaction of a lc3-gfp autophagosome with a vwf - mcherry positive wpb ( fig .
nonetheless , additional dual - color ( lc3-mcherry and vwf - gfp ) or single color ( lc3-gfp ) tirf imaging suggested these interactions were rare and that under normal circumstances , autophagosomes do not appear to directly fuse with the plasma membrane ( supplemental movie 2&3 ) .
to begin to address whether autophagy modulates vwf biology , we performed lentiviral shrna mediated knockdown of atg7 in human endothelial cells . in atg7 knockdown cells , western blot analysis confirmed a significant reduction in atg7 expression ( fig .
assessment of autophagic flux often relies on the simultaneous measurements of the level of expression of the multidomain , signaling adaptor protein p62/a170/sqstm1 , hereafter referred to simply as p62 , along with the ratio of lc3-ii to lc3-i . a rise in p62 levels and a fall in lc3-ii are consistent with impaired autophagic flux . as expected , knockdown of the essential autophagy gene atg7 had the expected effects on these well characterized markers of autophagic flux ( fig .
we then asked whether knockdown of atg7 altered the stimulated release of vwf . using an elisa - based method of the culture medium
, we detected an increase in vwf secretion following stimulation with histamine or vegf ( fig .
we saw similar effects on vwf secretion using an alternative and independent method for knockdown of atg7 that employed sirna ( supplemental fig .
the reduction in the stimulated release of vwf was further confirmed by direct western blot analysis of the endothelial cell supernatant ( fig .
2c , d ) . a recent report has suggested that disruption of autophagy might alter intracellular calcium signaling .
given the central role of calcium in stimulated exocytosis from the endothelium , we wondered whether such a mechanism might explain our observations . using a calcium sensitive fluorometric probe
, we could not however detect significant differences in ligand - induced calcium transients between control and atg7 knockdown cells ( fig .
similarly , other proximal signaling events such as extracellular regulated kinase ( erk ) activation appeared to be unaffected by atg7 knockdown ( supplemental fig .
4 ) . we next sought to establish whether other genetic or pharmacological inhibitors of autophagy had similar effects .
knockdown of the essential autophagy gene atg5 alone or in combination with atg7 also inhibited vwf release ( fig .
following histamine stimulation , the combined knockdown of atg5 and atg7 appeared slightly more effective than single knockdown of either atg7 or atg5 in blocking vwf secretion ( fig .
incubation of human endothelial cells with chloroquine or bafilomycin , two known inhibitors of autophagic flux , also resulted in a marked inhibition of ligand - stimulated vwf secretion ( fig .
in contrast , knockdown of atg5 or atg7 did not appear to alter ligand - stimulated release of endothelin-1 from endothelial cells ( supplemental fig .
5b ) . in addition , while knockdown of atg6 ( beclin 1 ) was quite effective in reducing atg6 expression ( fig 2i ) , it had only a very modest effect on markers of autophagic flux ( supplemental fig .
we next sought to attempt to get a better understanding of why knockdown of an essential autophagy gene such as atg7 might inhibit the secretion of vwf .
following synthesis , vwf undergoes a series of processing events in the er and golgi that eventually lead to maturation of the protein and incorporation into wpbs . using a quantitative elisa - based method we determined that the total intracellular level of vwf protein was not significantly different between control and atg7 knockdown endothelial cells ( fig .
similar results were observed following knockdown of atg5 or combined atg5 and atg7 knockdown ( supplemental fig .
we next sought to analyze whether differences might exist in the processing , maturation and secretion of vwf .
the processing of vwf involves a furin - dependent proteoltyic cleavage within the trans - golgi network with the pro - vwf precursor distinguishable from mature vwf based on electrophoretic mobility .
we observed that the steady state ratio of pro - vwf to mature vwf was significantly altered in atg7 or atg5 knockdown cell ( fig .
consistent with a defect in vwf processing , we also noted a reduction in the number of mature wpbs observed in atg7 knockdown cells ( fig .
we saw similar reduction in wpbs in endothelial cells with either atg5 knockdown or following pharmacological inhibition of autophagy using chloroquine or bafilomycin ( supplemental fig .
in contrast , knockdown of atg6 did not alter the number of mature wpbs ( supplemental fig .
these results suggest that one explanation for our observation with atg5 or atg7 knockdown is that these strategies , while not altering overall vwf expression , appear to inhibit the processing and maturation of vwf into mature wpbs .
the formation of wpbs is believed to occur at the trans - golgi network ( tgn ) and autophagy is known to be critical for the homeostatic maintenance of both the er and golgi .
furthermore , the ph of wpbs is known to be acidic and this low ph is essential for the shape of the wpbs , as well as vwf tubulation and function .
using previously described methods , we were able to establish that wpbs within atg7 knockdown cells exhibited a higher ph than wpbs found in control cells ( fig . 4a and supplemental fig .
consistent with previous results , when assessed by confocal microscopy , this higher ph in atg7 knockdown cells resulted in rounder and less rod - shaped wpbs ( fig .
this also translated into a statistically significant reduction in the length of wpbs found in atg7 knockdown cells ( fig .
similar observations were evident by electron microscopy where we observed that knockdown of atg7 resulted in wpbs that were shorter , rounder and had less distinct tubulation ( fig .
4e ) . to further assess whether in addition to a processing defect , there was also an effect of atg7 on the secretion of mature wpbs , we employed a previously described system using live cell imaging of the secretion of a vwf - gfp chimeric fusion protein .
expression of this tagged vwf results in assembly of wpbs containing the co - expressed fluorescently labeled vwf protein ( supplemental fig .
consistent with the alterations in processing described above , the number of gfp - positive wpbs was reduced in cells where atg7 was knockdown ( supplemental fig .
9b ) and the wpbs that formed , had an altered , more rounded morphology ( supplemental movies 4 , 5 ) . using this system , in conjunction with total internal reflection fluorescence ( tirf ) microscopy
, we could monitor individual secretion events of gfp - positive wpbs that localized near the plasma membrane ( fig .
our analysis demonstrated that knockdown of atg7 resulted in reduced wpb secretion after histamine stimulation ( fig .
in an effort to extend these observations to an in vivo situation , we conditionally deleted atg7 within endothelial cells by crossing mice harboring atg7 floxed alleles with transgenic mice expressing the previously described ve - cadherin cre recombinase .
parental mice both of which bore the genotype atg7 ; ve - cadherin cre were crossed to generate offspring that were either atg7 ; ve - cadherin cre ( hereafter denoted as atg7 ) or atg7 ; ve - cadherin cre ( hereafter denoted as control ) . given the relatively small contribution of endothelial cells to overall organ mass , we did not observe a significant reduction in atg7 expression in the tissues and organs isolated from atg7 mice ( supplemental fig .
in contrast , primary cultures of mouse endothelial cells isolated from atg7 mice exhibited the expected reduction in atg7 expression ( fig .
the residual expression of atg7 in these cells may represent a combination of incomplete deletion with ve - cadherin cre , as well as potentially some degree of contamination of non - endothelial cells in these primary cultures .
nonetheless , this level of atg7 expression was sufficient to induce a reduction in autophagic flux as evidenced by the lc3-i / ii ratio and p62 levels ( fig .
similar to our observations employing knockdown of atg7 in human endothelial cells , we did however observe a reduction in the number of mature wpbs in endothelial cells derived from atg7 mice ( fig 5b ) . while conditional deletion of essential autophagy genes in tissues such as brain led to marked alterations in tissue homeostasis and early mortality , atg7 mice exhibited no obvious vascular phenotypes .
similarly , atg7 mice exhibited no increase in mortality over the first year of life ( unpublished observations ) .
assessment of vascular density demonstrated that atg7 mice were indistinguishable from control animals ( fig .
5c ) . in addition , while autophagy has been implicated in angiogenesis , we saw no consistent or reproducible abnormalities in atg7 mice when post - natal retinal angiogenesis was assessed ( fig .
, the endothelium and underlying architecture of large vessels were similar in atg7 and control mice ( fig .
we also observed no reduction in the level of vwf expression in atg7 mice either by immunohistochemical ( fig .
we next asked whether atg7 mice exhibited defects in vwf secretion . in control mice , injection of epinephrine led to a significant rise in plasma vwf levels ( fig .
6a , fold increase : 1.5 + / 0.04 , n=27 mice , p<0.001 pre versus post ) .
this increase in vwf in response to subcutaneous epinephrine injection is similar in magnitude to previous reports .
in contrast , atg7 mice failed to respond in a significant fashion to an epinephrine challenge ( fig .
6a , fold increase : 1.17 + / 0.05 , n=13 mice , p = ns pre versus post ) .
this lack of stimulated epinephrine release was also evident in mice where atg5 had been conditionally deleted again using ve - cadherin cre .
similar to atg7 mice , atg5 mice also failed to respond to epinephrine ( fig .
6a , fold increase : 1.13 + / 0.07 , n=9 mice , p = ns pre versus post ) .
neither atg5 mice nor atg7 mice had a significant reduction in basal vwf ( fig .
basal levels of circulating p - selectin levels were not altered between control and atg7 mice ( supplemental fig .
to therefore further assess the role of autophagy in regulating basal vwf levels , we analyzed mice having a complete , whole body knockout of atg7 .
previous studies demonstrated that these total body atg7 knockout mice rarely survive for more than 48 hours after birth .
when we analyzed atg7 mice shortly after delivery , we did note a reduction in plasma vwf when compared to wild type littermates , perhaps reflective of a more complete knockout in this model then can be achieved using conditional approaches ( fig .
we next sought to further characterize the physiological importance of atg7 in vwf processing in vivo .
consistent with the observed in vitro deficit in vwf processing , we noted that on non - reducing gels , plasma vwf analyzed from atg7 mice contained diminished levels of the biologically active , higher molecular weight multimers ( fig .
consistent with this reduction in high molecular weight vwf , coupled with the decrement in stimulated vwf release ( fig .
6a ) , we noted a significant increase in measured bleeding time when control mice were compared to atg7 mice ( fig .
such differences occurred in the absence of any measurable differences in various hemostatic parameters ( supplemental fig .
furthermore , we did not see any obvious abnormalities in platelet morphology or platelet vwf levels that could have potentially contributed to the observed alterations in the measured bleeding time ( supplemental fig .
14 ) . given that one of the many effects for chloroquine is inhibition of autophagy and since this agent is approved for human usage , we next randomized mice to saline or chloroquine ( cq ) injections .
after 9 days , basal plasma vwf levels in cq treated and vehicle treated mice were similar ( supplemental fig .
nonetheless , direct assessment of hemostasis revealed that similar to atg7 mice , pharmacological inhibition of autophagic flux resulted in a significant prolongation of bleeding time ( fig . 6e and supplemental fig .
our data suggests that knockdown or deletion of atg5 or atg7 results in the impaired in vitro and in vivo stimulated secretion of vwf .
interestingly , we and others have demonstrated that conditional deletion of atg7 within the pancreatic -cell leads to impaired secretion of insulin .
similarly , impaired granular exocytosis is observed following disruption of autophagy within mast cells , the intestinal paneth cell and osteoclasts .
thus , our current results , in conjunction with these past observations , suggest that in addition to its known role in intracellular recycling , autophagy plays a fundamental role in the regulated secretion that occurs in certain specialized cell types .
it is currently unclear in the case of mast cells , paneth cells or osteoclasts exactly how autophagy regulates granule secretion .
our data suggests that autophagosomes and wpbs can directly interact and that vwf protein and wpb remnants appear to be found within autophagosomes .
recent evidence suggests that under certain conditions ( e.g. starvation ) , autophagic vacuoles can actually be directly secreted by endothelial cells .
consistent with our microscopy observations , proteomic analysis of these secreted endothelial autophagic vacuoles reveals they contain vwf .
nonetheless , although our data suggests an interaction between autophagosomes and wpbs , this interaction is relatively rare when assessed by electron microscopy , super resolution microscopy or dual - color tirf imaging .
one intriguing possibility is that the observed interaction represents a mechanism to remove damaged or dysfunctional wpbs , in a similar fashion to how mitophagy is thought to remove damaged mitochondria .
how autophagosomes could potentially distinguish a normal from a damaged wpb is unclear , although in this regard , the basis of mitophagic selectivity also remains unresolved .
finally , it is interesting to note that our observations may have important therapeutic implications .
this agent has many effects on cells since it can suppress the acidification of late endosomes , mvbs and lysosomes .
chloroquine is widely given for the treatment and prevention of malaria as well as for treatment of certain classes of autoimmune diseases .
interestingly , one known side effect of the drug is an increased risk for bleeding and the longer acting derivative hydroxychloroquine has been shown to be effective in humans as a prophylaxis against thromboembolic complications following surgery .
our data provides support to the notion that other agents that similarly target autophagic flux might also modulate vwf secretion . as such
, targeted pharmacological inhibition of the autophagic pathway may provide a novel approach to reduce thrombus formation .
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endothelial secretion of von willebrand factor ( vwf ) from intracellular organelles known as weibel - palade bodies ( wpbs ) is required for platelet adhesion to the injured vessel wall . here
, we demonstrate that wpbs are in some cases found near or within autophagosomes and that endothelial autophagosomes contain abundant vwf protein .
pharmacological inhibitors of autophagy , or knockdown of the essential autophagy genes atg5 or atg7 , inhibits the in vitro secretion of vwf .
furthermore , while mice with an endothelial specific deletion of atg7 have normal vessel architecture and capillary density , these animals exhibit impaired epinephrine - stimulated vwf release , reduced levels of high molecular weight vwf multimers and a corresponding elevation of their bleeding times .
endothelial deletion of atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis .
thus , autophagy regulates endothelial vwf secretion and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events .
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single nucleotide polymorphisms ( snps ) are dna sequence variations that occur when a single nucleotide ( a , t , c , or g ) in the genome is altered .
snps make up about 90% of all human genetic variation , occurring every 100300 bases along the 3-billion - base human genome , although their density vary between regions .
snps are found in both coding ( gene ) and noncoding regions of the genome .
many snps have no effect on cell function ; however , others could predispose people to disease or influence their response to a drug .
nonsynonymous snps ( nssnps ) that lead to an amino acid residue substitution in the protein product are of particular interest because they are responsible for nearly half of the known genetic variations related to human inherited disease . coding synonymous snps ( ssnps ) and snps occurring outside gene promoter or coding regions may nevertheless still have consequences for gene expression , splicing , or transcription - factor binding [ 3 , 4 ] .
the identification of snps responsible for specific phenotypes appears to be a problem that is very difficult to solve , requiring multiple testing of hundreds or thousands of snps in candidate genes .
however , the question of how to choose the set of snps to be screened is critical to the success of association studies . a possible way to overcome this problem would be to prioritize snps according to their functional significance [ 6 , 7 ] by using bioinformatics prediction tools , which may help discriminate neutral snps from snps of likely functional importance and could also be useful to reveal the structural basis of disease mutations . without any careful preselection of snps to be screened , a huge number of individuals might be required to detect association at a reasonable level of statistical significance .
although wetlab - based approaches used to identify disease - associated snps from a large number of neutral snps remain crucial evidence for the functional role of snps , numerous disease associations published could not be confirmed by subsequent independent studies [ 6 , 9 ] .
hence , independent evidence of functionality of snps obtained by using prediction tools could also serve as additional argument to discriminate true associations from false positives , as shown recently by the functional snp analysis of the brca1 , abl1 , erbb2 , cftr , and egfr genes [ 1014 ] .
insulin - like growth factor 1 receptor ( igf1r ) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival .
this receptor is implicated in several cancers , including both breast and prostate cancer [ 15 , 16 ] .
evidence suggests that igf1r signaling is required for survival and growth when prostate cancer cells progress to androgen independence , as increased levels of the receptor are expressed in the majority of primary and metastatic prostate cancer patient tumors .
there have also been studies showing associations of igf1r polymorphisms in dementia and ischemic stroke [ 19 , 20 ] . although there are presently several articles describing the association of snps in the igf1r gene with different types of diseases , computational analysis has not yet been undertaken on the functional consequences of snps in this gene .
we applied different publicly available computational algorithms , namely , sorting intolerant from tolerant ( sift ) , polymorphism phenotyping ( polyphen ) , and function analysis and selection tool for single nucleotide polymorphisms ( fastsnp ) to identify likely deleterious snps which could affect protein function .
the sift algorithm predicts whether an amino acid substitution affects protein function based on sequence homology among related genes and domains over evolutionary time , and the physical - chemical properties of the amino acid residues [ 2426 ] .
sequence conservation and the nature of the amino acid residues involved are also incorporated by polyphen , but it also values the location of the substitution within known structures and structural features of the protein available in the annotated database swissprot [ 5 , 27 ] . by accessing a variety of heterogeneous biological databases and analytical tools , fastsnp is able to identify snps most likely to have functional effects , such as changes to the transcriptional level and pre - mrna splicing .
sift and polyphen were approximately 80% successful in benchmarking studies employing amino acid substitutions assumed to have a major negative impact on the residual activity of the variant protein as the test set [ 22 , 25 , 2729 ] and it has been estimated that the false negative and false positive error rates of sift is 31% and 20% , and 31% and 9% for polyphen .
fastsnp was used to analyze 1569 snps from the snp500 cancer database , and results showed that snps with a high predicted risk exhibited low allele frequencies for the minor alleles , which is consistent with the finding that a strong selective pressure exist for functional polymorphisms [ 23 , 30 ] . as the majority of disease mutations affect protein stability [ 31 , 32 ] , we also proposed modeled protein structures for the mutant proteins and compared them with the native protein in order to evaluate stability changes .
sift takes a query sequence and uses multiple alignment information to predict tolerated and deleterious substitutions for every position of the query sequence ( http://sift.jcvi.org ) .
it is a multistep procedure that , given a protein sequence , ( 1 ) searches for similar sequences , ( 2 ) chooses closely related sequences that may share similar function , ( 3 ) obtains the multiple alignment of these chosen sequences , and ( 4 ) calculates normalized probabilities for all possible substitutions at each position from the alignment .
substitutions at each position with normalized probabilities less than a tolerance index of 0.05 are predicted to be intolerant or deleterious ; those greater than or equal to 0.05 are predicted to be tolerated [ 24 , 26 ] .
the analysis was performed by allowing the algorithm to search for homologous sequences using the default settings ( uniprot - trembl 39.6 database , median conservation of sequences of 3.00 , and allowance to remove sequences more than 90% identical to query sequence ) .
the igf1r fasta amino acid sequence of the ncbi protein accession i d np_000866.1 was used as the query sequence , and a total of 24 igf1r nssnps filtered from the dbsnp database were analyzed .
polyphen prediction is based on straightforward empirical rules which are applied to the sequence , phylogenetic and structural information characterizing the substitution .
the online input form available at http://coot.embl.de/polyphen was filled with the igf1r amino acid sequence in fasta format ( ncbi protein accession i d np_000866.1 ) , and the position and substitution of each of the 24 nssnps analyzed by sift were also submitted for polyphen analysis .
polyphen then searched for 3d protein structures , multiple alignments of homologous sequences and amino acid contact information in several protein structure databases , calculated position - specific independent counts ( psic ) scores for each of the two amino acid residues entered ( the original residue and the nssnp ) , and then computed the psic scores difference of the two residues .
the higher a psic score difference , the higher functional impact a particular amino acid substitution is likely to have . a psic score difference of 1.5 and above is considered to be damaging .
the online tool fastsnp was used to determine the impact of the ssnps , 3 utr regions snps and intronic snps on the regulation of the igf1r gene .
the fastsnp server ( http://fastsnp.ibms.sinica.edu.tw ) follows the decision tree principle with external web service access to tfsearch , which predicts whether a non - coding snp alters the transcription factor binding site of a gene .
the score is given on the basis of levels of risk with a ranking of 0 , 1 , 2 , 3 , 4 , or 5 .
this signifies the levels of no , very low , low , medium , high , and very high effect , respectively .
structural analysis was performed in order to evaluate and compare the stability of native and mutant structures .
information about mapping the nssnps in the protein structure was obtained from dbsnp . the highest resolution ( 2.00 ) native structure of the igf1r protein available in the protein data bank ( pdb ) has
the positions of the studied nssnps mutations on pdbid 2oj9 were confirmed by pairwise alignment between the fasta amino acid sequence of the igf1r protein obtained from the ncbi ( np_000866.1 ) and the 2oj9 fasta amino acid sequence , using the sequence manipulation suite .
the amino acid residue substitutions were performed using the swiss - pdb viewer , followed by energy minimization of the modeled 3d structures using the gromacs software version 4.0 .
the algorithms used for energy minimization were the steepest descent ( 1000 steps ) , followed by conjugate gradient ( 1500 steps ) alternating with the steepest descent every 100 steps .
the comparison between the resulting native and modeled structures was made by the calculation of the potential energy and rmsd values .
polymorphism data of the igf1r gene investigated in this paper was retrieved from the dbsnp database .
it contained a total of 2412 snps , out of which 32 ( 1.3% ) were nssnps , 58 ( 2.4% ) were ssnps , 83 ( 3.4% ) occurred in the mrna 3 utr , and 2225 ( 92.2% ) occurred in intronic regions .
it can be seen from the distribution in figure 1 that the vast majority of snps occur in the intronic region , and that there are more 3 utr region snps than nssnps or ssnps .
we selected missense nssnps , ssnps , 3 utr snps , and intronic snps for our investigation .
protein sequence with mutational position and amino acid residue variants associated to 24 missense nssnps were submitted as input to the sift server , and the results are shown in table 1 , along with the corresponding heterozygosity and validation status description for each snp , when available from dbsnp . according to the classification proposed by ng and henikoff and xi et al .
, the lower the tolerance index , the higher the functional impact a particular amino acid residue substitution is likely to have and vice versa . among the 24 nssnps analyzed ,
five nssnps ( rs61740868 , rs45578132 , rs45553041 , rs45526336 , and rs45504297 ) showed a highly deleterious tolerance index score of 0.00 .
the remaining deleterious nssnps showed tolerance index scores of 0.01 ( rs45524940 and rs45512296 ) and 0.03 ( rs45445894 ) .
four deleterious nssnps showed a nucleotide change from g / a , four a change from c / t , two a change from t / c , and one a change from a / g .
all the 24 protein sequences of missense nssnps submitted to sift were also submitted to the polyphen server . a psic score difference of 1.5 and above is considered to be damaging .
eight nssnps ( rs70958401 , rs61740868 , rs45578132 , rs45504297 , rs45553041 , rs45512296 , rs45524940 , and rs33958176 ) were considered to be damaging and exhibited a range of psic score difference between 1.503 and 2.609 ( table 1 ) . out of these damaging nssnps , two changed from positively charged amino acid in the native protein to hydrophobic amino acid in the mutant type , two from aliphatic nonpolar amino acid to non - polar amino acid , two from positively charged amino acid to aromatic positively charged amino acid , one from polar amino acid to non - polar amino acid , and one from
it can be seen from table 1 that there was significant correlation between the results obtained from the evolutionary - based approach sift and the structural - based approach polyphen for six nssnps predicted to be damaging by polyphen , suggesting that these nssnps may disrupt both the protein function and structure .
the most damaging nssnp ( rs61740868 ) showed a psic score of 2.609 , due to a mutation from arginine to cysteine . according to fastsnp , out of 58 ssnps in the igf1r gene ,
31 ssnps were predicted to be damaging with a risk ranking of 2 - 3 , and a possible functional effect on splicing regulation ( table 2 ) . among these
, the a / g polymorphism ( rs2229765 ) has been shown experimentally to affect the susceptibility to ischemic stroke in chinese population to be associated with higher plasma concentrations of circulating igf1r and premature pubarche [ 39 , 40 ] and adult height variation in the human population . out of 2225
snps which occur in the intronic region of the igf1r gene , 3 snps ( rs55895813 , rs36108138 and rs45495500 ) were predicted to affect the splicing site ( 3 - 4 risk ) ( table 2 ) .
it can be seen from table 2 that a coding nonsense snp ( rs45437300 ) due to a nucleotide change from a to t was detected and showed a very high ( 5 - 5 ) level of risk , as it can truncate and even inactivate the igf1r protein , causing disease as a result .
out of eight nssnps predicted to be deleterious by sift or polyphen , four ( rs61740868 , rs45526336 , rs45512296 , and rs45504297 ) were mapped to the pdb i d 2oj9 native structure .
the amino acid residue substitutions were performed by swiss - pdb viewer independently to get four mutant modeled structures ( 2oj9 r1216c , 2oj9 e1253k , 2oj9 r1216h , and 2oj9 l1211p , respectively ) . then , energy minimizations were performed by gromacs for the native structure ( 2oj9 ) and the mutant modeled structures . the total energy for the native structure ( 2oj9 ) and the four mutant modeled structures 2oj9 r1216c , 2oj9 e1253k , 2oj9 r1216h , and 2oj9 l1211p was 13841.67 , 13343.28 , 13887.05 , 13483.34 , and 13782.33 kj / mol , respectively ( table 3 ) .
three out of four mutant modeled structures ( 2oj9 r1216c , 2oj9 r1216h , and 2oj9 l1211p ) showed an increase in energy ( less favorable change ) in comparison with the native structure .
this result correlates with the structural homology method ( polyphen ) results , which predicted all these three mutants to be deleterious ( psic scores 2.609 , 2.128 , and 2.372 , resp . )
( table 1 ) . the mutant model 2oj9 r1216c showed the greatest increase in energy , which may be explained by the energetically unfavorable substitution of a positively charged arginine amino acid residue to a non - polar cysteine amino acid residue at the surface of the protein structure ( figure 2 ) .
it can be seen from table 3 that the rmsd values between the native structure ( 2oj9 ) and the mutant modeled structures are all similar , ranging from 0.22 to 0.48 . because these values are low
, we can suggest that these mutations do not cause a significant change in the mutant structures with respect to the native protein structure .
in this paper , we investigated the functional and structural impact of snps in the igf1r gene using computational prediction tools .
out of a total of 2412 snps in the igf1r gene , 32 snps were found to be non - synonymous , 58 were synonymous , 83 occurred in the mrna 3 utr , and 2225 were found in intronic regions . out of 24 missense nssnps , eight were found to be deleterious by sift , and eight were found to be damaging by the polyphen tool .
a total of six nssnps were found to be damaging by both sift and polyphen tools .
the structural analysis results showed that the amino acid residue substitutions which had the greatest impact on the stability of the igf1r protein were mutations 2oj9 r1216c ( rs61740868 ) and r1216h ( rs45512296 ) . among
the nssnps studied , a nonsense snp ( rs45437300 ) was found . out of 58 ssnps , 31
were predicted to affect splicing regulation by fastsnp , including an ssnp ( rs2229765 ) associated with several diseases . in the intronic region ,
3 snps ( rs55895813 , rs36108138 , and rs45495500 ) were predicted to affect splicing regulation .
based on our results , we conclude that these snps should be considered important candidates in causing diseases related to igf1r malfunction .
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insulin - like growth factor 1 receptor ( igf1r ) acts as a critical mediator of cell proliferation and survival .
many single nucleotide polymorphisms ( snps ) found in the igf1r gene have been associated with various diseases , including both breast and prostate cancer .
the genetics of these diseases could be better understood by knowing the functions of these snps . in this study , we performed a comprehensive analysis of the functional and structural impact of all known snps in this gene using publicly available computational prediction tools . out of a total of 2412 snps in igf1r retrieved from dbsnp , we found 32 nssnps , 58 ssnps , 83 mrna 3 utr snps , and 2225 intronic snps . among the nssnps
, a total of six missense nssnps were found to be damaging by both a sequence homology - based tool ( sift ) and a structural homology - based method ( polyphen ) , and one nonsense nssnp was found .
further , we modeled mutant proteins and compared the total energy values with the native igf1r protein , and showed that a mutation from arginine to cysteine at position 1216 ( rs61740868 ) on the surface of the protein caused the greatest impact on stability . also , the fastsnp tool suggested that 31 ssnps and 3 intronic snps might affect splicing regulation . based on our investigation , we report potential candidate snps for future studies on igf1r mutations .
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the gastrointestinal tract is the most common site of extranodal lymphomas , and colorectal lymphoma comprises between 10 to 20% of primary gut lymphomas in a larger published series .
most such lymphomas are b - cell origin , and t - cell lymphoma of the gastrointestinal tract is extremely rare .
there is a growing interest in the combination of gastrointestinal malignancy and chronic inflammatory bowel disease .
ulcerative colitis is probably best known to be associated with primary lymphoma of the colorectum .
recently , there have been reports suggesting an association of colorectal lymphoma with crohns disease .
because the clinical symptoms of chronic inflammatory bowel disease and malignant lymphoma of the colon can be very similar , diagnosis of lymphoma can be masked and correct treatment may be delayed .
a 40-year - old woman suffering from abdominal pain and diarrhea since september 1994 was admitted to another institution .
she had undergone colonoscopic examination and endoscopy of the colon and histological examination showed chronic inflammatory bowel disease with signs of crohns disease involving the entire colon and the terminal ileum .
differential therapeutic strategies , including corticosteroid , had improved the symptoms which were dominated by abdominal pain and bloody stools 5 times a day for 6 months . after stopping medication , including corticosteroid since februrary 1995
, she had experienced abdominal pain and bloody stool twice a day . when she visited our institute in april of 1995 due to ineffective long - term treatment with corticosteroid , she presented with bloody stool twice a day and 7 kg weight loss in a period of six months . among her complaints
her blood pressure was 130/80mmhg , pulse rate 85/min , respiratory rate 20/min and body temperature 36.c .
abdominal palpatation and percussion indicated a slightly painful abdomen and showed normal peristalsis , no superficial lymphadenopathy and no hepatospelomegaly .
the hematocrit was 38.6% and the white cell count was 7300/mm , with 89% polymorphonuclear cells and 5% lymphocyte .
the serum sodium was 138meq / l , potassium 4.0meq / l , chloride 99meq / l , bicarbonate 23.2meq / l .
the results of liver function tests were as follows : protein 7.3g / dl , albumin 4.4g / dl , cholesterol 189mg / dl , bilirubin 0.6mg / dl , alkaline phosphatase 69u / l , ast 17u / l and alt 13u / l . urinalysis did not reveal any evidence of proteinuria .
anti - hiv was negative , findings on the chest radiograph and simple abdomen were normal .
colon study revealed shallow and geographic ulcerations along the entire colon . small bowel series and enteroclysis revealed ulceration on the distal jejunum .
laboratory examination showed mild inflammatory activity , anemia , normal total and differential white blood cell counts and normal immunoglobulin values .
abdominal ultrasonography and computed tomography were both negative . in upper endoscopy , we were able to find only multiple erosions of the gastric mucosa .
histologic examination of the colon showed diffuse infiltration of monotonous small atypical lymphocytes in the laminar propria of the colon(fig .
these atypical lymphocytes invaded the epithelium of the crypts , mimicking a lymphoepithelial lesion of low grade b cell lymphoma of malt type .
2b ) and cd3 , negative for cd20 . thus , a diagnosis of primary t - cell lymphoma , small cell type , was made .
although there was reactive epithelial change including depletion , no histologic evidence of inflammtory bowel disease was found in the uninvolved mucosa . also , we reviewed the pathology of the colon which was taken from colonoscopy at another institute .
the histologic findings were very similar to those of the 2nd biopsy except for marked acute inflammatory reaction associated with mucosal ulceration .
there were dense atypical small lympohoid infiltrates in the lamina propria with destruction of colonic glands and crypts .
the histologic finding of bone marrow was negative . in conclusion , she had developed primary t - cell lymphoma of the colon .
to distinguish between primary and secondary gastrointestinal lymphoma , the definition of primary gastrointestinal lymphoma is quite applicable , which consists of no palpable superficial lymphadenopathy at presentation , a normal chest x - ray , no evidence of hepatosplenomegaly , normal white blood cell count(i.e .
, no evidence of leukemia ) and a predominant tumor mass in the bowel with only local lymphadenopathy .
this case fulfills these criteria and we considered it as a primary lymphoma which developed in the colon .
the most common site of involvement by gastrointestinal t - cell lymphoma is the small bowel and other sites are exceptional .
t - cell malignant lymphoma of the gastrointestinal tract , although relatively infrequent , may present a difficult diagnostic challenge to pathologists .
difficulties in diagnosing t - cell lymphomas of the gastrointestinal tract stem from several sources .
ideally , tissues from these tumors should be frozen for immunostaining of cell surface markers and for gene rearrangement studies , as well as being fixed routinely and embedded in paraffin . moreover , even when such tissues are frozen , the florid inflammatory infilatrate which usually accompanies these neoplasms , often with ulceration and necrosis , may be all that is present in the areas sampled , or inflammatory cells may greatly outnumber the tumor cells that the neoplasms are difficult to be detected in the frozen section . in this case ,
colonoscopic and histologic finding showed ulceration and inflammation of the entire colon mimicking crohns disease .
an ideal solution to these problems in diagnosis would be to use antibodies for detection of t - cell in routinely fixed , paraffin - embedded tissue .
this would enable appopriate blocks from resection specimens or biopsy specimens to be selected on a basis of well - preserved morphology , and would allow characterization of the neoplastic cells in their inflammatory background .
a number of antibodies reactive with routinely processed tissue have proven recently to be effective in detecting b - and t - cell and their malignant counterparts in routinely processed specimens from nodal and extranodal malignant lymphomas . also , there was the combination of gastrointestinal malignancy and chronic inflammatory bowel disease .
it is already well known that after 2530 years of ulcerative colitis , the cumulative risk of developing cancer is about 10% .
the nature of the relation between inflammatory bowel disease and intestinal lymphoma , however , is less certain .
the first instance of this association was evaluated by barken , in 1927 , who reported two cases of ulcerative colitis with malignant lymphoma .
the small number of reported cases makes it difficult to prove definite association between ulcerative colitis and colonic lymphoma .
today , the principle treatment for non - hodgkins lymphoma is a combination of chemotherapy with radiotherapy .
surgery is used chiefly as a diagnostic tool , with some unique exceptions . combination chemotherapy with cyclophosphamide , doxorubicin , vincristine and prednisone - chop regimen was introduced in the 1970 's . since then , a number of second generation and third generation regimens and combinations of non - cross - resisitant regimens for non - hodgkins lymphoma have been developed .
the benefit of these newer regimens is now in question , but some issues are not yet clearly resolved .
. however , combination chemotherapy with chop regimen should be the primary treatment in cases of lymphoma which is widespread throughout the gastrointestinal tract .
radiotherapy , chemotherapy , or both , may also be of use in cases of advanced stage and high grade lymphoma . however , prognosis is poor .
it is noteworthy that the two reported cases of t - cell lymphoma both pursued an aggressive and fatal course .
the traditional approach of the frozen section immunohistochemistry , so valuable for characterizing most maligant lymphomas , is often less helpful in the evaluation of the typically diffusely involved , inflammated and ulcerated resection specimens .
use of paraffin - reactive monoclonal antibodies facilitated the diagnosis . in conclusion , this is a report of primary t - cell lymphoma of the colon mimicking crohn s disease .
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a 40-year - old woman had been diagnosed with crohns disease in september 1994 , but later examinations revealed a primary t - cell lymphoma of the colon .
colonoscopic and histological examination showed ulcerative lesions simulating crohns disease involving the entire colon and the terminal ileum , and she was first diagnosed as having crohns disease .
differential therapeutic strategies , including corticosteroid , had improved the symptoms which were dominated by abdominal pain .
when she visited our institute in april 1995 , she presented with bloody stool twice a day , 7 kg weight loss in a period of six months and a slightly painful abdomen .
colonoscopic finding showed geographic ulceration on the entire colon , especially rectum and terminal ileum .
the histologic examination of specimens from colonoscopic biopsy showed primary peripheral t - cell lymphoma of the colon .
any dense lymphocyte infiltrates seen in the biopsy specimens obtained from lesions simulating ulcerative colitis or crohns disease should be assessed to exclude intestinal lymphoma
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dementia as a neurodegenerative disease has afflicted 24 million people in the world , and the victim toll is expected to climb to 42 million in 2020 and 80 million in 2040 . as most of these people will suffer from alzheimer 's disease ( ad ) and the treatment expenditure continues to rise , ad has been ranked as a major public health threat to people over 60 years old and remained a research focus in areas such as china , developing countries in western pacific region , and america .
currently , a clinical diagnosis of ad is only possible years after the appearance of typical clinical symptoms , which can be supported by the progressive disease process and increasing symptomatic severity and an accurate diagnosis can be confirmed by autopsy or biopsy , delaying an early provision of any effective treatment . since the 1990s ,
symptomatic ad treatment schemes , such as those with cholinesterase inhibitors and n - methyl - d - aspartate receptor antagonist , have been proposed but many clinical trials have failed to offer promised results .
recently , other treatments such as immunotherapy and those with secretase inhibitors , amyloid aggregators , tau aggregation inhibitors , and gsk3 inhibitors have been employed in animals but most of them are not clinically applied and others have failed in clinical trials .
this depressing situation has rendered it urgent to ascertain an early diagnosis and an early intervention targeting the risk factor .
an early diagnosis of ad involves a prompt identification of the early ad - related biomarkers , which will facilitate the screening of potential therapeutic agents and tracking the prodromal stages of ad and assist the doctors in deciding on an effective therapy to guarantee an optimal prognosis . in line with this concern
, many efforts have been made to establish methods to diagnose ad before the manifestation of typical clinical symptoms .
one possible approach to early ad diagnosis is to look into the olfactory sensory dysfunction .
research has showed that ad patients always have an impaired ability to detect , discriminate , and identify odors .
the scores of the ad groups in the odor identification ability tests were significantly lower than those of normal old - adult groups .
many clinical investigations have demonstrated that impairment in odor identification , superior to deficits in verbal episodic memory , is a better index in predicting cognitive decline in cognitively intact participants , who were followed - up 24 years before cognitive decline and ad dementia .
other observations also strongly argue for olfactory marker as a complementary tool for the early screening of ad patients .
these findings suggest that targeting the olfactory dysfunction may be rewarding in searching for a potential approach to an early diagnosis of ad . of the technologies currently employed in ad research , radiotracer for amyloid imaging of ( f)-florbetapen ( flor ) and ( 11c)-pittsburg compound b ( pib )
have been applied to observe in vivo a deposition in ad , for soluble a in the cytoplasm of neurons and extracellular amyloid plaques are the core pathological biomarkers of ad . however , because of the inherent limitations of the imaging principle , both of them can only reveal the extracellular amyloid plaque and can not display intracellular amyloid , meaning that the techniques are applicable only after the formation of extracellular a plague at the middle stage of ad .
actually , due to the limited sensibility of ( f)-flor and ( 11c)-pib , amyloid deposition can only be detected at the late stage of ad .
fortunately , many recent investigations have showed that positron emission tomography ( pet ) scanning with a radio - labelled glucose analog , 2-deoxy-2-(f ) fluoro - d - glucose ( [ f]-fdg ) , can precisely reveal the decreased glucose metabolism in the ad brain , which results from a deposition and its deleterious effects on neurons and astrocytes .
this scanning technology has also been used in ad patients and demonstrated classical glucose metabolic changes in temporal , parietal , posterior cingulate , and inferior parietal cortices .
but these significant changes occur only years after the appearance of clinical symptoms . up to date
, no research has employed this promising technology to investigate the glucose metabolism of the olfactory bulb in ad model mice .
recently , a new kind of transgenic animal model , which contains all 5 human mutations related to familial ad ( swedish , florida , and london human app mutations ; m146l and l286v ps1 mutations , designated as 5xfad ) , has been invented for ad research .
intracellular amyloid and gliosis and other neuropathological characteristics of ad can be detected in 5xfad mice at 2 months of age , and cognitive deficits are observed at 56 months of age , much earlier than in other transgenic mouse models .
here , we attempted to investigate whether the reduction of glucose metabolism in the olfactory bulb of 5xfad mice occurs earlier than that in other cerebral regions and prior to other incidents such as cognitive impairment and olfactory dysfunction .
mary jo ladu ( department of anatomy and cell biology , university of illinois at chicago , il , usa ) .
these transgenic hemizygous mice were bred to produce the subjects used in the study , and the genotype of the animals was determined by polymerase chain reaction analysis with dna from tail biopsy . as a result , a total of 14 male 5xfad mice ( 3 months of age , n = 7 , 6 months of age , n = 7 ) and 14 male wild - type ( wt ) counterparts ( 3 months of age , n = 7 , 6 months of age , n = 7 ) were enrolled for this study .
the animals were housed in cages ( 30 cm 20 cm 15 cm ; 34 mice per cage ) on a 12 h light cycle ( lights on at 6:30 pm ) at 22c .
the cage contained wood - chip bedding material and a small polyethylene tube and were covered with a metal cage top .
all protocols and procedures used in the study observed the national institute of health guidelines for the care and use of laboratory animals and were approved by the institutional animal care and utilization committee of fujian medical university .
morris water maze test is one of the most common methods to detect the symptoms of dementia and frequently employed to assess ad - related cognitive impairment .
n = 7 , 6 months of age , n = 7 , wt counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) are enrolled for this test .
the maze ( noldus information technology , wageningen , the netherlands ) was a black pool ( 120 cm in diameter ) filled with a mixture of water and milk ( 30 cm in depth ) . a colorless platform
( 10 cm in diameter ) was submerged 2 cm beneath the surface of the mixture and the temperature was set at 23 1c .
before each behavioral test , mice were given 1 h to adapt to the light - controlled testing room . during the testing
, the mice were placed in the pool at one of the four locations , which was changed on each trial .
the animals were allowed 60 s to locate the platform and to stay there for 30 s. if they failed to locate the platform within 60 s , they were guided to the platform , and left there for 30 s. after each trial , the animals were towel dried and returned to their home cages .
mice underwent four swimming trials daily for 4 days with a 20 min interval between trials .
all trials were videotaped and swim path tracked using ethovision video - tracking software ( v3.1.16 noldus information technology , wageningen ) . for each trial , escape latency and total times crossing the platform were calculated . on the fifth day
, the animals were placed in the pool for 60 s and tested in a probe trial with the platform removed .
escape latency and total times crossing the location of the removed platform were taken as a measurement of the memory of the platform position .
the first test was buried food pellet test ( shanghai xinruan , co. , ltd . , china ) . as described , before the test
, the mice were placed on a food - restricted diet of 0.2 g chow each day for 3 days but allowed free access to water .
in each trial , mice were placed in a black test cage ( 45 cm 24 cm 20 cm ) , in which a food pellet ( 0.5 g ) was buried 0.5 cm below the surface of the bedding material ( 3 cm in depth ) .
the latency was recorded from the moment the mice were placed in the cage to the moment they grasped the food pellet with the forepaws or teeth . after the mice had consumed the pellet , they were returned to the cages .
a visible pellet test with the food on top of the bedding was conducted , and mice were allowed to consume the food pellet after the test .
the second test was olfactory maze test ( shanghai xinruan , co. , ltd . ) .
the maze was a black platform pool ( 76.2 cm in diameter ) bounded by a circular wall ( 20 cm in height ) . located within this circular wall were 6 fixed , open corners with equal distance between one another [ figure 1 ] . within the corners ,
the food pellet was hidden from sight but could be found by smelling . before the test , mice were placed on a food - restricted diet of 0.2 g chow each day for 3 days but allowed free access to water .
for the subsequent 3 days , they underwent behavioral testing . before the behavioral trials , the animals were given 1 h to adapt to the testing room .
in each trial , a mouse chow pellet ( 0.5 g ) was placed behind one of the corners randomly , and then mice were placed at the center of the platform .
the latency was calculated from the time when the mice were placed at the center of the platform to the moment they grasped the food pellet .
the animals were returned to the cages after they consumed the pellet . as a control , a visible pellet test following each daily trial
was performed with the food pellet placed on a visible corner of the platform and the mice were allowed to consume the food before returning to the cages .
impaired olfaction in 5xfad mice shown by buried food pellet and olfactory maze tests ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 ; wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) . at 3 months , no significant difference in the latency between wild - type and 5xfad mice on each trial day . at 6 months , a significantly greater latency time for 5xfad mice on each trial day when compared with wild - type mice ( day 1 * p = 0.0012 , day 2 * p = 0.0031 , day 3 * p = 0.0160 in buried food pellet recovery test ; day 1 * p = 0.0002 , day 2 * p < 0.0001 , day 3 * p = 0.0045 in olfaction maze test ) .
no significant difference in the latency time was found in the visible food pellet test across the groups .
after pet - computed tomography ( ct ) scan , all mice were anesthetized with intraperitoneal chloral hydrate ( 125 mg / kg ) and perfused with 4% paraformaldehyde in pbs , and then with pbs .
after the mice had been decapitated , the whole brain containing the olfactory bulb was dissected and postfixed in paraformaldehyde at 4c for 24 h. after being dehydrated with increasing concentrations of sucrose , frozen in liquid nitrogen and stored at 80c , the dissected tissues were further sectioned with a freezing microtome ( cm1950 , leica , wetzlar , germany ) from the olfactory bulb to the caudal hypothalamus into slices ( 30 m in thickness ) and stored at 20c in cryoprotectant solution with 30% glycerin , 30% ethylene glycol ( sigma , st .
louis , mo , usa ) and 40% pbs ( 0.1 mol / l ) .
immunofluorescence was performed according to a published protocol of our laboratory . in brief , sections were washed with tris - buffered saline ( tbs ) , then blocked at room temperature for 1 h with tbs containing 0.3% triton x-100 and 5% bsa , and further incubated at 4c for 48 h with the polyclonal rabbit anti - a ( 6e10 , which recognizes the 116 amino acid sequence of human a , 1:5000 , santa cruz biotechnology , ta , usa ) in tbs containing 0.3% triton x-100 and 5% bsa .
after incubation , the sections were washed with tbs 6 times ( 10 min per wash ) and incubated with cy3-conjugated goat anti - rabbit igg ( 1:2000 ; abcam , cambridge , ma , usa ) in tbs at room temperature for 1 h. then the sections were further incubated with 4,6-diamidino-2-phenylindole for 5 min .
after a 30 min wash , the sections were mounted on poly - l - lysine - coated glass slides and coverslipped with prolong gold antifade reagent ( invitrogen , china ) .
these slides were examined under a confocal microscope ( zeiss , 780 , germany ) to detect a in consistent parameter settings including objectives , laser intensities , pmt voltages , offsets , and pinhole sizes . before the imaging procedure
, all the 5xfad and age - matched wt mice were weighed , and then restrained in a tailveiner restrainer and injected via the tail vein with ( f)-fdg ( beijing pet , co , ltd , china ) at a dose of 500600 ci in 100120 l saline according to their weights .
after 40 min for the uptake of ( f)-fdg , mice were anesthetized with a mixture of 96% o2 and 4% isoflurane , and placed into the animal dedicated pet - ct camera ( inveon micropet , germany ) for ct attenuation correction scan and high density pet image reconstruction .
the spatial restructuring was facilitated with ordered subset expectation maximization with three - dimensional resolution recovery ( osem3d , implementation of c michel ) .
the ct image of the skull of each animal was co - registered with the mouse brain resonance image template , in which the volumes of interest ( vois ) were already drawn .
after co - registration , the spatial mathematic data were transformed into the animals fused pet images to produce a correct match between pet image and the brain template .
for the purpose of quantification , the fdg uptake of the vois was obtained and shown in standard uptake value ( suv ) , and regional suv was quantified with pmod version 3.2 ( pmod technologies , zurich , switzerland ) . during data
acquisition , anesthesia was maintained with 1.5% isoflurane vaporized in o2 through an orofacial mask .
statistical analysis was performed with spss version 11.0 ( spss inc . , chicago , il , usa ) software package and diagrams were plotted with prism graphpad 5.0 software ( graphpad , san diego , usa ) .
indices in acquisition trails such as escape latency , total times crossing the platform in morris water maze test , latency time in olfaction behavioral test , and suv in pet - ct were analyzed with one - way analysis of variance ( anova ) of repeated measures .
all statistical analysis of 5xfad group in comparison with wt group was rechecked with student 's t - test . in all cases ,
mary jo ladu ( department of anatomy and cell biology , university of illinois at chicago , il , usa ) .
these transgenic hemizygous mice were bred to produce the subjects used in the study , and the genotype of the animals was determined by polymerase chain reaction analysis with dna from tail biopsy . as a result , a total of 14 male 5xfad mice ( 3 months of age , n = 7 , 6 months of age , n = 7 ) and 14 male wild - type ( wt ) counterparts ( 3 months of age , n = 7 , 6 months of age , n = 7 ) were enrolled for this study .
the animals were housed in cages ( 30 cm 20 cm 15 cm ; 34 mice per cage ) on a 12 h light cycle ( lights on at 6:30 pm ) at 22c .
the cage contained wood - chip bedding material and a small polyethylene tube and were covered with a metal cage top .
all protocols and procedures used in the study observed the national institute of health guidelines for the care and use of laboratory animals and were approved by the institutional animal care and utilization committee of fujian medical university .
morris water maze test is one of the most common methods to detect the symptoms of dementia and frequently employed to assess ad - related cognitive impairment .
all mice ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wt counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) are enrolled for this test . the maze ( noldus information technology , wageningen , the netherlands ) was a black pool ( 120 cm in diameter ) filled with a mixture of water and milk ( 30 cm in depth ) . a colorless platform
( 10 cm in diameter ) was submerged 2 cm beneath the surface of the mixture and the temperature was set at 23 1c . before each behavioral test , mice were given 1 h to adapt to the light - controlled testing room . during the testing , the mice were placed in the pool at one of the four locations , which was changed on each trial . the animals were allowed 60 s to locate the platform and to stay there for 30 s. if they failed to locate the platform within 60 s , they were guided to the platform , and left there for 30 s. after each trial , the animals were towel dried and returned to their home cages .
mice underwent four swimming trials daily for 4 days with a 20 min interval between trials .
all trials were videotaped and swim path tracked using ethovision video - tracking software ( v3.1.16 noldus information technology , wageningen ) .
for each trial , escape latency and total times crossing the platform were calculated . on the fifth day
, the animals were placed in the pool for 60 s and tested in a probe trial with the platform removed .
escape latency and total times crossing the location of the removed platform were taken as a measurement of the memory of the platform position .
the first test was buried food pellet test ( shanghai xinruan , co. , ltd . , china ) . as described , before the test
, the mice were placed on a food - restricted diet of 0.2 g chow each day for 3 days but allowed free access to water . for the subsequent 3 days , they underwent behavioral testing . before the behavioral trials
in each trial , mice were placed in a black test cage ( 45 cm 24 cm 20 cm ) , in which a food pellet ( 0.5 g ) was buried 0.5 cm below the surface of the bedding material ( 3 cm in depth ) .
the latency was recorded from the moment the mice were placed in the cage to the moment they grasped the food pellet with the forepaws or teeth . after the mice had consumed the pellet , they were returned to the cages .
a visible pellet test with the food on top of the bedding was conducted , and mice were allowed to consume the food pellet after the test .
the second test was olfactory maze test ( shanghai xinruan , co. , ltd . ) .
the maze was a black platform pool ( 76.2 cm in diameter ) bounded by a circular wall ( 20 cm in height ) . located within this circular wall were 6 fixed , open corners with equal distance between one another [ figure 1 ] . within the corners ,
the food pellet was hidden from sight but could be found by smelling . before the test , mice were placed on a food - restricted diet of 0.2 g chow each day for 3 days but allowed free access to water .
in each trial , a mouse chow pellet ( 0.5 g ) was placed behind one of the corners randomly , and then mice were placed at the center of the platform .
the latency was calculated from the time when the mice were placed at the center of the platform to the moment they grasped the food pellet .
the animals were returned to the cages after they consumed the pellet . as a control , a visible pellet test following each daily trial
was performed with the food pellet placed on a visible corner of the platform and the mice were allowed to consume the food before returning to the cages .
impaired olfaction in 5xfad mice shown by buried food pellet and olfactory maze tests ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 ; wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) .
at 3 months , no significant difference in the latency between wild - type and 5xfad mice on each trial day . at 6 months , a significantly greater latency time for 5xfad mice on each trial day when compared with wild - type mice ( day 1 * p = 0.0012 , day 2 * p = 0.0031 , day 3 * p = 0.0160 in buried food pellet recovery test ; day 1 * p = 0.0002 , day 2 * p < 0.0001 , day 3 * p = 0.0045 in olfaction maze test ) .
no significant difference in the latency time was found in the visible food pellet test across the groups .
after pet - computed tomography ( ct ) scan , all mice were anesthetized with intraperitoneal chloral hydrate ( 125 mg / kg ) and perfused with 4% paraformaldehyde in pbs , and then with pbs .
after the mice had been decapitated , the whole brain containing the olfactory bulb was dissected and postfixed in paraformaldehyde at 4c for 24 h. after being dehydrated with increasing concentrations of sucrose , frozen in liquid nitrogen and stored at 80c , the dissected tissues were further sectioned with a freezing microtome ( cm1950 , leica , wetzlar , germany ) from the olfactory bulb to the caudal hypothalamus into slices ( 30 m in thickness ) and stored at 20c in cryoprotectant solution with 30% glycerin , 30% ethylene glycol ( sigma , st .
louis , mo , usa ) and 40% pbs ( 0.1 mol / l ) .
immunofluorescence was performed according to a published protocol of our laboratory . in brief , sections were washed with tris - buffered saline ( tbs ) , then blocked at room temperature for 1 h with tbs containing 0.3% triton x-100 and 5% bsa , and further incubated at 4c for 48 h with the polyclonal rabbit anti - a ( 6e10 , which recognizes the 116 amino acid sequence of human a , 1:5000 , santa cruz biotechnology , ta , usa ) in tbs containing 0.3% triton x-100 and 5% bsa . the primary antibody was omitted in control sections .
after incubation , the sections were washed with tbs 6 times ( 10 min per wash ) and incubated with cy3-conjugated goat anti - rabbit igg ( 1:2000 ; abcam , cambridge , ma , usa ) in tbs at room temperature for 1 h. then the sections were further incubated with 4,6-diamidino-2-phenylindole for 5 min . after a 30 min wash , the sections were mounted on poly - l - lysine - coated glass slides and coverslipped with prolong gold antifade reagent ( invitrogen , china ) .
these slides were examined under a confocal microscope ( zeiss , 780 , germany ) to detect a in consistent parameter settings including objectives , laser intensities , pmt voltages , offsets , and pinhole sizes .
before the imaging procedure , all the 5xfad and age - matched wt mice were weighed , and then restrained in a tailveiner restrainer and injected via the tail vein with ( f)-fdg ( beijing pet , co , ltd , china ) at a dose of 500600 ci in 100120 l saline according to their weights .
after 40 min for the uptake of ( f)-fdg , mice were anesthetized with a mixture of 96% o2 and 4% isoflurane , and placed into the animal dedicated pet - ct camera ( inveon micropet , germany ) for ct attenuation correction scan and high density pet image reconstruction .
the spatial restructuring was facilitated with ordered subset expectation maximization with three - dimensional resolution recovery ( osem3d , implementation of c michel ) .
the ct image of the skull of each animal was co - registered with the mouse brain resonance image template , in which the volumes of interest ( vois ) were already drawn .
after co - registration , the spatial mathematic data were transformed into the animals fused pet images to produce a correct match between pet image and the brain template . for the purpose of quantification ,
the fdg uptake of the vois was obtained and shown in standard uptake value ( suv ) , and regional suv was quantified with pmod version 3.2 ( pmod technologies , zurich , switzerland ) . during data
acquisition , anesthesia was maintained with 1.5% isoflurane vaporized in o2 through an orofacial mask .
statistical analysis was performed with spss version 11.0 ( spss inc . , chicago , il , usa ) software package and diagrams were plotted with prism graphpad 5.0 software ( graphpad , san diego , usa ) .
indices in acquisition trails such as escape latency , total times crossing the platform in morris water maze test , latency time in olfaction behavioral test , and suv in pet - ct were analyzed with one - way analysis of variance ( anova ) of repeated measures .
all statistical analysis of 5xfad group in comparison with wt group was rechecked with student 's t - test . in all cases ,
at 3 months , no significant difference was found between wt and 5xfad mice in the escape latency , total times crossing the platform [ figure 2 ] . at 6 months ,
the latency of the 6-month - old 5xfad mice was significantly longer than that of 3-month - old 5xfad mice ( p = 0.0034 ) and of 6-month - old wt mice ( p = 0.0262 ) ; no significant difference was reported between the two groups of wt mice .
in addition , the total times crossing the platform of the 6-month - old 5xfad mice were significantly reduced when compared with those of 3-month - old 5xfad mice ( p = 0.0223 ) and of 6-month - old wt mice ( p = 0.0157 ) ; no significant difference was found between the two groups of wt mice . impaired memory function in 5xfad mice shown by water maze testing ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) .
( a ) : at 3 months , the latency was not significantly different between wild - type and 5xfad mice . at 6 months , the latency of 6-month - old 5xfad mice was significantly longer than that of 3-month - old 5xfad mice ( p = 0.0034 ) and of 6-month - old wild - type mice ( * p = 0.0262 ) ; ( b ) : at 6 months , the total times crossing the platform for 6-month - old 5xfad mice was significantly reduced as compared with that for 6-month - old wild - type mice ( * p = 0.0157 ) and 3-month - old 5xfad mice ( p = 0.0223 ) , and no significant difference between two wild - type groups .
the latency was recorded from the time when the mice were placed in the cage to the moment they grasped the food pellet . at 3 months ,
no significant difference in the latency was found between the groups in each daily trial [ figure 1 ] .
in contrast , at 6 months , a significantly greater latency time was found in 5xfad mice in each daily trial when compared with that of age - matched wt mice ( day 1 p = 0.0012 , day 2 p = 0.0031 , day 3 p = 0.0160 ) . as control , the latency time for recovery of a visible food pellet did not differ significantly across the groups regardless of age . in the olfactory maze test , the latency was calculated from the moment the animals were placed in the platform to the moment they recovered the hidden food pellet . in each daily trial , no significant difference in the latency was reported between wt and 5xfad mice at 3 months of age . however , at 6 months , a significantly greater latency time in 5xfad mice was found when compared with that of age - matched wt mice ( day 1 p = 0.0002 , day 2 p < 0.0001 , day 3 p = 0.0045 ) . in the control test ,
the latency time for recovery of a visible food pellet did not differ significantly across the groups regardless of age . in the present study , a deposit , including soluble a in the cytoplasm of neurons and extracellular amyloid plaques ,
was detected by a immunolabeling with a antibody 6e-10 . in wt mice at either age , no amyloid deposits were present [ figure 3 ] . in 3 months old 5xfad mice , intracellular a deposits were found , but not in extracellular regions of the neocortex , hippocampus , and olfactory bulb . in 6 months old 5xfad mice , intracellular a was detected throughout the brain , and olfactory bulb and extracellular a plagues were also observed in the neocortex , hippocampus , and olfactory bulb .
histological images of amyloid stained with 6e-10 ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) . at 3 months , intracellular amyloid deposit in the neocortex , hippocampus , and olfactory bulb was present and no plague was observed in the immunofluorescence ( original magnification 400 ) . at 6 months , amyloid deposits were found throughout the cerebral cortex and hippocampus , and olfactory bulb and amyloid plaques were distributed throughout the brain in the immunofluorescence ( original magnification 400 ) .
as shown , at the age of 6 months , ( f)-fdg uptake in the whole brain of 5xfad mice was obviously lower than that in age - matched wt mice . because of different injected doses and animal weights , and small size of the olfactory bulb , the differences of ( f)-fdg uptake in the olfactory bulb between 5xfad and wt mice could not be visually detected [ figure 4 ] .
however , image quantification of suv showed more details : at 3 months , no significant difference in suv between 5xfad , and wt mice was found in the hippocampus and cerebral cortex ; at 6 months , the uptake of ( f)-fdg in the hippocampus and cerebral cortex of 5xfad mice was remarkably reduced when compared with that of wt mice ( p = 0.0121 and p < 0.0001 , respectively ) .
representative images of the 2-deoxy-2-(f ) fluoro - d - glucose brain uptake of a wild - type and 5xfad transgenic mouse at 3 and 6 months ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) . at 3 months
, no significant difference was found between 5xfad and wild - type mice in the hippocampus and cerebral cortex ; at 6 months , the uptake was remarkably reduced in both hippocampus ( * p = 0.0121 ) and cerebral cortex ( * p < 0.0001 ) . in the olfactory bulb ,
the uptake of the 3-month - old 5xfad mice was significantly lower than that of age - matched wild - type mice ( * p = 0.023 ) ; and at 6 months , the uptake of the 5xfad mice was much lower than that of age - matched wild - type mice ( * p < 0.0001 ) .
however , different from that in the hippocampus and cerebral cortex , the uptake of ( f)-fdg in the olfactory bulb of the 3 months old 5xfad mice was significantly lower than that of wt mice of the same age ( p = 0.023 ) , which was exacerbated in the 6 months old 5xfad mice when they were compared with the age - matched wt mice ( p < 0.0001 ) .
this finding suggests that the decrease of ( f)-fdg uptake in the olfactory bulb occurs earlier than that in other cerebral regions .
at 3 months , no significant difference was found between wt and 5xfad mice in the escape latency , total times crossing the platform [ figure 2 ] . at 6 months ,
the latency of the 6-month - old 5xfad mice was significantly longer than that of 3-month - old 5xfad mice ( p = 0.0034 ) and of 6-month - old wt mice ( p = 0.0262 ) ; no significant difference was reported between the two groups of wt mice .
in addition , the total times crossing the platform of the 6-month - old 5xfad mice were significantly reduced when compared with those of 3-month - old 5xfad mice ( p = 0.0223 ) and of 6-month - old wt mice ( p = 0.0157 ) ; no significant difference was found between the two groups of wt mice . impaired memory function in 5xfad mice shown by water maze testing ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) .
( a ) : at 3 months , the latency was not significantly different between wild - type and 5xfad mice . at 6 months ,
the latency of 6-month - old 5xfad mice was significantly longer than that of 3-month - old 5xfad mice ( p = 0.0034 ) and of 6-month - old wild - type mice ( * p = 0.0262 ) ; ( b ) : at 6 months , the total times crossing the platform for 6-month - old 5xfad mice was significantly reduced as compared with that for 6-month - old wild - type mice ( * p = 0.0157 ) and 3-month - old 5xfad mice ( p = 0.0223 ) , and no significant difference between two wild - type groups .
the latency was recorded from the time when the mice were placed in the cage to the moment they grasped the food pellet . at 3 months ,
no significant difference in the latency was found between the groups in each daily trial [ figure 1 ] .
in contrast , at 6 months , a significantly greater latency time was found in 5xfad mice in each daily trial when compared with that of age - matched wt mice ( day 1 p = 0.0012 , day 2 p = 0.0031 , day 3 p = 0.0160 ) . as control , the latency time for recovery of a visible food pellet did not differ significantly across the groups regardless of age . in the olfactory maze test , the latency was calculated from the moment the animals were placed in the platform to the moment they recovered the hidden food pellet . in each daily trial , no significant difference in the latency was reported between wt and 5xfad mice at 3 months of age . however , at 6 months , a significantly greater latency time in 5xfad mice was found when compared with that of age - matched wt mice ( day 1 p = 0.0002 , day 2 p < 0.0001 , day 3 p = 0.0045 ) . in the control test , the latency time for recovery of a visible food pellet did not differ significantly across the groups regardless of age .
in the present study , a deposit , including soluble a in the cytoplasm of neurons and extracellular amyloid plaques , was detected by a immunolabeling with a antibody 6e-10 . in wt mice at either age , no amyloid deposits were present [ figure 3 ] . in 3 months old 5xfad mice , intracellular a deposits were found , but not in extracellular regions of the neocortex , hippocampus , and olfactory bulb . in
6 months old 5xfad mice , intracellular a was detected throughout the brain , and olfactory bulb and extracellular a plagues were also observed in the neocortex , hippocampus , and olfactory bulb .
histological images of amyloid stained with 6e-10 ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) . at 3 months , intracellular amyloid deposit in the neocortex , hippocampus , and olfactory bulb was present and no plague was observed in the immunofluorescence ( original magnification 400 ) . at 6 months , amyloid deposits were found throughout the cerebral cortex and hippocampus , and olfactory bulb and amyloid plaques were distributed throughout the brain in the immunofluorescence ( original magnification 400 ) .
the reconstruction of pet - ct images demonstrated remarkable ( f)-fdg uptake . as shown , at the age of 6 months , ( f)-fdg uptake in the whole brain of 5xfad mice was obviously lower than that in age - matched wt mice . because of different injected doses and animal weights , and small size of the olfactory bulb , the differences of ( f)-fdg uptake in the olfactory bulb between 5xfad and wt mice could not be visually detected [ figure 4 ] .
however , image quantification of suv showed more details : at 3 months , no significant difference in suv between 5xfad , and wt mice was found in the hippocampus and cerebral cortex ; at 6 months , the uptake of ( f)-fdg in the hippocampus and cerebral cortex of 5xfad mice was remarkably reduced when compared with that of wt mice ( p = 0.0121 and p < 0.0001 , respectively ) .
representative images of the 2-deoxy-2-(f ) fluoro - d - glucose brain uptake of a wild - type and 5xfad transgenic mouse at 3 and 6 months ( 5xfad mice , 3 months of age , n = 7 , 6 months of age , n = 7 , wild - type counterparts , 3 months of age , n = 7 , 6 months of age , n = 7 ) . at 3 months
, no significant difference was found between 5xfad and wild - type mice in the hippocampus and cerebral cortex ; at 6 months , the uptake was remarkably reduced in both hippocampus ( * p = 0.0121 ) and cerebral cortex ( * p < 0.0001 ) . in the olfactory bulb ,
the uptake of the 3-month - old 5xfad mice was significantly lower than that of age - matched wild - type mice ( * p = 0.023 ) ; and at 6 months , the uptake of the 5xfad mice was much lower than that of age - matched wild - type mice ( * p < 0.0001 ) . however , different from that in the hippocampus and cerebral cortex , the uptake of ( f)-fdg in the olfactory bulb of the 3 months old 5xfad mice was significantly lower than that of wt mice of the same age ( p = 0.023 ) , which was exacerbated in the 6 months old 5xfad mice when they were compared with the age - matched wt mice ( p < 0.0001 ) .
this finding suggests that the decrease of ( f)-fdg uptake in the olfactory bulb occurs earlier than that in other cerebral regions .
in the present study , we investigated a deposit , cognitive impairment , olfactory dysfunction , and the reduction of glucose metabolism in the olfactory bulb , cerebral cortex and hippocampus of 5xfad mice at the ages of 3 and 6 months . of note
, in 3 months old 5xfad mice , we found that the decline of ( f)-fdg uptake in the olfactory bulb occurred earlier than other incidents .
first , the odor input is transmitted to the olfactory bulb by sensory neurons , which is then decoded and recoded in the piriform cortex and finally flocks together in the hippocampus .
although the mechanisms of ad - related olfactory sensory loss have already been explored through the use of transgenic mouse models and studies have documented olfactory deficits in app transgenic mice , the role of a in contributing to ad - related olfactory impairment and the involvement of these cerebral regions remain largely obscure . the current study
employed buried food pellet test to investigate the olfactory behaviors of the animals and olfactory maze test to justify the findings .
other olfactory tests such as odor discrimination test and a 2-nozzle drink test have also been used in mice , but these tests involve training , and thus other sensory and higher cortical functions are implicated in the results , suggesting the potential presence of other cofounders . in current study , we observed similar cognitive outcomes to those of morris water maze test : the latency of 6 months old 5xfad mice was significantly longer than that of age - matched wt mice and 3 months old 5xfad mice ; no significant difference was found between wt and 5xfad mice at the age of 3 months . in both olfactory function tests , we observed improvement in the performance during the subsequent repeated 3 daily tests , probably due to the learning and conditioning effect . the latency time for recovery of a visible food pellet did not differ significantly in all groups , indicating that the prolonged acquisition for 5xfad mice is not due to visual acuity disorder .
these findings suggest that the olfactory dysfunction of 5xfad mice occurs between 3 and 6 months .
functional neuroimaging techniques have been widely used to assess nerve dysfunction . in particular , among all the functional brain imaging techniques , fdg - pet has the unique ability to assess the local cerebral glucose metabolic rate and can provide information on the distribution of neuronal death and synaptic dysfunction in ad in vivo . in our study ,
consistent with the result of a accumulation and memory impairment , ( f)-fdg uptake of the 3 months old 5xfad mice was not significantly different from that of age - matched wt mice but significantly lower than that of the 6 months old 5xfad mice in the region of hippocampus and cerebral cortex , implying that glucose uptake dysfunction or impaired mitochondrial energy metabolism and synaptic damage may play a significant role in the progression of ad . at the beginning of the ad ,
intracellular a starts to accumulate and damage the mitochondrial energy metabolism , but because of the possible compensation of other healthy cells , the phenomenon can not be detected by pet - ct until enough a deposits result in the observed reduction in ( f)-fdg uptake . on the contrary ,
the suv of the olfactory bulb in the 3 months old 5xfad mice was significantly lower than that in wt mice , which increased in severity with age .
this discrepancy between the olfactory bulb and other cerebral regions makes the reduction of ( f)-fdg uptake in the olfactory bulb an earlier in vivo ad - related biomarker in 5xfad mice .
olfactory dysfunction is a common and early symptom of many neurodegenerative diseases , particularly of ad , parkinson 's disease , and mild cognitive impairment heralding its progression to dementia .
a deposits are detected in the olfactory bulb in ad postmortem brains and are severer than in other cerebral regions , indicating that a deposition in the olfactory bulb may occur faster than other regions , resulting in an earlier reduction of ( f)-fdg uptake .
another reason is nonfibrillar a deposition within the olfactory bulb , which is another ad - related issue , also occurs earlier than that in other brain regions .
in addition , postnatal interneurons arise from the subventricular zone of the lateral ventricle and constantly mature into the neurons in the olfactory bulb while the differentiation and proliferation of those in other regions stop postnatally , which means that the olfactory bulb is more metabolically active and more easily damaged and needs more glucose than other regions of the brain .
taken together , our data indicate that the reduction of glucose metabolism in the olfactory bulb is an earlier ad - related biological marker in 5xfad mice , which provides a potential new therapeutic approach to early diagnose ad patients and to make an optimal prognosis possible . because of the distinct anatomic structure of human body , different from that of mice , the evaluation of suv in human olfactory bulb may pose some difficulties .
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background : early diagnosis assumes a vital role in an effective treatment of alzheimer 's disease ( ad ) .
most of the current studies can only make an ad diagnosis after the manifestation of typical clinical symptoms .
the present study aimed to investigate typical and other biomarkers of ad to find a possible early biomarker.methods:a total of 14 5xfad mice ( at 3 and 6 months old ) , with 14 age - matched wild - type ( wt ) mice as control , were enrolled in this case - control study .
morris water maze test was performed to evaluate the cognitive function ; buried food pellet test and olfactory maze test were employed to investigate the olfactory function ; immunofluorescence to detect amyloid deposition and positron emission tomography to examine 2-deoxy-2-(18 f ) fluoro - d - glucose ( [ 18 f]-fdg ) uptake in the hippocampus and cerebral cortex.results:with the increasing age , cognitive performance ( p = 0.0262 ) and olfactory function were significantly deteriorated ( day 1 p = 0.0012 , day 2 p = 0.0031 , day 3 p = 0.0160 , respectively ) and the ( 18 f)-fdg uptake was markedly decreased in multi - cerebral regions including the olfactory bulb ( p < 0.0001 ) , hippocampus ( p = 0.0121 ) , and cerebral cortex ( p < 0.0001 ) . of note , in 3-month - old 5xfad mice , a significant decline of ( 18 f)-fdg uptake in the olfactory bulb was found when compared with that of age - matched wt mice ( p = 0.023 ) while no significant difference was present when the uptakes in other cerebral regions were compared.conclusions:the decline of ( 18 f)-fdg uptake in the olfactory bulb occurs earlier than other incidents , serving as an earlier in vivo biological marker of ad in 5xfad mice and making early diagnosis of ad possibly .
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the rv144 immune correlates analysis was an important achievement , establishing the framework on which future prophylactic hiv-1 vaccines are being developed .
the analysis showed that a humoral immune response of the right immunoglobulin class to the v1v2 region of gp120 was associated with vaccine efficacy.1 the vaccine regimen induced t - cell responses , in particular cd4 polyfunctional t cells,2 but this was not associated with infection outcomes in the analysis of primary variables despite suggestions of an association among secondary variables.1 the association of t - cell responses and reduced infection risk is controversial . inducing t cells using an adenovirus - vectored gag / pol / nef vaccine failed to protect human vaccines from hiv-1 infection and
may have increased infection risk.3,4 in contrast , results from cytomegalovirus - vectored simian immunodeficiency virus vaccination of nonhuman primates showed vaccine - elicited cd8 t cells , which associated with viral suppression.5 we have also recently shown a reduced infection risk associated with naturally acquired t - cell responses in the iprex trial.6 more recently , a reanalysis of the rv144 t - cell response data suggested that hiv-1 gp120 envelope - specific t - cell responses are part of the protective immune response in vaccines .
this reanalysis used the novel , open source analytical tool compass , enabling a more thorough dissection of the complexities of t - cell polyfunctionality and overall response in rv144 .
the results indicated that the cd4 t - cell responses had protective associations that were comparable in magnitude with the previously reported humoral responses.7 the totality of the rv144 data indicated that vaccine - induced hiv-1 prophylactic efficacy is linked to both humoral and cellular immunity and that the qualitative characteristics are critical in determining outcomes .
the results also suggested that a response against a single protein antigen , with the optimal qualitative characteristics , may be sufficient for protection .
the rv144 analyses were constrained to immune responses mechanistically related to the vaccination and study - related variables and thus not designed to address other mechanisms of reduced hiv-1 infection risk .
one mechanism of potential - reduced infection risk , vaccine - induced hiv-1specific cd8 t - cell responses , could not be rigorously tested in the correlates analysis because of the low frequency , consistent with the vaccine 's design .
we have shown that hiv-1specific t - cell responses are present in some hiv-1exposed seronegative ( hesn ) subjects and that certain responses were associated with reduced hiv-1 infection risk.6 in that initial study , responses to the gp120 protein antigen were not assessed . in light of the rv144 results and evidence of protective naturally acquired t - cell immunity , it may also be possible that cd8 gp120-specific t - cell responses could contribute to protection . in support of this
are reports of naturally induced t - cell responses specific for gp120 described in cohorts of hiv-1 exposed but persistently seronegative individuals , both from sexual and occupational hiv-1 exposure.811 we hypothesized that gp120-specific cd8 t - cell responses , quantitatively or qualitatively , would be associated with infection risk among comparably exposed placebo - treated individuals in the global pre - exposure prophylaxis initiative ( iprex ) chemoprophylaxis trial.12
our study was designed as a case control cross - sectional analysis of gp120-specific ifn cellular immune responses with phenotypic characterization of positive responses .
we examined gp120-specific cellular immune responses from preinfection - cryopreserved peripheral blood mononuclear cells ( pbmcs ) from 25 subjects who seroconverted during the follow - up period of the iprex trial ( median time - on - study at the time of documented infection was 561 days for this group ) .
the most proximal hiv-1negative time point relative to diagnosis of hiv-1 infection was used for the analyses with hiv-1negative status at this time point determined by a combination of hiv-1 antibody and hiv-1 rna testing .
each sc - bi was matched to 3 persistently hiv-1negative trial participants ( hesn ; n = 75 ) from the same enrollment site with comparable time - on - study as the sc - bi participant ( n = 75 ) .
all pbmc specimens used in the study were obtained from consented participants of the iprex trial completed in 2010 ( registered with clinicaltrials.gov , nct00458393 ) .
we used a prespecified 2-step process first screening with an ifn- elispot assay followed by phenotypic characterization and response confirmation with multiparametric flow cytometry ( mfc ) where pbmcs were available from the same aliquot .
ifn- elispot assays were conducted with peptide pools consisting of 15-mer peptides overlapping by 11 amino acids ( aa ) from nih aids research and reagent program ( rockville , md ) that spanned gp120 protein antigen with sequences corresponding to hiv-1 consensus b sequence .
a gp120 pool and series of matrix peptide pools along with individual peptide preparations were used in the experiment .
all peptides were reconstituted , pooled , and used at a final concentration of 5 g / ml .
pbmcs were plated at a concentration of 1 10 cells / well and incubated overnight ( 1618 hours ) . two positive controls ( phytohaemagglutinin , and a peptide pool consisting of cytomegalovirus , epstein barr virus , and influenza virus peptides )
no reassays were permitted and all data included in the analyses to ensure objectivity . a positive gp120 response in elispot
was defined as 55 spots per million pbmcs after subtracting twice the spot count in the negative control well .
mfc was performed on the same pbmc aliquot from which elispot responses were detected and within 1 day of thawing .
costimulatory anti - cd28 and cd49d mabs were added for every 1 10 cells in all conditions .
peptides were added at 5 g / ml incubated for 1 hour at 37c after which 50 l of brefeldin a was added then incubated over night .
cells were stained in the presence of human igg ( sigma ) with pacific blue anti - cd3 ( biolegend ) , brilliant violet 605 anti - cd8 ( biolegend ) , brilliant violet 650 anti - cd4 ( biolegend ) , and allophycocyanin / cyanine 7 anti - cd14 and anti - cd19 ( biolegend ) .
amine aqua ( life technologies ) was included in each stain to exclude dead cells .
cells were then stained intracytoplasmically with allophycocyanin anti - ifn ( bd pharmingen ) , tnf- with alexa fluor ( bd pharmingen ) , and mip-1 with pe - a ( bd pharmingen ) .
samples were analyzed on a 4-laser lsr ii flow cytometer ( bd biosciences , san jose ) .
data analysis was performed using flowjo version 9.6.1 software ( treestar , ashland , or ) .
all subjects mounted ifn- responses to either the positive peptide pool or phytohaemagglutinin control ; no statistically significant differences were noted between sc - bi and hesn .
median ( interquartile range ) spots per million counts for sc - bi and hesn were 775 ( 188995 ) and 160 ( 106569 ) , respectively ( fig .
the difference in magnitude of response for sc - bi and hesn did not meet statistical significance .
elispot and polyfunctionality . a , elispot results . of the 25 sc - bi tested , 6 ( 24% ) had counts above the 55 spot per million threshold ( indicated by the dotted line ) with a median [ interquartile range ( iqr ) ] of 775 ( 188995 ) .
an equal frequency ( 24% ) of hesn , or 18/74 , had counts above the threshold with a median ( iqr ) of 160 ( 106569 ) .
median ( iqr ) percent of ifn+tnf+mip-1+ t cells of 76.5 ( 69.884.4 ) in 9 hesn and 66.5 ( 51.589.8 ) in 5 sc - bi .
ifn- responses to the gp120 peptide pool were confirmed by mfc in each of the 5 sc - bis and 9 hesns who had a positive t - cell response in elispot and sufficient pbmc to conduct the testing ( fig .
nonspecific ifn- responses were low after incubation with cell culture medium alone in the presence of costimulation .
responses were cd8 t cell polarized in all cases tested with a trend of stronger responses in hesn when tested in mfc .
responses were detected on subsequent visits indicating persistence of responses in those tested ( fig .
t - cell responses were more uniformly polyfunctional as measured by tnf- and mip-1- coproduction in hesn in comparison with sc - bi with a median ( interquartile range ) frequency of 76.5 ( 69.884.4 ) and 66.5 ( 51.589.8 ) , respectively ( fig .
confirmation of elispot responses 55 spots per million ( spm ) with multiparametric flow cytometry .
cryopreserved pbmcs were tested first in and ifn- elispot assay with gp120 responses confirmed in mfc .
hesns or sc - bis shown are individual subjects ; flow data in this figure for each hesn or sc - bi were used once . a , example of sc - bi and hesn responses in the absence of stimulating peptide pools and in the presence of gp120 peptide pool at 5 g / ml , proportion of cd4 and cd8 ifn-positive cells , and proportion of cd8 ifn-positive cells also positive for tnf- and mip-1 ( cd4 ifn-positive t - cell frequency was too low to examine for polyfunctionality ) .
b , time dependency of response to gp120 peptide pool in a hesn , tested at 5 g / ml .
the hesn individual demonstrated 2 distinct positive responses , and the sc - bi individual from the same enrollment site at the same protocol - defined visit had 2 positive responses to overlapping 15-mer peptides .
we mapped epitope - specific responses in 1 sc - bi and 1 hesn from the same enrollment site , both at day 144 of follow - up .
one epitope comprised aa102116 ( based on the hxb2 sequence ) lying proximal to the cd4 binding site and v1 loop region ( fig .
the other epitope was located within the v2 loop region comprising hxb2 aa178 - 192 , containing a glycosylation site at aa186 - 188 .
in contrast , the sc - bi subject demonstrated a weak reactivity to 2 overlapping 15-mer peptides on the gp120 pool . both peptides spanned aa213 - 231 , outside the v1v2 loop .
the weaker response was against the peptide with a partial overlapping of an nkt glycosylation site ( fig . 2c ) .
a second hesn from another enrollment site responded to a v2 loop epitope , hxb2 aa171 - 185 ( fig .
the importance of gp120 responses ( both humoral and cellular ) in the protection from a productive systemic infection with hiv-1 has been established . in the rv144 trial ,
t - cell responses in vaccines were cd4 , targeted the v2 loop , and were associated with a predominance of il2 or ifn- production , or a polyfunctional combination of the 2 in vaccinated subjects.2 the compass analysis shed further light on the qualitative characteristics strongly suggesting a cd4 t - cell response to be contributing to reduced infection risk .
a corroborating study in rhesus monkeys also showed the importance of gp120 responses to protection.13 these studies were not designed to assess other potentially protective immune responses . leveraging
the placebo - controlled iprex trial with its 3-year follow - up period together with limiting our analysis to subjects receiving placebo only provided a naturalistic setting in which to investigate risk - associated gp120 t - cell responses .
this unique approach enabled an assessment of hiv-1specific immunity that was not biased by vaccination or pharmacologic interventions . in our study
, we confirmed that hiv-1 gp120-specific ifn- responses were present in both sc - bi and hesn .
the prevalence did not differ between the groups but the medians suggested a 4.8-fold greater response magnitude in sc - bi .
such results corroborate the potential of t - cell responses to increase infection risk.3 however , the qualitative characteristics of the responses differentiated with infection outcomes similar to the compass analysis and affirming the importance of qualitative factors of immune response in the overall impact of an immune response . we observed a more homogenously polyfunctional response in hesn with a greater frequency of mip-1+ cells in hesn .
this observation is analogous to the dominance of cd4 t - cell polyfunctionality observed in rv144 vaccines,2 although the range of detected cytokines differed between the 2 studies .
our result also mirrors analyses conducted in hiv-1 vaccines immunized with a dna prime / rad5 boost that showed that cd8 polyfunctionality , inclusive of mip-1 and cd107a , was correlated to antiviral activity.14 although our subject numbers were limited , which impacted our ability to conduct rigorous statistical testing and may have obscured the contribution of cd4 t - cell responses in the larger population , the overall trend suggests that a more uniformly polyfunctional t - cell response may be associated with the relative resistance to hiv-1 infection .
the most impressive corroborative link between our study and the rv144 analyses is the overlap of targeted epitopes . in rv144
, responses were predominantly directed to the v2 loop region and specifically hxb2 aa172 - 204 containing the 47-binding motif and glycosylation sites at aa186 - 188 and aa197 - 199.2 in the mapped iprex participants , we observed similar regional targeting for 2 hesn but not the sc - bi .
importantly , one of the hesn came from the same enrollment site and the pbmc samples from the same protocol - defined visit as the sc - bi .
however , the overlap with the findings in the rv144 analyses suggests that gp120 v2 loop - specific cd8 specific t - cell responses together with humoral and cd4 t - cell immune responses may be important for protection from hiv-1 infection .
we show that qualitative characteristics of gp120-specific cd8 t - cell responses differentiate with infection outcomes .
the study was principally designed to assess elispot ifn- responses and to phenotypically characterize those that were positive .
thus , the number of studied responses overall and peptide - mapped responses in particular were small , and the range of cytokines was less expansive relative to those of previous studies.1,2,6,7 in addition , 15-mer peptides are used for detection of both class - i and class - ii t - cell responses so the absence of cd4 t - cell responses is likely stochastic .
the study was also not designed to assess protection ; therefore , the gp120-specific responses could be a marker of exposure and not related to mechanisms of resistance .
however , the striking overlap between the observations in this study , particularly in a case control pairing , to that of others linked with vaccine efficacy may suggest that polyfunctional cd4 and cd8 gp120-specifc t - cell responses could enhance the protective effect of a prophylactic hiv-1 vaccine .
therefore , a larger study of naturalistic exposure to hiv-1 with additional polyfunctionality variables is needed to make robust conclusions . in conclusion ,
we observed strong , polyfunctional cd8 t - cell responses specific for the v2 loop of hiv-1 gp120 in hiv-1 exposed but persistently seronegative individuals . in the subjects tested , the qualities and regional epitope specificity differed in persistently negative hesn individuals relative to those individuals who eventually became infected .
the observations made in this study corroborate the ground - breaking analyses and observations of the envelope - based rv144 vaccine study regarding cd4 t - cell polyfunctionality .
the concordance of the findings suggests that cd8 t - cell responses may also contribute to protection from infection and should be further studied in sufficiently powered studies , inclusive of prospectively designed retrospective studies of vaccine , and pre - exposure prophylaxis trials .
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abstract : association of hiv-1specific t - cell responses to infection risk in seronegative individuals is controversial . we quantified and phenotypically characterized gp120-specific t - cell responses in hiv-1 exposed , but uninfected subjects enrolled in the global pre - exposure prophylaxis initiative ( iprex ) chemoprophylaxis trial .
ifn elispot responses were detected in 24% of subjects irrespective of infection outcome .
hiv-1
gp120 envelope - specific t - cell responses were more uniformly ifn-+tnf-+mip-1+ in persistently seronegative subjects relative to subjects who later seroconverted ( median frequency of 76.5% and 66.5% , respectively ) .
ifn responses targeted the v2 loop for subjects who remained seronegative .
hiv-1
gp120 envelope v2 loop - specific cd8 + t - cell responses may help to protect against hiv-1 acquisition .
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mixed reality technologies , including augmented reality ( ar ) and virtual reality ( vr ) , either supplement or completely replace the real world with computer - generated data to facilitate user interaction via one 's natural senses . both vr and ar technologies have been shown to be beneficial in providing contextual learning experiences , enabling the exploration of complex networks of information , and constructing new models of understanding .
vr technology , which was all the rage among technophiles in the early 1980s and 1990s , has been utilized in various applications from gaming to engineering . however , early prototypical vr systems came with a hefty price tag .
additionally , early vr systems often featured large , cumbersome head - mounted displays ( hmd ) , which were hampered by narrow fields of view ( fov ) , low frame rates , and suboptimal latencies .
as a result , the vr experience offered by such systems were not very immersive and rather uncomfortable to wear for extended periods of time . thus , work with vr technology was essentially limited to researchers and developers with access to these proprietary tools .
more recently , oculus vr , a company acquired by facebook following a successful kickstarter campaign , is on the verge of releasing the first consumer version of it 's low - cost , lightweight vr platform , the rift .
the oculus rift is a vr hmd with a wide fov ( 110 ) , high - definition quality video resolution ( 2160x1200 ) , and a high frame rate ( 90hz ) , ensuring little to no lag with head movement tracking .
in addition , the oculus rift relies on an embedded infrared tracking system that enables positional and rotational tracking of the hmd , which has implications for clinical research .
putting on the headset replaces an individual 's field of vision with a digital image , where each eye looks through a different lens to create a stereoscopic three - dimensional ( 3d ) effect .
a few studies have investigated the role of vr and ar technologies as tools for medical education and training , where rich contextual learning environments are required .
much of this work stems back to the early 2000s , when these technologies were still in their infancy . a handful of studies have purported investigating the role of vr technology in pathology ; however , there are practically no investigations that leverage the current iterations of vr or ar technology .
several pathology laboratories are currently in the process of transitioning to using digital imaging systems , such as whole slide imaging ( wsi ) .
wsi platforms emulate traditional light microscopy by digitizing glass slides using specialized hardware ( e.g. , whole slide scanners ) and software ( e.g. , vendor - specific or vendor - agnostic digital slide viewers ) .
digital slides obtained from wsi platforms are typically viewed in two dimensions either using a desktop personal computer monitor / display or mobile device ( e.g. , tablet ) .
current complaints from users of wsi are that navigation of digital slides is slow and tedious , especially with input devices such as a computer mouse . indeed , employing gaming controllers such as those used by sony playstation to create an ultra - high - speed wsi viewing system appear to improve user viewing satisfaction .
pathologists also complain that when viewing a digital slide on a computer monitor , they lose the benefit of peripheral vision they are used to when looking at a glass slide with a conventional light microscope .
this may partly explain why pathologists like using very large displays made up of many monitors ( e.g. , power wall ) to interact with virtual slides .
to the best of our knowledge , we are not aware of any studies regarding regarding digital pathology slides being viewed in a vr environment .
therefore , we sought to explore the use of the oculus rift for examining digital pathology slides .
an oculus rift development kit 2 ( dk2 ) was connected through usb cables to a 64-bit computer ( hp z440 workstation , intel xeon cpu e5 - 1650 v3 , 32 gb ddr4 - 2133 , nvidia geforce gtx titan x gpu , 512 gb pcie ssd , 1 tb 7200rpm hdd ) running microsoft windows 10 ( 64-bit ) .
whole slide images acquired for this study were stored on a remote server and viewed using imagescope ( leica biosystems , wetzlar , germany ) software .
additional software was necessary to display two - dimensional ( 2d ) whole slide images in a stereoscopic 3d virtual environment .
initially , we investigated the possibility of creating our own vr software using the development kit provided by oculus ( sdk 0.8.0.0 ) and a unity 3d plugin ( http://www.unity3d.com/unity ) , a popular cross - platform game engine . a base example was built in the form of an empty , cubic room with static images used to wallpaper the walls [ figure 1 ] . for ease of use , we opted to instead use commercially available virtual desktop software ( v0.9.34 ) ( http://www.vrdesktop.net/ ) for navigation of digital slides .
navigation of digital whole slides with the oculus rift was tested using keyboard controls , an optical mouse , and a leap motion controller ( https://www.leapmotion.com/product/vr ) .
the leap motion controller relied on two monochromatic infrared cameras and three infrared light - emitting diodes to generate 3d positional data .
leap motion enabled users to perform hand gesture - controlled navigation of digital slides in a vr environment .
example of a stereo rendering constructed using the oculus sdk and unity three - dimensional plugin de - identified glass slides from twenty cases ( n = 20 ) were obtained from a lymph node teaching set .
cases were randomly selected by two pathologists who were nonparticipants in the viewing phase of the study .
ten of the twenty cases harbored malignant diagnoses including metastatic ductal carcinoma , lobular carcinoma , signet ring cell carcinoma , leiomyosarcoma , papillary thyroid carcinoma , malignant melanoma , squamous cell carcinoma , prostatic adenocarcinoma , endometrioid endometrial adenocarcinoma , and hodgkin 's lymphoma .
the remaining ten cases harbored benign diagnoses including reactive paracortical lymphoid expansion , normal lymph node , fatty infiltration of an inguinal and pelvic lymph node , tuberculous lymphadenitis , epstein
barr virus - associated lymphadenitis , cat scratch disease , hiv - associated lymphadenitis , sarcoidosis , and sinus histiocytosis .
a single glass slide from each of the twenty cases was digitized using an aperio scanscope xt wsi scanner ( leica biosystems , wetzlar , germany ) with an olympus uplansapo 20x/0.75 objective .
the resultant whole slide images were acquired as jpeg2000 encoded svs files and were not further compressed .
digital slides were first viewed using the oculus rift dk2 and virtual desktop application . after a washout period of 2 weeks
, the participants again evaluated the same digital slides on a 27-inch in - plane switching ( ips ) 5k display ( hp zr27q ) with a resolution of 4096 2160 pixels , or a 24-inch ips display ( hp zr2440w ) with a resolution of 1920 200 , both with a landscape orientation .
three , board - certified , attending pathologists with subspecialty expertise in cytopathology , breast / gynecologic , and gastrointestinal pathology were recruited for review of the aforementioned digital slides . each of the three participants had prior experience in viewing digital pathology slides using traditional wsi modalities .
the pathologists were first trained to navigate whole slide images in our vr environment [ figure 2 ] .
the primary endpoints assessed included diagnostic concordance and time to read ( interpret ) digital slides using both modalities .
diagnostic concordance was defined as ( 1 ) accurate classification of cases as either benign or malignant and ( 2 ) rendering a specific pathologic diagnosis ( i.e. , metastatic carcinoma ) .
the pathologists were also asked to rate image quality ( on a scale of 110 ) , ease of navigation ( on a scale of 110 ) , and their diagnostic confidence ( on a scale of 110 ) for both modalities .
all primary and secondary endpoints were collected on a per slide / case basis , and the average values for each pathologist were tabulated as overall " values . virtual reality - based digital slide navigation using oculus rift development kit 2 , virtual desktop , and a web - based whole slide imaging browser
an oculus rift development kit 2 ( dk2 ) was connected through usb cables to a 64-bit computer ( hp z440 workstation , intel xeon cpu e5 - 1650 v3 , 32 gb ddr4 - 2133 , nvidia geforce gtx titan x gpu , 512 gb pcie ssd , 1 tb 7200rpm hdd ) running microsoft windows 10 ( 64-bit ) .
whole slide images acquired for this study were stored on a remote server and viewed using imagescope ( leica biosystems , wetzlar , germany ) software .
additional software was necessary to display two - dimensional ( 2d ) whole slide images in a stereoscopic 3d virtual environment .
initially , we investigated the possibility of creating our own vr software using the development kit provided by oculus ( sdk 0.8.0.0 ) and a unity 3d plugin ( http://www.unity3d.com/unity ) , a popular cross - platform game engine . a base example was built in the form of an empty , cubic room with static images used to wallpaper the walls [ figure 1 ] . for ease of use , we opted to instead use commercially available virtual desktop software ( v0.9.34 ) ( http://www.vrdesktop.net/ ) for navigation of digital slides .
navigation of digital whole slides with the oculus rift was tested using keyboard controls , an optical mouse , and a leap motion controller ( https://www.leapmotion.com/product/vr ) .
the leap motion controller relied on two monochromatic infrared cameras and three infrared light - emitting diodes to generate 3d positional data .
leap motion enabled users to perform hand gesture - controlled navigation of digital slides in a vr environment .
example of a stereo rendering constructed using the oculus sdk and unity three - dimensional plugin
de - identified glass slides from twenty cases ( n = 20 ) were obtained from a lymph node teaching set .
cases were randomly selected by two pathologists who were nonparticipants in the viewing phase of the study .
ten of the twenty cases harbored malignant diagnoses including metastatic ductal carcinoma , lobular carcinoma , signet ring cell carcinoma , leiomyosarcoma , papillary thyroid carcinoma , malignant melanoma , squamous cell carcinoma , prostatic adenocarcinoma , endometrioid endometrial adenocarcinoma , and hodgkin 's lymphoma .
the remaining ten cases harbored benign diagnoses including reactive paracortical lymphoid expansion , normal lymph node , fatty infiltration of an inguinal and pelvic lymph node , tuberculous lymphadenitis , epstein
barr virus - associated lymphadenitis , cat scratch disease , hiv - associated lymphadenitis , sarcoidosis , and sinus histiocytosis .
a single glass slide from each of the twenty cases was digitized using an aperio scanscope xt wsi scanner ( leica biosystems , wetzlar , germany ) with an olympus uplansapo 20x/0.75 objective .
the resultant whole slide images were acquired as jpeg2000 encoded svs files and were not further compressed .
after a washout period of 2 weeks , the participants again evaluated the same digital slides on a 27-inch in - plane switching ( ips ) 5k display ( hp zr27q ) with a resolution of 4096 2160 pixels , or a 24-inch ips display ( hp zr2440w ) with a resolution of 1920 200 , both with a landscape orientation .
three , board - certified , attending pathologists with subspecialty expertise in cytopathology , breast / gynecologic , and gastrointestinal pathology were recruited for review of the aforementioned digital slides .
each of the three participants had prior experience in viewing digital pathology slides using traditional wsi modalities .
the pathologists were first trained to navigate whole slide images in our vr environment [ figure 2 ] .
the primary endpoints assessed included diagnostic concordance and time to read ( interpret ) digital slides using both modalities .
diagnostic concordance was defined as ( 1 ) accurate classification of cases as either benign or malignant and ( 2 ) rendering a specific pathologic diagnosis ( i.e. , metastatic carcinoma ) .
the pathologists were also asked to rate image quality ( on a scale of 110 ) , ease of navigation ( on a scale of 110 ) , and their diagnostic confidence ( on a scale of 110 ) for both modalities .
all primary and secondary endpoints were collected on a per slide / case basis , and the average values for each pathologist were tabulated as overall " values . virtual reality - based digital slide navigation using oculus rift development kit 2 , virtual desktop , and a web - based whole slide imaging browser
technically , digital images were able to be viewed in a vr environment wearing the oculus rift dk2 .
all input methods to navigate slides worked satisfactorily . however , the leap motion method resulted in the most fatigue after prolonged hand gestures .
all three reviewers confirmed that digital pathology slides were easily viewable in a vr environment using the oculus rift dk2 .
there was 90% diagnostic concordance between the traditional method of reviewing wsi on a flat computer monitor and the vr method .
the variation in time to interpret digital pathology slides did reach statistical significance when tested using an unpaired t - test ( two - tailed p = 0.0009 ) .
variations in image quality and ease of navigation also reached statistical significance when tested using unpaired t - tests ( two - tailed p = 0.0001 and 0.027 , respectively ) .
the variations in diagnostic confidence , however , did not reach statistical significance ( two - tailed p = 0.15 ) .
there were no significant side effects ( e.g. , motion sickness ) associated with the use of the oculus rift ; however , the participants did note that the vr headset did feel heavy after prolonged usage .
this proof of concept study represents the first attempt to examine pathology whole slide images in a vr environment .
it is important to note , however , that the aim of this initial study was on proof - of - concept and feasibility as opposed to a comprehensive vr validation study . as such ,
the small sample size of only twenty cases likely limited our ability to discern meaningful differences between vr and traditional modalities .
nonetheless , our findings demonstrate that digital pathology slides can be successfully interpreted using vr .
vr technology today has become readily accessible so that it no longer requires the use of very expensive , complicated , or proprietary hardware or software .
therefore , pathology departments currently using wsi can readily leverage vr technologies , such as the oculus rift , without significant investment .
a widely available , commercial version of the oculus rift is expected in 2016 . in order for digital slides to be viewed with the oculus rift dk2 , we had to integrate a wsi viewer into a virtual environment , map the wsi viewer navigation controls into this environment , and utilize the virtual environment to create a novel 3d interaction pattern for navigating digital slides .
navigation was possible using standard input devices ( e.g. , keyboard , mouse ) and natural senses ( e.g. , hand gestures ) .
we utilized the well - known aspects of vr classification scheme to determine which software to employ in this study .
this classification includes six major factors of vr : interaction , immersion , simulation , visualization , real space , and autonomous agents . to create a truly immersive and engaging vr experience
a high frame rate and low hmd latency are perhaps the most essential factors in the creation of a truly immersive vr experience .
the frame rate is the frequency at which a device displays continuous images for human visual systems to perceive . while the majority of current media is run at 3060 frames per second , vr media requires higher frame rates to ensure smooth movements and prevent motion sickness .
the dk2 makes virtual objects feel almost tangible by supposedly maintaining a frame rate of up to 75 frames per second .
unfortunately , the frame rate was varied significantly as users navigated the digital slides , resulting a noticeable judder .
latency , in the context of vr , refers to the total time between movement of the user 's head and the updated image being displayed on the screen .
thus , a lower latency results in a more immersive and comfortable user experience . low latency head tracking is part of what sets the rift apart from other vr technologies .
first , the image quality on the oculus rift dk2 was limited by the resolution of the organic light - emitting diode display panel in the headset , which only has a resolution of 960 1080 per eye .
in addition , there are hardly any medical applications available for use with vr . while the vr environment we developed provides pathologists with a wide fov because the digital images themselves were not acquired in 3d , the vr environment only created a virtual projection of the 2d whole slide image floating in a simulated 360 environment .
this may explain why the oculus rift did not significantly enhance the navigation of digital slides .
moreover , navigation may have been negatively impacted by the input devices ( mouse and keyboard ) and pathologists were confined to use for the comparative study .
oculus is currently developing a pair of lightweight , wireless , handheld motion controllers that may perform better than traditional input devices .
in addition , our participants did not experience any serious negative side effects largely because they did not spend much time immersed in the vr environment .
given the well - documented side effects associated with vr immersion ( e.g. , nausea , eye - strain , and seizures ) , the long - term usage of vr in clinical practice is an area that deserves further investigation .
unfortunately , many pathologists have encountered frustration when attempting to review digital pathology slides because of navigation difficulties encountered with certain input devices or viewing limitations with mobile computing devices such as laptops .
hence , the availability of an immersive , private , and more natural means of digital slide review would be very helpful .
a vr headset such as oculus rift could allow pathologists to more easily perform telepathology consultations in a secure environment .
vr also has a great potential use in pathology teaching , especially if combined with 3d images ( e.g. , 3d models of gross pathology specimens ) .
our group is investigating the methods for generating 3d digital images of tissue blocks that may be benefited from stereoscopic 3d visualization in a vr environment .
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background : digital slides obtained from whole slide imaging ( wsi ) platforms are typically viewed in two dimensions using desktop personal computer monitors or more recently on mobile devices . to the best of our knowledge , we are not aware of any studies viewing digital pathology slides in a virtual reality ( vr ) environment .
vr technology enables users to be artificially immersed in and interact with a computer - simulated world .
oculus rift is among the world 's first consumer - targeted vr headsets , intended primarily for enhanced gaming . our aim was to explore
the use of the oculus rift for examining digital pathology slides in a vr environment.methods:an oculus rift development kit 2 ( dk2 ) was connected to a 64-bit computer running virtual desktop software .
glass slides from twenty randomly selected lymph node cases ( ten with benign and ten malignant diagnoses ) were digitized using a wsi scanner .
three pathologists reviewed these digital slides on a 27-inch 5k display and with the oculus rift after a 2-week washout period .
recorded endpoints included concordance of final diagnoses and time required to examine slides .
the pathologists also rated their ease of navigation , image quality , and diagnostic confidence for both modalities.results:there was 90% diagnostic concordance when reviewing wsi using a 5k display and oculus rift .
the time required to examine digital pathology slides on the 5k display averaged 39 s ( range 10120 s ) , compared to 62 s with the oculus rift ( range 15270 s ) .
all pathologists confirmed that digital pathology slides were easily viewable in a vr environment .
the ratings for image quality and diagnostic confidence were higher when using the 5k display.conclusion:using the oculus rift dk2 to view and navigate pathology whole slide images in a virtual environment is feasible for diagnostic purposes .
however , image resolution using the oculus rift device was limited .
interactive vr technologies such as the oculus rift are novel tools that may be of use in digital pathology .
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it is usually due to thrombosis of the main adrenal vein or secondary to microvascular thrombosis in the adrenal parenchyma .
recently it has also been described in conditions like heparin - induced thrombocytopenia and myelodysplastic syndrome .
we describe a case of a 25-year - old female patient with history of crohn 's disease who presented with bilateral adrenal infarcts without any computerized tomography ( ct ) or magnetic resonance imaging ( mri ) evidence of hemorrhage . to our knowledge , this is the first case with association between crohn 's disease and bilateral adrenal infarction .
a 25-year - old female patient presented with gradual onset right shoulder pain for 1 week , which was constant and achy in nature and radiated to the left shoulder , down to her back and bilateral flanks .
she also complained of weakness , decreased oral intake and nausea but did not have any fever , vomiting or diarrhea .
she was diagnosed with crohn 's disease since the year 1995 with extensive perianal disease complicated by abscess formation and rectovaginal fistula .
she had also been put on filgrastim , a granulocyte colony - stimulating factor ( g - csf ) analog for chronic leucopenia secondary to azathioprine treatment for crohn 's disease .
physical examination at the time of current admission revealed pallor , mild tachycardia with a heart rate of 115/minute and blood pressure of 96/56 mm hg .
although the patient did not have significant electrolyte abnormalities ( sodium : 137 meq / l , potassium : 4.6 meq / l ) , there was postural tachycardia and hypotension .
mri of the spine and sacroiliac joints did not reveal any evidence of infection / inflammation / disc disease that could be attributed as a cause of the pain .
ct scan of abdomen and pelvis with contrast showed some edema in the rectum in keeping with her crohn 's disease as well as some mild terminal ileitis .
there was also increased thickening and hypodensities in both the adrenals [ figure 1a ] . further evaluation with mri [ figure 1b - d ] revealed that the adrenals were bulky bilaterally .
they showed hypointense signal on t1wi and hyperintense signal intensity on t2wi without any demonstrable hemorrhage .
following administration of gadolinium - based contrast , there was no enhancement of the parenchyma signifying a lack of viable parenchyma , based on which a diagnosis of adrenal infarction without hemorrhage was made .
( a ) axial contrast - enhanced ct image at the level of adrenal glands reveals bilateral thickened , hypodense poorly enhancing infarcted adrenal glands .
( b ) axial t1w , ( c ) axial t2w mr images show t1 hypointense , t2 hyperintense adrenal gland secondary to edema , ( d ) postcontrast image shows poor parenchymal enhancement with minimal peripheral capsular enhancement in keeping with adrenal infarction biochemical confirmation of adrenal insufficiency was made by low plasma cortisol ( 21 nmol / l ) and acth stimulation test that failed to raise the plasma cortisol levels to more than 25 nmol / l .
she did not have any altered skin pigmentation or any history of hypotensive episodes in the past making the possibility of a long standing primary adrenal insufficiency unlikely .
bilateral adrenal infarction is a rare , but a well - recognized cause of adrenal insufficiency .
adrenals are highly vascular with arterial supplies from the inferior phrenic artery , the aorta , and the renal artery .
these alterations in flow dynamics in the medullary sinusoids may predispose the gland to microvascular thrombosis in procoagulable states and during episodes of hypotension , and may lead to infarction .
usually this may be complicated by hemorrhage due to ischemic necrosis , reperfusion injury , anticoagulant therapy or a combination of these factors . the patient presented with biochemical evidence of adrenal adrenal insuffieciency , pain in back , flanks and right shoulder .
though the flank pain has traditionally been ascribed to hemorrhage in the gland , a recent study suggests that it is more likely due to ischemic necrosis and inflammatory swelling .
t1wi in our patient did not show hyperintense areas to suggest any hemorrhage and t2wi showed no blooming .
t2-weighted images however , showed hyperintense signal which denoted edematous change related to the inflammatory swelling of the adrenal gland .
demonstration of hemorrhage is important as it may alter the treatment , especially the need for anticoagulation .
bilateral adrenal infarctions have previously been described in a setting of antiphospholipid - antibody syndrome .
antiphospholipid antibodies target the late endosomal and lysosomal lysobisphosphatidic acid ( lbpa ) in the cholesterol - rich cells of the adrenal cortex leading to secretion of lysosomal proteinases like cathepsin d which leads to a local procoagulant state .
upregulation of vascular cell adhesion molecule-1 ( vcam-1 ) and e - selectin by antiphospholipid antibodies leading to endothelial damage as well as displacement of annexin v , an anticoagulant protein by antiphospholipid antibodies can further lead to microthrombosis .
crohn 's disease is recognized to be a hypercoagulable state which can be attributed to a variety of factors including thrombocytosis as well as increased levels of clotting factors v and viii , fibrinogen , accelerated thromboplastin generation , acquired antithrombin iii deficiency , and decreased protein c and protein s. however , no single consistent coagulation abnormality has been identified .
a wide range of infectious agents ( mycobacterial , bacterial , fungal and viral agents ) can also lead to adrenal insufficiency .
though our patient had chronic leucopenia and was on filgrastim , the normal wbc count at presentation as well as absence of fever or any other sign of sepsis makes infectious etiology unlikely in our case .
the absence of antiphospholipid antibodies in our patient might suggest that pathways other than those elucidated previously may be involved in local microthrombosis and resulting adrenal infarctions .
thus , we herein report an unusual extraintestinal complication occurring as a bilateral adrenal infarction with resultant adrenal insufficiency in crohn 's disease .
ct and mri serve as useful aids in diagnosis as well as identifying hemorrhage which could alter the management .
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adrenal infarction is an uncommon cause of adrenal insufficiency .
we herein present unique occurrence of bilateral adrenal infarction detected on imaging in a young female with known history of crohn 's disease .
the patient responded well to steroids and is on follow up . to our knowledge , this is the first case reported in english literature of adrenal infarct associated with crohn 's disease as extraintestinal manifestation .
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