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A. A bar graph showing highest incidence and mortality of PCa in Eastern, Middle, Western and Southern regions of Africa as well as the Caribbean regions. B. and C. are maps demonstrating high incidence and mortality of prostate cancer in sub-Saharan Africa respectively. Even with a high incidence in South Africa, there is still a relatively high mortality of prostate cancer in this region in comparison to the western world. (Maps and bar graphs were adapted from the online cancer fact sheets of the WHO/IARC GLOBOCAN database 2012 at http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx).
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other
| 99.9 |
The current conventional regimen for PCa diagnosis, which includes PSA measurement, is unreliable in the diagnostic grey area of the reference ranges (2-10ng/mL). Hence, identifying biomarkers which tally accurately with disease risk and staging, as well as providing for evidence based treatment, is key in the reduction of PCa burden in men of African descent. Although PCa may be more aggressive in men of African descent , lack of access to care and delay in diagnosis has obfuscated evidence of a biologically more aggressive disease in African descent men. Among speculated causes of aggressive PCa disease in African men, only age [7, 8] and genetic factors [9, 10] are incontrovertible. Men over the age of 40 and men of African descent are at greater risk of developing PCa compared to their Caucasian counterpart. Men of Asian origin have the least risk of developing PCa, albeit their risk has been reported to increase when they migrate to North America . Few aetiologic and risk factors have been suggested to be associated with PCa development. Modifiable risk factors for PCa have been difficult to identify. Obesity, smoking, alcohol consumption, androgens, diet, diabetes mellitus, and hereditary factors, inter alia, have not been consistently associated with PCa etiology . PCa susceptibility gene loci on 1q24-25 (HPC1), 8p22 (MSR1), 1q25 (RNASEL), and 17p11 (ELAC2) have been recognized by genome wide association studies (GWAS) [12, 13]. Some biological pathways that have been enriched for genetic variants in PCa GWAS were JAK2, IGF-1, prolactin, and androgen signaling pathways . A better understanding of the role of these putative genetic markers is needed among African men with PCa.
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review
| 99.9 |
Despite the benefit that the discovery of PSA provided in PCa detection, it remains an imperfect biomarker and there is room to add omics-based biomarkers to improve PCa detection. A number of cancer-related biomarkers of PCa have been identified that may play a role in early PCa detection or prognosis, including: PTEN, PI3K, PCA7 gene panel, PSGR, MME, PSCA, PCA3, TMPRSS2-ERG gene fusion, CD98, EPCA, CD276, prostate-specific membrane antigen (PSMA), caveolin-1, EN-1, and annexin A3 .
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review
| 99.8 |
Acid Phosphatase (ACPP) is one of the oldest biomarkers used for PCa diagnosis in serum, however the drawback of this biomarker is that ACPP is expressed by both normal and malignant prostatic tissues, as well as extraprostatic tissues . Prostate-Specific Antigen (PSA), also known as kallekrein 3 (KLK3) is the most widely used serum biomarker of PCa and has tremendously improved the diagnosis of PCa. PSA screening was widely adopted in the USA in early 1990s as gold standard investigation for PCa . However, recent evidence indicates that PSA falls short in its diagnostic ability in the lower reference ranges (2-10ng/mL). This led to its contraindication in the USA for men greater than 75 years in 2008 ; and for all men in 2012 . Even though highly sensitive, it is not so specific and has led to a high false positive rate, false negatives and overtreatment of PCa patients. Despite this down side to PSA, very few biomarkers are currently poised to replace PSA in clinical practice [20, 21]. To improve the diagnostic ability of PSA, several related parameters such as PSA doubling time, PSA velocity, free-to-total PSA ratio and prostate health index (PHI) have been explored .
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review
| 99.9 |
Prostate cancer antigen 3 (PCA3) which is otherwise known as DD3 or differential display clone 3 is a noncoding mRNA which is found to be highly abundant in malignant prostatic tissues in comparison to benign [23, 24]. It is the most widely used non-PSA based biomarker for PCa diagnosis . One limitation of PCA3 test is that it is dependent on the urinary PSA transcript expression level. TMPRSS2-ERG gene fusions (Transmembrane Protease, Serine 2- ETS fusion) are members of the ETS family of genes, and can be highly expressed in malignant prostatic tissue but not expressed in benign tissue. Emerging research evidence suggests TMPRSS2-ERG fusions are unlikely to be good predictors of PCa outcomes or aggressiveness. They are expressed in only about 50% of all PCa patients, and varies substantially by race, with African-Americans having much lower staining rates . It suffers the same drawback as PCA3 in that it depends on urinary PSA transcript levels for meaningful interpretation of result. Alpha methylacyl-CoA racemase (AMACR) is a highly sensitive and specific diagnostic biomarker often used in PCa tissues. Low levels of AMACR in biopsy tissues have been associated with biochemical recurrence and PCa metastasis .
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review
| 99.7 |
Emerging proteomics approaches such as mass spectrometry (MS), protein microarrays, interactomics, proteogenomics as well as posttranslational modification proteomics have also been very useful in the development of biomarkers for personalized/individualized therapy of PCa (Figure 2). It is plausible that interplay of various other omics-based approaches would benefit personalized PCa therapies. For example, a recently described classification of the seven possible subtypes of PCa [28, 29] based on TMPRSS2: ERG translocations may represent a more useful molecular classification of PCa (in terms of therapeutic options) than histologic classification. Even though these emerging biomarkers (PCA3, TMPRSS2-ERG, and AMACR) have been validated in Western populations [30, 31], fewer such studies have primarily focused on African-American or African populations [32, 33]. A few other good reviews of genomic biomarkers of PCa can be found elsewhere [34–36].
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review
| 99.9 |
Various proteomics approaches have improved the individualization of prostate cancer therapy. An integrative approach using these proteomics methodologies would improve the identification of proteomics biomarkers of prostate cancer. As shown here, proteomics approaches such as MS-based proteomics, protein microarrays-based proteomics, interaction network proteomics, proteogenomics and well as posttranslational modification proteomics have all been of great benefit in biomarkers development for personalized/individualized therapy of Prostate cancer.
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review
| 99.9 |
The field of proteomics is a high-throughput approach to large-scale identification of the full complement of proteins in an organism, tissue, cells or body fluid. These methods are also potentially able to investigate the functional states of proteins including, post-translational modifications, protein-protein interactions; and protein interaction with other biomolecules such as carbohydrates, lipids and other metabolites. Proteomics can provide insight into 3-D protein structures, alternative splicing events, as well as aiding genome annotation. There are variations to the proteome of a cell depending on the time point, stage of disease, diet and a host of other factors. Currently, proteomics has been employed to identify cancer-related signatures between disease and healthy cohorts of patient [37–42]. Notably, the most common proteomics methodologies are MS-based proteomics and protein microarray technology based proteomics. Using these methodologies, a gamut of proteomics biomarkers of PCa have already been identified [43–49], and some were demonstrated to potentially predict progression and aggressiveness of PCa [50–52]. However, successful application of omics based approaches is heavily dependent on available bioinformatics and computational biology resources, which remain limited in Africa.
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review
| 99.9 |
The preclinical phase of biomarker developed is the foundation upon which translational and clinical validation can be built. Even though tissue-based proteomics has been widely performed [53–56], body fluid-based proteomics, albeit challenging, offers a non- or minimally-invasive alternative [57, 58] with comparable or even superior diagnostic accuracy to tissue-based proteomics, combined with greater suitability for large-scale screening or early detection methods. Potentially suitable body fluids for PCa biomarker discovery include urine, blood and semen. Liquid biopsy has been used for diagnosis of various cancer types [59–63], including PCa [64, 65]. Additionally, this technique has been found useful in cancer patient stratification, monitoring and screening .
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review
| 99.9 |
As an ultrafiltrate of blood, urine possesses analogous protein profiles as are found in peripheral blood and provides a usable catalog of proteins for interpretation of pathophysiologic events in the human body . Sampling urine as compared to blood or prostatic tissue biopsy is a less invasive approach for PCa diagnosis and treatment monitoring. The service of skilled personnel is not required for urine sample collection. In addition to this, urine is abundantly available and permits repeated sampling . Notably, the urinary proteome is less complex compared with the blood proteome and is comparatively stable, the proteins having undergone any likely proteolysis in blood or during storage in the bladder . Hence, there is no need to use protease inhibitors during urine storage . Despite the fact that urine is a very promising disease biomarker source, a major caveat is that its composition is variable depending on the time of the day, dietary intake and the state of health of the individual. Many urinary biomarkers of various diseases have been previously described , albeit only a few of these biomarkers have entered into clinical use [70–73].
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review
| 99.9 |
Considering its extensive contact with body structures, human blood is an attractive source for biomarker discovery. Increased discohesiveness of cancerous tissues sometimes makes tumour cells more mobile and is transported in the blood stream as tumour markers. Even though limited in volume, it is the most frequently used sample for clinical diagnosis of many disease conditions. Blood samples have been used to identify biomarkers of many human diseases including: Alzheimer's disease , Parkinson's disease , breast cancer , preeclampsia , and PCa . Importantly, it is also known that disease conditions such as cancer are able to stimulate humoral immune response leading to the generation of auto-antibodies ; and this has been demonstrated in many different human cancers, including renal , colorectal , lung , and PCa [82–86]. These autoantibodies generated, have potential theranostic utilities for cancer diagnosis and therapeutic vaccine target development.
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review
| 99.9 |
Both urine and blood are very attractive preclinical biomarker sources for PCa in Africa. PCa proteomics has been carried out in African-American populations [87, 88] but there are a limited number of studies among indigenous men of African descent. One such study identified 82 novel potential urinary biomarkers of PCa in a heterogeneous cohort of 45 South African patients composed of Indigenous, Mixed-Ancestry and Caucasian African PCa patients via label-free MS . Among these 82 identified biomarkers, nine biomarkers were identified that suggested racial differences among the ethnicities in the cohort. Verification and prevalidation of the 82 biomarkers using both experimental ‘parallel reaction monitoring’ and in silico computational methods enabled identification of the top performing 12 potential biomarkers , ready for translation through a large scale, multiplatform and multicenter targeted proteomics validation study. Furthermore, relating to the ability of cancers to stimulate humoral immune response in the body, 41 novel potential serological autoantibody profiles have been identified in a South African PCa patient cohort (N = 67) . These identified serological autoantibody responses could potentially be used in the construction of mini-arrays as point of care diagnostic tools as well as for therapeutic vaccine development for PCa in Africa. Many of these potential biomarkers have been reported in literature as being associated with other diseases, PCa as well as cancers in other human body organs; however, many are still yet to be described in PCa . A comprehensive list which highlights the emerging potential biomarkers of PCa from those studies in terms of their source, methods used for their discovery and how far down the biomarker discovery pipeline they have moved, is provided in Tables 1A, 1B, 1C, 1D. Even though several potential preclinical biomarkers of PCa have been discovered in Western studies, very few studies have identified and validated novel potential biomarkers of PCa in Africa.
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review
| 99.9 |
The essence of translational research in the biomarker pipeline is to create a bridge from bench-derived or preclinical biomarkers to clinical utility. The clinical validation pipeline is comprised of various stringent phases through which preclinical biomarkers are tested before they can be certified for clinical utility. These biomarkers are applicable to various stages of PCa diagnosis and treatment as described below. Many of the currently emerging biomarkers have theranostic capabilities, implying that they possess predictive, diagnostic and prognostic potential [92–94] (Figure 3). Such double-edged “theranostic” biomarkers, which are capable of aiding diagnosis and well as serving as a means of treatment are very much needed in Africa where early diagnosis, treatment costs and patient compliance play a major role in PCa management outcome. Considering that the burgeoning biomarkers of PCa in the literature are poorly validated , more effort is needed to establish innovative ways to improve the success of emerging biomarkers through the validation phases and into clinical application.
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review
| 99.9 |
The need for systematic validation of proteomics biomarkers and lack of standardization of validation methods among researchers [71, 96, 97] has led to the establishment of the Early Detection Research Network (EDRN) and the Prospective Specimen Collection, Retrospective Blinded Evaluation (PRoBE) collaboration with the National Cancer Institute (NCI) [25, 98]. One important identified cause of unsuccessful biomarker validation is the lack of concordant experimental outcome from independent research groups . Prensner et al therefore suggested a three-phased biomarker validation pipeline, starting with a discovery phase, followed by validation in a retrospective cohort, and then final validation in a prospective cohort . Mandrekar et al emphasized that critical planning is essential in biomarker validation design and that the use of prospective randomized controlled trials as a gold standard enables a distinction between prognostic and predictive biomarkers . Analytic validation, clinical qualification/validity and clinical utilization has been recommended as vital steps in the rigorous evaluation of biomarkers and surrogate endpoints in chronic diseases [72, 99].
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review
| 99.9 |
Using these biomarker validation steps, features including high-grade prostatic intraepithelial neoplasia (HGPIN), PSA level, apoptosis and proliferation may be considered as surrogate endpoint biomarkers of PCa . In addition, Brown et al suggested that bone-related peptides like N- and C-terminal peptide fragments of type I collagen, and bone-specific alkaline phosphatase can be used as endpoint biomarkers in PCa . Despite the fact that the drug development pipeline is well established, biomarker validation pipeline is still a controversial subject among biomarker researchers. Through the efforts of the EDRN, Pepe et al have developed a 5-phased biomarker validation pathway [97, 102] that extends the ideas of Prensner et al : The first stage is the preclinical exploratory phase where promising potential biomarkers are identified; Phase II involves clinical assay and validation to identify disease establishment; Phase III is a retrospective longitudinal study for preclinical detection of disease; Phase IV involves a prospective screening of the characteristics and extent of disease; and Phase V is aims at cancer control by assessing the effect of screening with biomarker on the burden of disease in the population (Table 2). Several standard guidelines for reporting and evaluating biomarker studies have emerged: reporting recommendation for tumour markers (REMARK); biospecimen reporting for improved study quality (BRISQ); minimum information about a microarray experiment (MIAME); standard for reporting diagnostic accuracy (STARD); and the level of evidence (LoE) based tumor marker guideline (TMUG) proposed by the American Society of Clinical Oncology (ASCO) in 1996 . These guidelines have promoted transparency and rigour in the way biomarker discovery is being reported and evaluated; albeit most are yet to be implemented in African cancer biomarker studies.
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review
| 99.9 |
It is clear that the burden PCa on the African continent has increased significantly over the recent years, with the highest mortality rates reported in sub-Saharan Africa . It has also been demonstrated that PCa incidence rates in African-American and West African populations differ, even though they share a common genetic ancestry [7, 9]. The bases for such disease disparities are largely unknown, and it has been difficult to develop state-of-the-art research on prostate tumorigenesis and biology in Sub-Saharan Africa. Barriers to knowledge about PCa in Sub-Saharan Africa include sociocultural issues such as poor funding [102, 103], insufficient manpower and skilled health personnel , poor access to healthcare , religious and cultural beliefs , lack of well-updated cancer registries , poor research and healthcare infrastructure [102, 107], low educational level , prevalence of infection [109, 110], poor governance structures and fiscal policies . Although these factors are not unique to the African continent, the underlying genetic, hereditary and environmental basis of PCa aggressiveness in men of African descent still warrants further research.
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review
| 99.8 |
The advent of proteomics and other high throughput omics-based technologies has highlighted the need for computational biology, as well as for state-of-the-art banking of experimental biospecimens. Improper documentation and storage of biologic specimen may result in skewed biochemical inferences, histopathologic analysis and predicted therapy. Hence, a good specimen biorepository is an essential infrastructure for development of high throughput omics based personalized medicine in Africa. Most ground breaking projects in the field of molecular biology such as the Human Genome Project (HGP) , The Cancer Genome Atlas (TCGA) , Human Proteome Project (HPP) and Chromosome Centric Human Proteome Project (CHPP) have benefitted immensely from specimen biorepositories. However, in Africa, it is apparent that there is a limited biobanking capacity and that procedures such as fresh snap frozen tissue sampling cannot be performed in most places because liquid nitrogen is largely unavailable. Furthermore, there has traditionally been a paucity of bioinformatics infrastructure and bioinformaticians in Africa, although this is now being addressed through National Institutes of Health (NIH)-funded training and capacity development initiatives, such as the H3ABionet consortium. The National Cancer Institute has described a biorepository as human specimen collection including relevant data for the purpose of research, and subject to relevant processes, ethics and policies [116, 117]. Despite the gamut of biorepositories established in the Americas, Europe, Asia and Australia [118–120], very few such biobanks have been established in sub-Saharan Africa . Most biorepositories in Africa are established within investigator's research group and most are yet to be standardized and centralized. Several challenges have plagued biorepository development and regulation in sub-Saharan Africa [121–124], albeit modest progress has been made in a few sub-Saharan African countries. Notably, the emergence of the NIH and Wellcome Trust-funded H3Africa consortium, has improved the centralization and standardization of biospecimen collection in Africa . Further collaborative effort is required by clinicians and scientists to standardize and improve the development of a centralized biorepository in Africa, such that high throughput ‘omics technologies can be backed by adequate research materials/resources, enabling them to play a central role in improving our current understanding of PCa in Africa.
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review
| 99.9 |
In silico prevalidation is required in early phase of biomarkers development study [21, 90], to optimize potential biomarkers prior to large scale studies. The cost implication and infrastructure needed for large scale validation of omics based biomarkers can be prohibitive , hence collaborative efforts such as the EDRN are necessary to achieve this goal . In addition, standard validation procedures often require validated surrogate prognostic biomarkers that have been tested in multiple phase III trials . Due to prohibitive costs, only important proteins or antibodies from the discovery phase would typically be validated . Proteogenomic integration is thus an important multipronged approach to identify viable diagnostic and treatment signatures for PCa. For example, androgen receptors (AR) and EGFR has been identified as correlating with PCa progression using gene expression data integrated with protein interaction networks . In line with an integrated proteogenomic approach, other emerging liquid biopsy biomarkers such as long non-coding RNA and exosomes holds future prospects for PCa . Sixteen putative proteomics biomarkers of PCa validated on a well annotated tissue microarray (TMA) containing ca. 2500 PCa samples has been used to establish a prediction nomogram for PCa . Several genomic classifiers have also emerged for clinical metastasis and biochemical recurrence, as well as for patient stratification . Hitherto, most discovered biomarkers have focused on diagnostic rather than prognostic and predictive potentials . As emphasized earlier, there is an urgent need for larger biorepository establishment to facilitate disease biomarker discovery and validation . Emerging biomarkers such as circulating tumour cells (CTC) in blood may enable the evaluation of disease prognosis and survival . Advances in PCa biomarker development has resulted in the clinical utility of laboratory tests such as Prolaris score, Oncotype Dx, Confirm MDx, Prostarix and 4K score . These tests have helped to improve diagnosis of PCa and decipher the level of evidence (LoE) assigned for biomarker validation . Urine and blood sample testing for biomarkers is a reasonable approach with high prospect for mass screening of populations for disease , particularly in African populations that are at greater risk of developing aggressive PCa.
|
review
| 99.9 |
This review has demonstrated the prospects for discovery and validation of potential biomarkers in Africa. Currently available biomarkers and biomarkers sources were highlighted, as well as the application of high throughput methodologies for candidate biomarker discovery. Being a continent with a huge burden of cancer and mostly populated by low and middle income countries (LMIC), several challenges have been identified that militate against the routine application of proteomics to PCa research. The most promising urinary biomarkers which require further validation are the top 12 identified by targeted proteomics ; while the best serological biomarkers are the top 41 tumour associated antigens . All potential urinary and serological biomarkers that demonstrated ethnic trends are also worth investigating further for potential used for personalized management of PCa in Africa. There is a pressing need to develop cancer research initiatives and collaborations with partners within and outside of Africa. Potential PCa biomarkers discovered in African studies requires research and systematic validation as practiced in the developed world, with urine and blood based theranostic biomarkers offering a multi-pronged approach to diagnosis and treatment of PCa in Africa. Ethnic-tailored biomarkers for PCa management are required if personalized medicine is to become a reality among African populations. Collaborative research directed towards understanding the potential role of PCa immunotherapy in African patients should also be encouraged, both locally and internationally. Importantly, diagnostic focus should be redirected towards less invasive approaches to PCa management, to improve early screening and intervention in Africa. It is also suggest that a centralized specimen and proteome repository should be built for PCa in Africa, as this would provide a resource for better research into PCa burden among men of African descent.
|
review
| 99.9 |
Pancreatic cancer is a malignant neoplasm of the digestive system. The incidence has increased and it is now the 10th most prevalent malignant tumor worldwide [1–3]. The median survival time of pancreatic cancer patients is 3–6 months and the 5-year survival rate is less than 5%. Because pancreatic cancer does not exhibit obvious clinical symptoms in the early stages, it is frequently diagnosed at a late stage (85% of patients) when surgical treatment is no longer effective . Therefore, adjuvant therapy (particularly radiotherapy and chemotherapy) is critical for management.
|
review
| 99.9 |
Gemzar (gemcitabine) single-agent chemotherapy is the standard treatment for metastatic or locally advanced pancreatic cancer, but it has a modest survival benefit . FOLFIRINOX, a combination of chemotherapeutic agents (folinic acid, fluorouracil, irinotecan, and oxaliplatin), was recently shown to nearly double the median survival of pancreatic ductal adenocarcinoma (PDAC) patients compared to gemcitabine (11.1 vs. 6.8 months). However, patients must be carefully selected and managed because of toxicity . Nab-paclitaxel (NPT) is a water-soluble, cremophor-free, albumin-bound formulation of paclitaxel. It was initially developed to circumvent the toxicity associated with cremophor, which is required to solubilize paclitaxel. NPT in combination with gemcitabine resulted in a median survival time of 8.5 months compared to 6.7 months after gemcitabine treatment alone [8–9]. Given the moderate improvements in PDAC patient prognosis, there is an urgent requirement for novel therapeutic strategies to improve overall survival.
|
review
| 99.9 |
Previous studies have demonstrated that Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA-MSHA) in combination chemotherapy functions through mannose-modified proteins to inhibit cancer cell proliferation, induce apoptosis, and promote an anti-tumor immune response. PA-MSHA also has displayed anti-tumor activity in breast, liver, lung, gastric, and bladder cancer [10–12]. EGFR overexpression and/or enhanced activity has been reported in many solid tumors including pancreatic cancer . Activation of EGFR results in phosphorylation and activation of downstream signaling components and leads to cell proliferation . In this study, we investigated the mechanisms underlying PA-MSHA function. We assessed whether PA-MSHA induced apoptosis in pancreatic cancer cells, and evaluated the association between PA-MSHA-mediated anti-tumor effects and EGFR signaling. Additionally, we evaluated the therapeutic potential of PA-MSHA.
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study
| 100.0 |
We analyzed the effects of PA-MSHA on the proliferation of pancreatic cancer and normal ductal epithelial cell lines using CCK-8 assays. PA-MSHA at concentrations of 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 × 109/mL inhibited the growth of PANC-1 and SW1990 pancreatic cancer cells in a time- and dose-dependent manner. In addition, treatment of HPDE6-C7 normal pancreatic ductal epithelial cells with PA-MSHA for 24 h and 48 h had little/no effect on proliferation (Figure 1). The IC50 values for the two pancreatic cancer cell lines were significantly higher than those of the normal pancreatic ductal epithelial cells (Table 1) (p < 0.05).
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study
| 100.0 |
Values represent the percentages of untreated control cells. The data are averages of triplicate results from representative experiments. P < 0.05 for (A) SW1990 or (B) PANC-1 cells treated with PA-MSHA vs. controls. P > 0.05 for (C) HPDE6-C7 cells treated with PA-MSHA vs. controls.
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study
| 99.94 |
We next examined changes in cellular ultrastructure in response to treatment of pancreatic cancer cells with PA-MSHA by transmission electron microscopy. Treatment of PANC-1 and SW1990 cells with 0.5 × 109/mL PA did not alter the cellular morphology. However, treatment with 0.5 × 109/mL PA-MSHA for 24 hours resulted in cytoplasmic condensation, shrinkage of the cell body, nuclear fragmentation, mitochondrial enlargement, and the detachment and fusion of the endoplasmic reticulum with the plasma membrane to form multiple vacuoles. The presence of budding apoptotic bodies was indicative of the initiation of cellular breakdown in response to PA-MSHA treatment. Vacuole size and number increased after 48 hours of PA-MSHA treatment. At this time-point, most of the cells had died and the cytoplasmic structures had collapsed (Figure 2 and Figure 3).
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study
| 100.0 |
Changes in PANC-1 cell ultrastructure after treatment with PA-MSHA (10 × 108/mL) for 24 h A. and 48 h B. visualized by electron microscopy (×5,000), (x10,000), and (x20,000). Changes in SW1990 cell ultrastructure after treatment with PA-MSHA (10 × 108/mL) for 24 h C. and 48 h D. visualized by electron microscopy (×5,000), (x10,000), and (x20,000).
|
study
| 99.94 |
The percentage of apoptotic PANC-1 and SW1990 cells was higher after PA-MSHA treatment compared to the controls (Figure 4). The rates of apoptosis were 8.17 ± 0.53% and 7.62 ± 0.89% in PANC-1 and SW1990 cells, respectively, after treatment with 0.25 × 109/mL PA-MSHA. These rates were much higher than those observed in the control group (4.18 ± 1.12% and 2.49 ± 0.74% for PANC-1 and SW1990 cells, respectively). Furthermore, the rate of apoptosis increased with increasing concentrations of PA-MSHA. Thus, our data demonstrate that PA-MSHA induces apoptosis in pancreatic cancer cell lines.
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study
| 100.0 |
A. The ratio of apoptotic cells (i.e. the annexin V-positive/PI-negative fraction) was measured in PANC1 and SW-1990 cells after treatment with the indicated concentrations of PA-MSHA in serum-free media for 24 h. The results are representative of three independent experiments. B. The percentage of apoptotic cells showing the annexin V-positive/PI-negative fraction. The columns represent the mean ± SD of the three independent experiments. *p < 0.05 for PA-MSHA vs. controls.
|
study
| 100.0 |
We next analyzed cell cycle progression in PANC-1 and SW1990 cells after treatment with PA-MSHA by flow cytometry. After 24 hours of PA-MSHA treatment, the population of pancreatic cancer cells in both the G0/G1 and G2/M phases was significantly lower than in the control group, while the population of cells in in S phase was significantly higher (p < 0.01) (Figure 5). These data indicated that PA-MSHA caused cell cycle arrest in S phase and induction of apoptosis.
|
study
| 100.0 |
A. The columns represent the mean ± SD of the three independent experiments. PANC-1 and SW-1990 were treated with the indicated concentrations of PA-MSHA in serum-free medium for 24 h. The results are representative of three independent experiments. B. The percentages of cells in G1, S, and G2-M are shown as histograms
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study
| 99.94 |
To evaluate the effects of PA-MSHA on EGFR signaling in pancreatic cancer cells, we examined the expression of key downstream proteins in the EGFR pathway (t-EGFR, t-AKT, t-ERK, p-EGFR, p-AKT, and p-ERK) by western blotting. Interestingly, the levels of p-EGFR, p-AKT, and p-ERK decreased with increasing concentrations of PA-MSHA, while total EGFR, AKT, and ERK protein levels did not change (Figure 6). Thus, PA-MSHA inhibited EGFR signaling in pancreatic cancer cells by blocking phosphorylation of several downstream effectors of EGFR.
|
study
| 100.0 |
To examine the anti-tumor effects of PA-MSHA in vivo, we subcutaneously injected nude mice with PANC-1 cells into the right hind limb. The mice were then injected intraperitoneally with NPT for 7 weeks. The mice were euthanized 42 days after transplantation and the tumor tissue collected. We found that the average tumor volume was larger in the control group than in the PA-MSHA group (714.51 ± 101.73 mm3 vs. 187.64 ± 28.58 mm3, p < 0.01). Overall, the tumors in the PA-MSHA treatment group were significantly smaller than those in the control group (Figure 7B). Finally, an additive effect was observed when the two agents were combined (Supplementary Figure 1A). Thus, PA-MSHA and NPT could reduce tumor growth in a xenograft model of pancreatic cancer.
|
study
| 100.0 |
A. Tumor volume measured at the indicated time-points. Treatment was initiated 10 days after implantation of pancreatic cancer cells into the mammary fat pads of mice. The mice were injected with either PA or PA-MSHA for 7 weeks and the tumor volumes measured. *p < 0.01. B. Caspase-3 and cleaved caspase-3 expression in xenograft mice treated with either PA or PA-MSHA.
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study
| 100.0 |
Next, we extracted the total protein from tumor tissue samples to examine the effects of PA-MSHA on apoptosis in the xenograft mice. Interestingly, we observed a reduction in caspase-3 levels in PA-MSHA- compared to PA-treated mice (p < 0.05). In contrast, cleaved caspase-3 levels were higher in PA-MSHA- compared PA-treated mice (p < 0.01; Figure 7). Combined treatment with PA-MSHA and NPT had a synergistic effect on apoptosis (Supplementary Figure 1B). These data indicated that PA-MSHA induced caspase-dependent apoptosis in pancreatic cancer cells in vivo.
|
study
| 100.0 |
Pancreatic cancer incidence and mortality have increased in recent years . Surgery is currently the most effective treatment option. However, pancreatic cancer is typically diagnosed at an advanced stage when surgical treatment may no longer be effective. Adjuvant chemotherapy is the standard of care for patients after pancreatic cancer surgery and for patients with advanced-stage disease . Gemcitabine as single-agent chemotherapy has traditionally been the standard treatment regimen, but it has a modest survival benefit . More recently, FOLFIRINOX and Nab-paclitaxel plus gemcitabine have become the standard of care [18–19]. However, poor clinical outcomes are still observed as a result of drug resistance and the side effects of chemotherapy. Therefore, new chemotherapeutic agents are required to improve patient outcomes .
|
review
| 99.9 |
In this study, we demonstrated that PA-MSHA inhibited the proliferation of two pancreatic cancer cell lines (PANC-1and SW1990) in a time- and dose-dependent manner. Treatment of pancreatic cancer cells with PA-MSHA resulted in cytoplasmic condensation, cell body shrinkage, nuclear fragmentation, disorganized cellular structures, and the detachment and fusion of the endoplasmic reticulum with the plasma membrane to form multiple vacuoles. These morphological changes indicated PA-MSHA induced apoptosis in pancreatic cancer cells.
|
study
| 100.0 |
Apoptosis was further analyzed in pancreatic cancer cells by flow cytometry. These results confirmed that PA-MSHA induced pancreatic cancer cell apoptosis in a dose-dependent manner. Additionally, we determined that PA-MSHA treatment blocked cell cycle progression at S phase.
|
study
| 100.0 |
EGFR is widely expressed in mammalian epithelial cells. Previous studies have demonstrated higher EGFR and EGF expression in pancreatic cancer tissue compared to normal pancreatic tissue. The expression of EGF/EGFR was correlated with tumor proliferation, invasion, and metastasis . EGFR inhibitors can inhibit the growth and proliferation of tumor cells . Our results indicated that PA-MSHA treatment did not affect the total levels of EGFR signaling pathway components compared to controls. However, the levels of pEGFR, pAKT, and pERK decreased in response to treatment with increasing concentrations of PA-MSHA, suggesting that PA-MSHA inhibited phosphorylation of these key downstream signaling proteins. Thus, PA-MSHA inhibits EGFR signaling and cell proliferation in pancreatic cancer cells.
|
study
| 100.0 |
Caspase-3 functions in the last step of caspase activation and plays an important role in apoptosis . PA has been shown to induce apoptosis through the caspase pathway . However, heat-inactivated PA does not have the capacity to induce apoptosis [26–27]. Consistent with previous reports, we found that inactivated PA did not induce apoptosis in pancreatic cancer cells. However, inactivated PA-MSHA was capable of inducing apoptosis in vitro. Therefore, we hypothesized that PA-MSHA could induce caspase-dependent apoptosis in tumor cells in vivo. Our data indicated that caspase-3 expression was reduced in pancreatic tumor tissue compared to normal pancreatic tissue, and that the level of cleaved caspase-3 increased in response to PA-MSHA treatment. The combination of PA-MSHA and NPT had a synergistic inhibitory effect on tumor growth. Overall, our data provide insight into the molecular mechanisms underlying PA-MSHA-induced apoptosis in pancreatic cancer cells. Because PA-MSHA inhibits pancreatic cancer cell proliferation and induces apoptosis, it may be a novel treatment for pancreatic cancer that can improve patient prognosis.
|
study
| 99.94 |
Pancreatic cancer (PANC-1 and SW1990) and normal human pancreatic duct epithelial (HPDE6-C7) cell lines were obtained from the American Type Culture Collection. Cells were maintained in DMEM medium supplemented with 10% FBS (Gibco, USA) at 37°C in a humidified incubator containing 5% CO2 (Shanghai Medical Instruments, China). PA-MSHA and wild type PA were obtained from Beijing Wanter Bio-Pharmaceutical (China). The following antibodies were used in this study: anti-pEGFR, -EGFR, -pERK, -ERK, -pAKT, -AKT, -caspase-3, -cleaved caspase-3, and -GAPDH (Cell Signaling Technology, USA).
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study
| 99.94 |
The cytotoxic effects of PA-MSHA were examined using CCK-8 assays (Cell Counting Kit-8, Dojindo Molecular Technologies Inc., USA). Cells were treated with 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, or 8.0 × 109/mL PA-MSHA and then maintained at 37°C for 24, 48, or 72 hours. The IC50 values were calculated using CalcuSyn (Biosoft, USA).
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study
| 100.0 |
Cells were treated with either 0.5 × 109/mL PA or PA-MSHA for 24 and 48 hours. They were then washed in twice in phosphate-buffered saline (PBS). Cells were fixed in glutaraldehyde for 2 hours, washed three times with PBS, and then fixed in osmium tetroxide for 2 hours. The samples were washed three times in PBS, dehydrated in a stepwise gradient of ethanol, and then embedded in resin. Samples were sectioned, stained, and imaged using a Hitachi Transmission Electron Microscope (Hitachi, Japan).
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study
| 99.94 |
Cells were treated with 0.5 × 109/mL PA or 0.25, 0.5, or 1.0 × 109/mL PA-MSHA for 24 hours, and then resuspended in serum-free media at a concentration of 1 × 106/mL. The cells were treated with 5 μL FITC-annexin V and 5 μL propidium iodide (PI; 50 μg/mL), mixed gently, and incubated at room temperature for 15 minutes in the dark. We then added 400 μL 1× Binding Buffer to each sample. Apoptosis was analyzed by flow cytometry using a FACSCalibur system (BD Biosciences, USA). For cell cycle analysis, cells were resuspended as described previously and then fixed in 5 mL of 70% ethanol at 4°C overnight. Samples were then washed twice with PBS and PI added to a final concentration of 50 μg/mL. After incubating the samples at 4°C for 30 minutes, cell cycle profiles were analyzed by flow cytometry.
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study
| 99.94 |
Cells were treated with 0.5 × 109/mL PA or 0.25, 0.5, or 1.0 × 109/mL PA-MSHA, and total protein extracted and quantified using BCA assays. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with the primary and secondary antibodies listed above. The amount of protein was estimated using the Quantity One software (Bio-Rad, USA) and the relative expression of the target proteins calculated as the amount of the target protein divided by that of GAPDH.
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study
| 100.0 |
Four-week-old female Balb/c-nu/nu nude mice were obtained from the Shanghai Institute of Materia Medica at the Chinese Academy of Sciences. Mice were randomly assigned to either the control or experimental group, and 5 × 106 PANC-1 cells subcutaneously injected into the right hind limbs. Subcutaneous injections of 0.1 mL PA (2 × 1010/ml) and 0.1 mL PA-MSHA (2 × 1010/mL) were administered daily to the control and experimental groups, respectively. The mice were injected intraperitoneally with Nab-paclitaxel (10 mg/kg, twice a week) for 7 weeks. Tumor size was measured twice a week using a vernier caliper. The following formula was used to calculate tumor size (V): V = π/6 × (W2 × L), and the values used to generate tumor growth curves. After the experiment, tumor tissues were collected and proteins analyzed by western blotting.
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study
| 100.0 |
Statistical analyses were performed using SPSS13.0 (SPSS Inc. Chicago, IL, USA). Data are presented as the mean ± standard deviation (x¯ ± SD). Differences between the control and experimental groups were analyzed using Student's t tests. Tumor growth curves were analyzed using Mann-Whitney U tests. A p value < 0.05 was considered statistically significant.
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study
| 99.9 |
The giant panda is known as a rare, vulnerable species that is extremely popular worldwide. As a special herbivore, the giant panda has retained a typical carnivorous digestive system, which is easily afflicted by various intestinal diseases, especially ETEC infection which affects the intestinal barrier when the feed structure changes (Nataro and Kaper, 1998; Chen et al., 2015). Controlling this pathogen and its associated diarrhea heavily relies on antibiotics, of which their disadvantage could also threaten the animals. Probiotics are considered as one of the most effective alternatives to antibiotics because of their abilities to maintain or restore the normal microbiota, inhibit pathogen adhesion to intestinal walls, prevent inflammation, and protect intestinal barrier function (Geier et al., 2010; Archambaud et al., 2012; Yu et al., 2015).
|
review
| 99.9 |
Few reports are available on the role of probiotic strains in preventing ETEC invasion in giant panda. In a previous work, we isolated L. plantarum G83, a novel probiotic strain, from the feces of healthy captive giant panda. The microbe survived well at low pH, was tolerant to high bile-salt concentrations and resistant to antibiotics, and antagonized pathogenic bacteria in vitro (data not shown). For the next research step, the safety of this strain was assessed in vivo. Our laboratory also analyzed the intestinal microfloral structure of captive giant pandas at different age (Peng et al., 2016). The above-mentioned results served as the foundation for using the L. plantarum G83 as an antibiotic alternative and motivated the present study in mice. Similar to giant pandas, mice are omnivorous and easily infected by ETEC. Thus, mice are adopted as models for the study of the protective effects of L. plantarum G83 against ETEC invasion in giant panda.
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study
| 99.94 |
L. plantarum G83 was grown in De Man, Rogosa, and Sharp broth at 37°C for 24 h. Meanwhile, the ETEC K88 strain (O8:H19:F4ac+, LT+, STa−, STb+) was obtained from the China Institute of Veterinary Drug Control (Beijing, China) and grown in Luria–Bertani broth containing 5% fetal bovine serum at 37°C for 12 h.
|
study
| 99.9 |
The L. plantarum G83 cells were harvested by centrifugation at 3,000 × g for 10 min at 4°C after growing in an anaerobic environment at 37°C for 24 h. The cell pellet was washed with sterile physiological saline twice. Finally, the cells were diluted to 5.0 × 108, 5.0 × 109, and 5.0 × 1010 colony-forming units (cfu) mL−1 in 200 μL of PBS for 14 consecutive days.
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study
| 99.94 |
Meanwhile, the ETEC cells were harvested by centrifugation at 3,000 × g for 10 min at 4°C after being incubated at 37°C for 12 h with vigorous shaking. Next, the cell pellet was washed with sterile physiological saline twice. The cells were then diluted to 1 × 109 cfu mL−1 in PBS for the infectious challenge.
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study
| 99.94 |
Ninety male ICR mice (16 ± 2 g average weight) were purchased from Chengdu Dashuo Biological Institute (Chengdu, China) and fed with commercial chow. All mice were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle and allowed free access to food and water.
|
other
| 98.56 |
The animals were randomly divided into five groups of 18 mice each. Each group was given different treatments as follows: (1) oral administration of sterile PBS from day 1 to day 15 (CONT), (2) oral administration of sterile PBS from day 1 to day 14 followed by oral challenge with ETEC on day 15 (ETEC), (3) oral administration of low-dose L. plantarum G83 (5.0 × 108 cfu mL−1) from day 1 to day 14 followed by oral challenge with ETEC on day 15 (LDLP), (4) oral administration of intermediate-dose L. plantarum G83 (5.0 × 109 cfu mL−1) from day 1 to day 14 followed by oral challenge with ETEC on day 15 (MDLP), and (5) oral administration of high-dose L. plantarum G83 (5.0 × 1010 cfu mL−1) on day 1 to day 14 followed by oral challenge with ETEC on day 15 (HDLP). Simultaneously, individual body weights were recorded daily.
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study
| 100.0 |
After blood sampling, the animals were killed by cervical dislocation. All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (No. SYXKchuan 2014-187).
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other
| 99.94 |
At 0, 24, and 144 h after ETEC challenge, the blood samples were centrifuged at 2,000 × g for 20 min at 4°C. The resultant sera were obtained and stored at −80°C. The levels of immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), D-lactate, and diamine oxidase (DAO) activity were then analyzed by using ELISA kits (Shanghai MLBIO Biotechnology Co. Ltd., China).
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study
| 100.0 |
Total RNA was extracted from liquid-nitrogen-frozen jejuna using RNAiso plus (TaKaRa, Dalian, China) in accordance with the manufacturer's instructions. Meanwhile, RNA integrity and purity were assessed using 1% agarose gel electrophoresis and Nano Drop spectrophotometry (Nano Drop Technologies, Wilmington, DE, USA), respectively. Then, 1 μg of the total RNA was reverse transcribed using the PrimeScript® RT reagent Kit with gDNAEraser (TaKaRa, China) in compliance with the manufacturer's protocol. The generated cDNA was stored at −80°C until real-time PCR analysis.
|
study
| 99.75 |
All the genes detected and specific primers used are listed in Table 1. Quantitative real-time PCR was performed using a CFX Connect™ Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) with a SYBR Premix Ex Taq™ II PCR kit (TaKaRa, China). The PCR conditions were as follows: 95°C for 1 min, 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Melting curve analyses were performed to monitor the purity of the PCR product. All reactions were run in triplicates. Relative gene expression levels were evaluated through the 2−ΔΔCt method, where ΔΔCt = (Ct, target Ct, β−actin)treated group (Ct, target Ct, β−actin)control group.
|
study
| 100.0 |
IL-1β, interleukin-1 beta; IL-6, interleukin-6; IL-8, interleukin-8; IL-10, interleukin-10; TLR2, toll like receptor 2; TLR4, toll like receptor 4; MyD88, myeloid differentiation primary response gene 88; MCP-1, monocyte chemotactic protein 1, ZO-1, zoluna occludens protein 1.
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other
| 99.94 |
The sample of total DNA for colon microflora (n = 6, each group, randomly) was extracted by using the E.Z.N.A.® Stool DNA kit (Omega Biotechnology, USA). The DNA concentration was detected by a Nano Drop spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). The primers, annealing temperatures, and product sizes for the different bacterial and total bacterial quantifications were displayed in Table 2. The PCR conditions were as same as those stated under Relative Quantitative Real-time PCR but with different annealing temperatures. The same approach (Rinttilä et al., 2004) was also used to quantify the LT.
|
study
| 100.0 |
All data were expressed as means and standard deviations. Statistical analysis was performed by one-way ANOVA in SPSS 19.0. Differences among treatments were compared using the Student-Newman-Keuls multiple comparison test. Statistical significance was set at P ≤ 0.05.
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study
| 99.94 |
Before the ETEC challenge, no clinical sign was observed in the mice of any experimental group. After the ETEC challenge, only one mouse from the ETEC group presented with mild diarrhea, whereas most of the mice initially became dull and depressed and then lost weight. All groups showed no significant difference (P > 0.05) in body weight gain throughout the entire trial period (Figure 1). However, the mice given L. plantarum gained more body weight than those by others. After ETEC challenge, the ETEC, LDLP, MDLP, and HDLP groups presented similar weight changes (Figure 1).
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on the body weight of mice during pre- and post-challenge periods (n = 6). CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. There was no significant (P > 0.05) in all groups.
|
study
| 100.0 |
Figure 2 shows the changes in the number of leukocytes, lymphocytes, intermediate cells (including monocyte, eosinophils, and basophils), and granulocytes numbers in the five groups after ETEC challenge. At 24 h after ETEC challenge, the lymphocyte concentrations decreased (P < 0.05) and the granulocyte concentrations increased (P < 0.05) in the ETEC group and Lactobacillus-supplemented groups relative to those in the CONT group. No significant difference (P > 0.05) was noted among the ETEC group and the Lactobacillus-supplemented groups. At 0, 24, and 144 h after ETEC challenge, the levels of leukocyte, lymphocyte, intermediate cell, and granulocyte did not change significantly (P > 0.05) in all groups.
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on blood leukocyte count and population distribution of mice during pre- and post-challenge periods. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. At 24 h after ETEC challenge, lymphocyte concentrations decreased (P < 0.05) and granulocyte concentrations increased (P < 0.05) in ETEC group and Lactobacillus-supplemented groups as compared to CONT group.
|
study
| 100.0 |
The levels of DAO and D-lactate after ETEC challenge are shown in Figure 3. At 24 h after ETEC challenge, the DAO and D-lactate concentrations in the ETEC group were significantly higher (P < 0.05) than those in the CONT and MDLP groups but did not differ (P > 0.05) from those of the HDLP group. At 0 and 144 h after ETEC challenge, no difference (P > 0.05) in DAO and D-lactate concentrations was noted in all the groups.
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on DAO activity and D-lactate concentration in serum of mice during pre- and post-challenge periods. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. Bars with different letters are significantly different (P < 0.05). Bars share the same letters do not differ significantly (P > 0.05).
|
study
| 100.0 |
Before challenge, the serum IgA levels in the LDLP and MDLP groups significantly increased (P < 0.05) with respect to that in the CONT group. Meanwhile, the IgM concentrations in the MDLP and HDLP groups were higher (P < 0.05) than that in the CONT. However, only the IgG level in HDLP increased (P < 0.05) with respect to that in the CONT. At 24 h after ETEC challenge, the IgA and IgM contents in the LDLP, MDLP, and HDLP groups were higher (P < 0.05) than those in the CONT. At 144 h post-challenge, no significant difference in IgA, IgM, or IgG level (P > 0.05) was observed among the groups (Figure 4).
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on concentrations of IgA, IgM and IgG in serum of mice during pre- and post-challenge periods. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. Bars with different letters are significantly different (P < 0.05). Bars share the same letters do not differ significantly (P > 0.05).
|
study
| 100.0 |
Before challenge, the MDLP group exhibited an up-regulated expression (P < 0.05) of occludin and zonula occluden 1 (ZO-1), whereas the LDLP group showed only an increased expression (P < 0.05) of occludin, with respect to those in the CONT group. At 24 h after ETEC challenge, the expression of all the TJ-associated proteins was down-regulated, and the ETEC group achieved lower claudin-1 and occludin levels than those in the CONT group (P < 0.05). However, the difference among the CONT, LDLP, and MDLP groups was not significant (P > 0.05). At 144 h after challenge, the expression of claudin-1 and occludin in all groups was not significant (P > 0.05). The expression of ZO-1 in ETEC and HDLP groups was down-regulated (P < 0.05) compared to CONT group (Figure 5).
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on tight junction proteins mRNA expression in jejunum of mice during pre- and post-challenge periods. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. Bars with different letters are significantly different (P < 0.05). Bars share the same letters do not differ significantly (P > 0.05).
|
study
| 100.0 |
Interleukin (IL)-1β and IL-8 levels in the HDLP group obviously rose (P < 0.05) relative to those of the CONT group at 0 h after ETEC challenge. At 24 h after ETEC challenge, the IL-1β, IL-8, IL-6, TLR4, and MyD88 levels in the ETEC group were significantly higher (P < 0.05) than those of the CONT, LDLP, and MDLP groups. Meanwhile, the IL-8 contents did not significantly change (P > 0.05) between the ETEC and HDLP groups. At 144 h, the IL-1β expression in the ETEC group was higher (P < 0.05) than those of the other groups (Figure 6).
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on cytokines and Toll-like receptor mRNA expression in jejunum of mice during pre- and post-challenge periods. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. Bars with different letters are significantly different (P < 0.05). Bars share the same letters do not differ significantly (P > 0.05).
|
study
| 100.0 |
At 0 h, the Lactobacillus concentration of the HDLP group and the Bifidobacterium spp. content of the MDLP group was significantly higher (P < 0.05) compared to the CONT group. Meanwhile, the Bacteroidetes level in the LDLP, MDLP, and HDLP group and the Enterobacteriaceae level in the MDLP decreased (P < 0.05). At 24 h, the Enterobacteriaceae concentration in the ETEC group grew (P < 0.05) relative to those of the other groups. At 144 h, all bacterial concentrations apart from Bacteroidetes and Lactobacillus did not considerably vary among the groups (Figure 7).
|
study
| 100.0 |
Dose effect of Lactobacillus plantarum on composition of the mice colonic microbiota during pre- and post-challenge periods. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. Bars with different letters are significantly different (P < 0.05). Bars share the same letters do not differ significantly (P > 0.05).
|
study
| 100.0 |
Amount of LT gene in colon of mice at 24 h after ETEC challenge. CONT, control group, treatment with PBS at all period; ETEC, negative group, oral challenge with ETEC on day 15; LDLP, low dose (5.0 × 108 cfu mL−1) group; MDLP, intermediate dose (5.0 × 109 cfu mL−1) group; HDLP, high dose (5.0 × 1010 cfu mL−1) group; LDLP, MDLP, and HDLP groups were infected with ETEC at day 15. Bars with different letters are significantly different (P < 0.05). Bars share the same letters do not differ significantly (P > 0.05).
|
study
| 100.0 |
Severe gut disease can damage host health. However, in giant panda, this kind of disease must be prevented and treated. The relationship between probiotics and improved gut health has received considerable scientific interest for more than a century. To protect animal guts, scientists have shown that probiotics, such as Lactobacillus, Bifidobacterium, and Bacillus, prevent gut bacterial disease (Barba-Vidal et al., 2017; Nishida et al., 2017; Wu et al., 2017). In the present study, L. plantarum G83 was isolated from the feces of healthy giant panda and confirmed beneficial in vitro. This current work aimed to evaluate the protective effect of L. plantarum G83 in mice challenged with ETEC. To accomplish this objective, we assessed mice for their weights, as well as chemical and biological intestinal barriers.
|
study
| 99.94 |
In the present study, the weights and gain ratios in the ETEC group were certainly lower than those of the other groups. However, the growth performance did not significantly change in the mice supplemented with L. plantarum G83 either before or after ETEC infection. This result was similar to those of previous works (Nguyen et al., 2007; Wang et al., 2009, 2016) but can otherwise be explained by the shortage of weight gain evaluation period for weight gain. After ETEC infection, the mice only showed weight loss and slow growth rather than the clear clinical signs of diarrhea and death. Such presentation may be due to the differences in sensitivity of the model animal and in virulence of the strain (Duchet-Suchaux et al., 1990; Porter et al., 2011). These results revealed that the strain did not obviously affect the gain weight within a short period.
|
study
| 100.0 |
Maintaining the intestinal epithelial barrier integrity is important for the defense against pathogen invasion and inflammation (Fasano and Shea-Donohue, 2005). In this regard, plasma DAO and D-lactate have been proposed as circulating markers for the extent of damage and repair of the intestinal mucosa (Wijtten et al., 2011; Zhao et al., 2014). These markers can reflect the intestinal permeability and barrier function in peripheral blood (Ewaschuk et al., 2005). Furthermore, TJs, such as claudin-1, occludin and ZO-1, have been proven to seal the lateral intercellular space and achieve an intact layer of epithelial cells that guard against pathogens (Groschwitz and Hogan, 2009; Suzuki, 2013). In the present study, the intermediate- and low-dose groups notably inhibited the increase in plasma DAO and D-lactate levels after ETEC infection. ETEC invasion has been reported to increase the release of DAO and D-lactate in the plasma and damage the intestinal epithelial cell membrane (Roselli et al., 2007; Yang et al., 2014; Xun et al., 2015). Hence, our results indicated that the strain exerts a protective effect on the host during ETEC invasion. Different doses of L. plantarum G83 prevented ETEC-induced membrane damage by inhibiting the delocalization of ZO-1 and consequently raising occludin and claudin-1 amounts. This effect is consistent with that observed by Roselli et al. (2007), who found higher occludin and ZO-1 mRNA expression in piglets fed with Lactobacillus sobrius than in those not fed with the strain. In the former piglets, ETEC-induced membrane damage was prevented by inhibiting the rearrangement of F-actin and dephosphorylation of occludin. As Mennigen and Bruewer (2009) reported, there is a close link between probiotics and regulation of intestinal permeability and barrier function. The above-mentioned findings collectively indicated that L. plantarum G83 exerts a protective effect against ETEC-induced membrane damage.
|
study
| 99.94 |
During infection, innate or acquired immunity is activated. Immunocytes then play a key role in the innate and adaptive immune responses. Immunocyte number and variation reflect the immunity or infection status (Demissie et al., 2013; Hunt et al., 2015). Furthermore, the immunocyte induction of cytokine and antibody production participates in immune regulation and the inflammatory response (Baehner, 1975; Cassatella, 1999). One signaling pathway that promotes the expression of pro-inflammatory genes is the TLR- and MyD88-dependent pathways (Zughaier et al., 2005). In the current study, the L. plantarum G83 strain did not significantly alter the leukocyte amount and variation in the mouse peripheral blood among the groups at different time points. Besides the granulocyte, the amount of leukocytes, lymphocytes, and intermediate cells decreased at 24 h after ETEC inoculation. This effect may have been caused by the high expression of IL-8 and MCP-1, which take part in the immunocytes migration to the site of infection in the intestine. Such results were similar to those of previous reports (Zhu et al., 2014). Lactobacillus supplementation enhanced serum IgG, IgM, and IgA levels and indicated that probiotics could stimulate systemic or mucosal antibody response (Frece et al., 2006; Mizumachi et al., 2009). In the present study, the L. plantarum G83 obviously increased the IgA, IgM, and IgG levels before or after ETEC challenge. IgA has an important role in the protection of mucosal surfaces against pathogens (Galdeano and Perdigon, 2004). The secretory IgA of intestinal mucosa will be detected in next study. Low and intermediate doses of the strain also decreased the mRNA levels of IL-1β, −6, and −8 in the jejunum, probably by modifying the immunocyte profile. Early studies confirmed that lactobacilli lower serum IL-6 concentrations after acute ETEC challenge in piglets (Zhang et al., 2010). The aforementioned observations suggested that inflammation was ameliorated in the LDLP and MDLP groups and were consistent with those of previous studies where probiotics exerted dose-dependent effects (Mileti et al., 2009). Lee et al. (2012) found that high-dose L. plantarum CJLP243 (108–1010 cfu kg−1) most substantially improved the growth and health performance of weaning pigs, especially those with bacterium-induced acute bowel inflammation. A recent trial of piglets challenged with F4+ ETEC demonstrated that administering LGG (1012 cfu day−1) failed to prevent F4+ ETEC infection. This failure was probably due to the disruption of microbial and inflammatory responses by excessive probiotic levels (Li et al., 2012). Same probiotic in different doses have different modulation effect on T-cell immune response and mucosal IgA response (Wen et al., 2012, 2015). Thus, only intermediate- and low-dose L. plantarum G83 positively affects acute inflammatory bowel disease.
|
study
| 99.9 |
Besides promoting nutritional digestion, intestinal microflora is also involved in host immunity and pathology. Bacterial infections or enteritis, antibiotic treatment, and immunosuppression may alter the population, quantity, or habitat of gut microflora and may lead to the excessive growth of opportunistic pathogens as well as microbial dysbiosis (Bouhnik et al., 2004; Taur and Pamer, 2013). Numerous studies showed that probiotics can regulate and maintain the balance of intestinal flora by enhancing indigenous bacterial colonization and/or competitive exclusion to battle pathogens (Gareau et al., 2010). The present results revealed that administering L. plantarum G83 not only increased the abundance of Lactobacillus and Bifidobacterium, but also decreased the number of Bacteroidetes and Enterobacteriaceae in the colon. Our results were also similar to those of the reports where LGG and Lactobacillus Bar13 greatly reduced the colonization of pathogenic E. coli and Salmonella (Collado et al., 2007; Candela et al., 2008). Probiotics secrete some bacteriocin that can kill or inhibit pathogens (Nakamura et al., 2013; Pandey et al., 2013). Furthermore, Lactobacillus and Bifidobacterium can also create a biomembrane on intestinal mucosa to serve as a barrier against colonizing pathogen and improve gut immunity (Collado et al., 2008; Jones and Versalovic, 2009; Chen et al., 2014). Probiotics can also compete with harmful bacteria for nutrition (Elli et al., 2000; Hooper, 2009). Overall, our results demonstrated that L. plantarum G83 is beneficial to host health.
|
study
| 99.94 |
The probiotic L. plantarum G83 had a positive effect against pathogens mainly by chemical barrier and biological barrier, not only increasing the abundance of Lactobacillus and Bifidobacterium, but modulating immune response. In addition, different dose of L. plantarum G83 had various effects in modulating immune response. The results of this research may suggest that there is an opportunity to develop a new probiotic product for use in giant panda. And more research is needed for fully guarantee the safety and efficacy of this strain.
|
study
| 99.94 |
Designed the experiments: QL, XN, and ZP. Conceived and supervised the study: XN, QW, ZP, and LN. Performed the experiments: QL, YZ, and HS. Data analyzed by: QL, ZP, KP, and BJ. Wrote the manuscript: QL, ZP, and DZ. Proofread the manuscript: DZ and HW. All authors read and approved the final manuscript.
|
other
| 99.94 |
Glaucoma can result in irreversible vision loss. At this time, the only proven treatment is lowering intraocular pressure (IOP) . IOP is elevated in glaucoma due to increased resistance to fluid outflow from the eye . Methods for IOP lowering include topical medicines, lasers, and invasive surgeries. Today, a new category of glaucoma surgery, Minimally Invasive Glaucoma Surgeries (MIGS) , is of great interest. MIGS offer a small wound, safe, and fast surgical approach to enhance aqueous humor outflow (AHO) drainage. With some of the early MIGS procedures, there has been inconsistent success [7–10]. Therefore, the importance of furthering our understanding of how aqueous humor exits from the eye is of increasing interest. Using new and emerging technologies, AHO structure and function is being re-examined with a goal of improving current and enhancing future therapeutics.
|
review
| 99.9 |
To understand the full AHO outflow pathways, one has to understand how it was discovered. Historically, one of the earliest clues for AHO came from Lauber’s dogs. Taking blood from ciliary veins, it was noted that the ciliary vein blood hematocrit was less than the peripheral vein hematocrit . This resulted in the hypothesis that the eye was diluting the blood by exuding some fluid into the systemic circulation leading to the discovery of the trabecular/conventional outflow pathway. While today we know that two AHO pathways exist (trabecular/conventional and uveoscleral/unconventional) [12–15], the purpose of this review is to focus on the conventional AHO route and the role of MIGS.
|
review
| 99.9 |
To figure out the physical structures that carried aqueous, outflow pathway mapping was done via casting experiments. Polymerizing agents such as neoprene latex or vulcanizing silicone were injected retrogradely into distal outflow structures identified either by direct visualization or anterior filling with trypan blue . Enzymatic removal of surrounding ocular tissue resulted in beautiful branching three-dimensional casts denoting the potential AHO anatomy. These casts, in addition to histology (Figure 1), suggested that aqueous humor flowed from the anterior chamber, passed the trabecular meshwork (TM) into Schlemm’s Canal (SC) through collector channels, into an intrascleral venous plexus, and eventually to aqueous and episcleral veins . While fundamentally critical and illuminating regarding basic anatomy, casting studies were limited based on their static (non-real-time) assessments of AHO and concerns that results were non-physiologic because of high and “steady” pressure required to push polymerizing agents through the eye .
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study
| 99.94 |
To complement structural studies, seminal aqueous humor dynamic physiological experiments were conducted by Morton Grant and others . Using a basic aqueous humor perfusion rig, outflow facility was measured in both normal and glaucomatous enucleated human eyes before and after TM removal . We have re-organized Morton Grant’s results into one table based on outflow resistance to better summarize the results. First, the TM was the primary resistor for AHO (~70–75% incorporating data from Morton Grant and others ) (Table 1; blue arrow). Second, glaucoma eyes showed more AHO resistance compared to normal eyes (Table 1; green arrow). These two observations provided the basis for conventional AHO research and clinical thought in normal and glaucomatous eyes for the next half-century. Changes to extracellular matrix , cytoskeletal changes , secreted pro-fibrotic factors , theories of funneling , and TM biomechanics have all contributed to a model explaining impaired outflow at the level of the TM.
|
study
| 99.94 |
However, careful examination of the AHO resistance measurements resulted in two more observations. First, even after TM removal, some level of resistance was still recorded implying potential sources of resistance distal to the TM (Table 1; ”Resistance after Trabeculotomy” values). Second, glaucoma eyes showed greater post-TM resistance values (2.53 mm Hg × min/microliters) compared to normal eyes (1.26 mm Hg × min/microliters) (Table 1; red arrow) implying that the post-TM AHO regions were also effected in glaucoma. In fact, the increase in TM (Table 1; green arrow; 2.7 fold) and post-TM (Table 1; red arrow; 2 fold) resistance in glaucoma were nearly equivalent. This suggested that glaucoma was not only a TM outflow resistance problem but potentially a whole-eye disease where mechanisms to explain impaired outflow would have to explain both TM and post-TM changes.
|
study
| 100.0 |
Minimally invasive glaucoma surgeries (MIGS) and their results from the last decade have re-invigorated glaucoma anterior segment thought and research. Trabecular MIGS moved from basic goniotomies/trabeculotomies to using electrocautery or bypass shunts to overcome TM-related resistance by connecting the anterior chamber directly to Schlemm’s canal . While IOP lowering has occurred, inconsistent results have naturally led to further consideration of either post-TM resistance seen in the physiologic experiments above (Table 1; green and red arrows) or a re-evaluation of AHO patterns [7–10].
|
review
| 99.8 |
This review surveys AHO research with an emphasis on the entire conventional outflow pathway, trabecular and post-trabecular. Research will be roughly organized into balancing the study of AHO structure (anatomy) and function (physiology of fluid flow) (Table 2). Glaucoma clinicians are already accustomed to structural (optical coherence tomography) and functional (visual field) relationships in the posterior segment. Our goal is to delineate the relationship between structure and function for the anterior segment as well.
|
review
| 99.9 |
Today, understanding AHO structure is more sophisticated than traditional casting methods. Initially, pathological/histological and electron microscopic methods were used . TM, SC, and distal outflow structures were observed. Sampling of these structures across the eye revealed an overall impression of non-360 degrees uniform luminal sizes and presence . Electron microscopic results implied the presence of “plaque” material in glaucomatous eyes not found in normal eyes that may have been related to increased outflow resistance. Limitations of these approaches were associated with variability in tissue procurement, possible fixation artifacts, and sample preparations differences .
|
review
| 99.56 |
Recently, structural assessments, particularly of the TM, have occurred in a more native three-dimensional context with two-photon multimodal imaging. Two photon-microscopy (TPM) enabled high-resolution deep tissue optical sectioning in whole fixed or live tissue . Different modalities, such as autofluoresence and second harmonic generation demonstrated native trabecular anatomy and allowed for segregation of different extracellular matrix components such as collagen and elastin [31–33]. This type of tissue-based trabecular analyses has led to exploration of live cell dynamics and study of drug effects for IOP modulation. TPM of AHO anatomy, however, is currently not applied in living human individuals.
|
review
| 99.6 |
To evaluate post-TM AHO structures, multiple approaches have been used. Three-dimensional micro-computed tomography (3D micro-CT) in enucleated eyes identified AHO pathways as lumens of low radiographic signal . SC, collector channels, and large intrascleral vessels were successfully identified. Results 360 degrees around the limbus resembled AHO outflow casts with approximately 24–29 channels counted.
|
study
| 100.0 |
To study AHO structure in living subjects, anterior segment optical coherence tomography (OCT) has been indispensable. Like 3D micro-CT, AHO lumens appeared as regions of low reflectivity on OCT scans (Figure 1). Spectral domain OCT (SD-OCT) successfully identified the TM, SC, collectors channels and intrascleral venous plexus. Image processing allowed for creation of three-dimensional in silico casts of small segments of distal AHO pathways in normal individuals . Pilocarpine in live patients (Skaat et al. IOVS 2014; 55: ARVO Abstract 5682) and mice has been reported to increase the size of these AHO lumens while IOP elevation has been shown to do the opposite . Automated segmentation has been proposed (Murakami Y et al. IOVS 2014; 55: ARVO Abstract 927).
|
study
| 99.44 |
Phase-based OCT (ph-OCT) has shown pulse-dependent TM motion in enucleated non-human primate and live human eyes . In live human imaging, there was significant correlation with the digital/cardiac pulse with TM motion. This correlation allowed assessment of the phase lag and time delay between TM motion and the cardiac pulse where the digital pulse would occur prior to the TM motion. A significant linear relationship was also present between the TM phase lag and the heart rate.
|
study
| 100.0 |
While illuminating, current imaging-based structural analyses of AHO pathways were limited by understanding the relevance of the anatomy observed. It was unclear if large lumens necessarily represent increased or decreased AHO. The trabecular meshwork itself, oft described as a hypo-reflective interface shadow could connect to SC and be confusingly included as a portion of the AHO pathways . Structure-to-structure correlation of OCT to histology in the anterior segment has not yet reached the stage of what has been done in the posterior segment and retina [41–43]. Therefore, the biological relevance of the AHO pathway motion is unclear.
|
study
| 99.94 |
Since the structure of a physiologic system often controls and predicts its function, without an exact working relationship between the two, the meaning of structural variations can on its own be unclear. Thus, functional assessment sometimes takes the primary role in patient care especially because it is usually the function of the organ that is what's clinically relevant. Glaucoma physicians are accustomed to this for posterior segment evaluation. Visual fields are a functional assessment of the structure (retinal ganglion cells as assessed by histology or OCT). Visual fields and defects therein are what a glaucoma patient actually experiences, as opposed to the absolute number of ganglion cells or thickness of the retinal nerve fiber layer on an OCT scan. Thus for glaucoma diagnosis and management, functional assessment of vision with visual fields historically preceded that of more structural evaluations that eventually became prominent today. Recent improvements in posterior segment structure and function relationship was required for this to happen [41–43].
|
review
| 99.9 |
For AHO, functional assessment is equal to assessing the flow itself. This can be determined experimentally with outflow facility measurements in enucleated eyes or calculated from clinically measured variables with help from the Goldmann equation . Simultaneously, another functional assessment of AHO is to actually visualize where and how aqueous humor flows in the eye. This is important as, instead of considering the eye as one single unit of outflow, regional variations can be tested and visually appreciated. Imaging of AHO can be divided into non-real time and real time methods.
|
review
| 47.75 |
Non-real time methods to visualize AHO means adding a tracer to the eye, waiting a set period of time, and then capturing this information, typically in an enucleated eye after fixation for histological or whole mount study. Labeled tracers can include gold particles, fluorescent microspheres, or quantum dots (Qdots) among others.
|
other
| 99.9 |
Gold particles or cationic ferritin were initially used with electron microscopic analyses [44–46]. Segmental uptake of gold particles was seen at the cellular level for non-human primate inner-wall SC cells . Biological relevance was demonstrated through a conversion of segmental into a more homogenous pattern using a serine-threonine kinase inhibitor (H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine)) known to block actomyosin-driven contractility .
|
study
| 100.0 |
For a more global AHO assessment, larger and fluorescently tagged tracers were delivered into either live or enucleated eyes in several species. Fluorescent microspheres ranged in size from 0.2 to 20 microns [22,44,47–51]. Alternatively, 0.01 micron quantum dots were used . Imaging was done with laboratory fluorescent or confocal microscopes. Segmental patterns were seen, and combining tracer studies with histology, estimates of TM utilization could be calculated from a percent effective filtration length . Two color tracer studies allowed for demonstrating reduced AHO with increasing IOP . Tracer collection in the TM localized near collector channels in enucleated human eyes .
|
study
| 99.9 |
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