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To assess the role of the tested miRNAs in the regulation of NKG2DL expression, BCap37 cells were transfected with control miRNAs (Ctrl), mimics or inhibitors of the tested miRNAs (Supplementary Figure 2a). Mimics of miR-20a, miR-20b, miR-93 and miR-106b decreased MICA/B and ULBP2 protein expression, whereas inhibitors of the tested miRNAs increased their expression in BCap37 cells (Figure 2e and f). However, the tested miRNAs showed limited influence on the expression of ULBP1/3. Among the four tested miRNAs, miR-20a showed the strongest influence on NKG2DL regulation in BCap37 cells (rMFI, mean±S.D., mimic versus Ctrl 47.2±5.7%, inhibitor versus Ctrl 202.0±10.1% for MICA/B, mimic versus Ctrl 76.1±2.3%, inhibitor versus Ctrl 136.4±3.3% for ULBP2, Figure 2f). In addition, these miRNA-mediated downregulations of NKG2DLs were typically associated with a decrease in related mRNA transcripts (Figure 2g). This finding indicated that the miRNA-mediated downregulation of NKG2DL expression was partially caused by enhancing degradation of related mRNA transcripts. We confirmed these results by assessing the effects of miR-20a and miR-93 on the BC cell line MDA-MB-231 and the normal breast cell line HBL-100 (Supplementary Figure 2b and c). Taken together, the tested miRNAs specifically downregulated MICA/B and ULBP2 expression in both BC and normal breast cell lines.
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| 100.0 |
We next confirmed the specific interaction between the tested miRNAs and the mRNA of MICA/B. All tested miRNA have the same predicted binding sites in MICA or MICB mRNA. We used miR-20a and miR-20b as models. We generated four firefly luciferase reporter vectors: two containing the wild-type MICA or MICB 3'-UTR and the other two containing the MICA or MICB 3'-UTR with a mutated binding site (Figure 3a). Transfecting BCap37 cells with wild-type MICA or MICB in combination with the miR-20a or miR-20b mimic revealed a clear downregulation of luciferase activity compared with the control miRNA group (Ctrl). In contrast, the miR-20a or miR-20b mimic did not effectively reduce the luciferase activity of the MICA or MICB construct with a mutated binding site (Figure 3b). These results indicated that miR-20a and miR-20b directly targeted MICA/B mRNA at the predicted 3'-UTR-binding sites.
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study
| 100.0 |
We found that ULBP2 had no predicted binding sites but was still effectively regulated by the tested miRNAs in different breast cell lines (Figures 2e–g and Supplementary Figure 2b and c). Both ERK and AKT are known to be involved in the regulation of ULBP2 expression.24, 25, 26 In addition, members of the miR-17-92 cluster target and inhibit the MAPK/ERK signaling pathway,27, 28 and miR-20a directly targets MAPK1 (ERK2) and inhibits its expression in BC cells (Wengong Si et al. unpublished data). For these reasons, the expression and activation status of the MAPK/ERK pathway were studied in untreated and miRNA-treated BCap37 cells. A western blotting assay showed that compared with the control treatment, the miR-20a mimic effectively inhibited the expression of p-ERK1/2 (mainly p-ERK2) and total ERK1/2. Moreover, the miR-20a inhibitor enhanced the expression of p-ERK1/2 and total ERK1/2, whereas neither the miR-20a mimic nor inhibitor affected AKT and p-AKT expression (Figure 3c). This miRNA-mediated downregulation of ERK2 was typically associated with a decrease in ERK2 (MAPK1) mRNA transcripts (Figure 3d). We confirmed these results by assessing the effects of miR-20b, miR-93 and miR-106b on ERK2 protein and mRNA expression in BCap37 cells. (Supplementary Figure d and e). To clarify the role of the MAPK/ERK signaling pathway in ULBP2 regulation, we silenced ERK2 expression by siRNA or increased constitutive ERK2 expression with ERK2-pcDNA transfection in BCap37 cells (Figure 3f). Flow cytometry analysis showed that the inhibition of ERK2 effectively downregulated the expression of ULBP2 but not MICA/B, whereas the overexpression of ERK2 increased the ULBP2 expression (Figure 3g). Moreover, ERK2 overexpression could reverse miR-20a-mediated ULBP2 downregulation in BCap37 cells (Figure 3e). These findings suggest that downregulated ULBP2 expression in miRNA-treated BC cells results from the MAPK/ERK signaling pathway inhibition.
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study
| 100.0 |
We next examined the effect of miRNA-mediated NKG2DL downregulation on NK cell recognition. BCap37 cells were transfected with control miRNA (Ctrl) or a miR-20a mimic or inhibitor. NK cells separated from the buffy coat of healthy donors were used in cytotoxicity assays at various effector-to-target (E:T) ratios. Transfection with a miR-20a mimic caused a remarkable reduction in the cytotoxicity activity at different E:T ratios. However, treatment with miR-20a inhibitor could effectively reverse these inhibitory effects (Figure 4a). These results implied that silencing NKG2DL-targeting miRNA could enhance NK cell-mediated cytotoxicity. Importantly, these differences between various treatments were abolished after NKG2D in NK cells was blocked with an anti-NKG2D monoclonal antibody (mAb) (Figure 4c). These results confirmed that the miRNA-mediated NK cell cytotoxicity was mediated through the NKG2D–NKG2DL pathway. The results were then verified using another human BC cell line (MDA-MB-231) as a target cell (Figure 4b) or LAK cells expanded from healthy donors as an effector cell (Supplementary Figure 2f).
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study
| 100.0 |
To further investigate the effects of NKG2DL-targeting miRNAs on BC immunogenicity in vivo, we conducted a short-term lung clearance assay using C57BL/6 mice (Figure 5a). Although human NKG2DLs are different from mouse NKG2DLs,29 it is feasible to detect the activity of NKG2D-associated functions on human malignant cells in an immunocompetent mouse model.30 BCap37 cells treated with miR-20a mimic (which decreased NKG2DLs expression) were less susceptible to immune cell targeting. Thus, fewer cells were killed and more cells were detected in the harvested lungs than in those from the group transfected with control miRNA (Ctrl group) (fluorescence intensity (FI), mean±S.D. 143.1±10.53%). On the other hand, when NKG2DLs were increased with a miR-20a inhibitor, fewer labeled BCap37 cells were detected in the lungs compared with the Ctrl group (33.7±6.2%, Figure 5b top and 5c left). These results were confirmed using another BC cell line (MDA-MB-231) (mimic versus Ctrl 216.9±18.9%, inhibitor versus Ctrl 58.2±5.5%, Figure 5b bottom and 5c right). Importantly, the differences between different groups were abolished when NKG2Ds were blocked in vivo with an anti-NKG2D mAb in the BC cells (Figure 5d). The differences were reduced when NK cells were depleted with an anti-NK1.1 mAb in the BC cells. Interestingly, in the anti-NK1.1 group, BCap37 cells showed an increase of detection after treated with a miR-20a inhibitor, which required further observation. Taken together, these results confirmed that the miRNA-mediated regulation of immune recognition was through the NKG2D–NKG2DL pathway.
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study
| 100.0 |
The half maximal inhibitory concentrations (IC50) values of 48 h HDACis exposure in the tested cells were shown in Supplementary Figure 3a. To explore the impact of HDACis on BC cell immunogenicity, we exposed BCap37 cells to different concentrations of SAHA or VPA. HDACis dose-dependently increased MICA/B and ULBP2 expression (Figures 6a and b), whereas ULBP1/3 was not affected (data not shown). SAHA and VPA effectively increased MICA/B and ULBP2 expression, even at concentrations lower than 1/10 of the IC50 (100 nM for SAHA and 400 μM for VPA), relative to control treatment (rMFI, mean±S.D. SAHA: 249.6±32.7% for MICA/B and 113.4±4.1% for ULBP2; VPA: 247.7±25.5% for MICA/B and 180.5±8.7% for ULBP2, Figure 6b). The results were then verified using another human BC cell line (MDA-MB-231) (Supplementary Figure 3b). Interestingly, the MICA/B and ULBP2 expression levels of the human normal breast cell line HBL-100 could not be effectively increased by HDACis, even at a concentration as high as the IC50 (rMFI, SAHA: 128.2±14.2% for MICA/B and 113.9±9.3% for ULBP2; VPA: 127.6±8.3% for MICA/B and 113.9±9.4% for ULBP2, Figure 6c). We found that this upregulation of NKG2DL expression was accompanied by the decreased expression of pri-miR-17-92, miR-20a, miR-20b, miR-93 and miR-106b in a dose-dependent manner (Figure 6d). The result indicated that HDACis might elevate the NKG2DLs expression in BC cell lines by inhibiting NKG2DL-targeting miRNAs. Moreover, a miR-20a mimic reversed HDACi-mediated MICA/B and ULBP2 upregulation in BCap37 cells (Figure 6e). To evaluate the effects of HDACi-mediated NKG2DL upregulation on NK cell recognition, we treated BCap37 or MDA-MB-231 cells with low concentrations of SAHA (50 nM or 100 nM) or VPA (200 μM or 400 μM) (Supplementary Figure 3a, c and d). Cells treated with HDACi became more sensitive to NK cell-specific cytotoxicity than cells cultured under standard conditions. In addition, these upregulation effects were reversed when cells were exposed to HDACis together with miR-20a mimics (Figure 6f left and Supplementary Figure 3e). Importantly, these differences between various treatments were abolished after NKG2Ds in NK cells was blocked with an anti-NKG2D mAb (Figure 6f right). Thus, by increasing NKG2DLs expression on BC cells, low concentrations of HDACis sensitized BC cells to NK cell-mediated cytotoxicity.
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study
| 100.0 |
Previous studies have revealed that the immune system influences tumor development and clinical outcomes. NKG2DLs have an important role in cancer immunosurveillance and have clinicopathological significance.31, 32, 33, 34, 35 In BC, however, conflicting results have been reported. Some researchers implied that high expression of MICA/B resulted in a favorable outcome concerning the relapse-free period in early BC patients.35 However, higher MICA expression was found as an indicator of poor prognosis in BC.36 In addition, most previous studies were focused on the association of total MICA/B or MICA expression with clinical outcomes, whereas few considered the clinicopathological significance of MICB. In this study, we confirmed that MICA/B expression levels in BC tissues were inversely associated with TNM stage (Figures 1a–c). Importantly, we found that patients with high MICB expression showed longer overall survival than patients with low MICB expression. However, the different expression levels of MICA showed no significant influence on the survival of BC patients (Figures 1d and e). Together, these results suggest that MICB may act as a predictive factor in BC.
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study
| 99.94 |
Members of the miR-17-92 cluster promote BC cell growth, migration and invasion.37, 38 Recently, miR-17-92 members were also reported to downregulate MICA/B protein expression in ovarian tumors,23 glioma17 and other tumors,16 contributing to their immune escape. In our study, we provided new evidence that members of the miR-17-92 cluster and its paralogs miR-20a, miR-20b, miR-93 and miR-106b specifically downregulated not only MICA/B but also ULBP2 expression in BC and normal breast cell lines (Figure 2). Importantly, in BC cells, miR-20b is newly reported in NKG2DL regulation. Unlike other studies,17 we found that this miRNA-mediated downregulation of MICA/B and ULBP2 was partly due to the enhanced degradation of related mRNA transcripts. The 3'-UTRs of MICA and MICB contain binding sites for the tested miRNAs, which was verified in BC cells using a dual-luciferase reporter gene assay (Figures 3a and b). Interestingly, we first found that ULBP2 had no predicted binding sites for the tested miRNAs but could still be effectively regulated in BC cells. This miRNA-mediated ULBP2 inhibition may result in miRNA-induced MAPK/ERK signaling pathway downregulation (Figures 3c–g and Supplementary Figure 2d and e). Members of the miR-17-92 cluster were previously reported to downregulate the expression of the MAPK/ERK signaling pathway.27, 28 In this study, western blot and quantitative PCR showed that the protein and mRNA levels of ERK2 (MAPK1) were inversely regulated by miR-20a, miR-20b, miR-93 and miR-106b. The MAPK/ERK pathway was reported to influence NKG2DLs expression in some other tumors.39, 40 For instance, the overexpression of constitutively active ERK (especially ERK1) in ARK cells was found increased MICA/B and ULBP2 expression.25 We found that ERK2 inhibition effectively downregulated the expression of ULBP2 but not MICA/B, whereas ERK2 overexpression increased the ULBP2 expression (Figures 3f and g). Our study further demonstrated the functional importance of miRNA-dependent regulation of NKG2DLs in BC cells using NK cell cytotoxicity assays (Figure 4). A short-term lung clearance model confirmed that silencing NKG2DL-targeting miRNA contributed to immune recognition in vivo (Figure 5). These findings emphasize the functional significance of the tested miRNAs in MICA/B and ULBP2 regulation.
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study
| 99.94 |
However, miRNA candidates are based on modified nucleic acids and therefore still present delivery challenges in clinical applications.41 In contrast, SAHA and VPA are HDACis that have already been used in clinical scenarios. The present results revealed that treating BC cells with HDACis effectively sensitized tumor cells to NK cell recognition (Figure 6f). HDACis increase the expression of NKG2DLs in myeloma25 and colon cancer cells.42 In this study, we demonstrated that in BC cells, HDACis upregulated the expression of not only MICA/B but also ULBP2. This upregulation of NKG2DLs is typically accompanied by a decrease in the expression of miR-17-92 members (Figure 6d). The HDACi-induced overexpression of MICA/B and ULBP2 was reversed with a miR-20a mimic (Figure 6e). Previous study reported that HDACis butyrate, SAHA and VPA inhibited miR-17-92 transcription by reducing c-Myc expression.42 However, H Yang et al.43 proposed that SAHA downregulated the miR-17-92 cluster by abolishing tyrosine phosphorylation of STAT3 and decreased MCM7 transcription. Thus, the mechanisms for how HDACis downregulate the expression of miR-17-92 require further observation. In addition, we found that SAHA and VPA did not effectively elevate the NKG2DL expression of the normal breast cell line HBL-100 (Figure 6c). The basis for normal breast cell resistance to HDACis is not fully understood, although cancer cells with multiple defects cannot reverse the critical effects of HDACis similar to normal cells.44, 45 This resistance of normal breast cells presumably functions to avoid the autoimmunity caused by treatment with HDACis. Taken together, our study indicates that BC patients who receive HDACi treatment might show increased immune activation in addition to tumor cell inhibition.
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study
| 99.94 |
The present study reports the following novel findings: (1) high MICB expression may be an indicator of a good prognosis in BC; (2) members of the miR-17-92 cluster inversely regulate ULBP2 and MICA/B in BC cells, which influences the immunogenicity of BC; (3) miRNAs downregulate the expression of MICA/B by targeting the mRNA 3'-UTR and downregulate ULBP2 by inhibiting the MAPK/ERK signaling pathway; (4) the HDACis SAHA and VPA may increase the expression of MICA/B and ULBP2 by inhibiting the miR-17-92 cluster; and (5) SAHA and VPA cannot effectively increase the expression of NKG2DLs in normal breast cells. In conclusion, this study is the first to comprehensively assess the miRNA-mediated NKG2DL regulation in the context of BC (Figure 6g). Our results indicate that targeting miRNAs with antisense inhibitors or by HDACis might therefore represent a novel approach to increasing the immunogenicity of BC.
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study
| 99.94 |
Male C57BL/6 (8–9 weeks old) mice were purchased from Vital River (Beijing, China). The human breast cell lines (BCap37, Hs 578T, MDA-MB-231, MDA-MB-468, BT-474, SK-BR-3, MCF-7, Hs 578Bst and HBL-100) were purchased from the cell bank of the Chinese Scientific Academy. BCap37, BT-474 and MCF-7 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, 31800105, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Biological Industries, 04-0101-1, Cromwell, CT, USA). MDA-MB-231 and MDA-MB-468 cells were cultured in Leibovitz's L-15 medium (Gibco, 11415114) with 10% FBS. Hs 578T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, 12430047) supplemented with 0.01 mg/ml bovine insulin (Solarbio, I8040, Beijing, China) and 10% FBS. SK-BR-3 cells were cultured in McCoy's 5A medium (modified, Gibco, 16600082) with 10% FBS. Hs 578Bst cells were cultured in DMEM with 50 ng/ml epidermal growth factor (Gibco, PHG0311) and 15% FBS. HBL-100 cells were cultured in DMEM with 10% FBS. The cell culture medium was changed every 2–3 days, and the cells were passaged with 0.25% trypsin-EDTA (Gibco, 25200056) and grown to 90% confluence. The cultures were kept at 37 °C with 5% CO2 in a water-jacketed incubator (Thermo Scientific, Waltham, MA, USA).
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study
| 99.94 |
TCGA is available from the website of the Cancer Genomics Browser of the University of California, Santa Cruz (https://genome-cancer.ucsc.edu/). MICA and MICB mRNA expression and miRNA-sequencing level 3 data in BC patients were extracted from TCGA's data portal. Only patients with fully characterized tumors, TNM stage, overall survival, complete miRNA information were included. In total, data on 1096 BC patients with detailed miR-20a, MICA and MICB expression were collected from TCGA's data portal. Read counts for both mRNAs and miRNAs were used as input for survival analysis with the R package edgeR.46, 47 Kaplan–Meier plots for MICA or MICB expression in association with overall survival were calculated with the R program. Patients were split into high and low expression groups based on the median expression of MICA or MICB. For correlation analysis, the patients with values that were too low (<100) or too high (≥4000) for MICA/B mRNA or miR-20a expression were excluded (N=1042).
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study
| 100.0 |
Dual-luciferase assays were performed using 1 × 104 BCap37 cells per well in a 96-well plate (Corning/Costar, 3499, Acton, MA, USA). After the cells attached for 8 h, they were co-transfected with 50 ng of respective reporter constructs with either 50 nM of miRNA mimics or control miRNA. After 48 h, a Reporter Assay System Kit (Promega, 017319, Beijing, China) was used to measure the luciferase activity. There were three replicates for each transfectant. Firefly luciferase activity was normalized to constitutive Renilla luciferase activity.
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study
| 100.0 |
Respective sensitivity of BC cells to NK cells was determined using the CytoTox 96 cytotoxicity assay kit (Promega, 017317) according to the manufacturer's instructions. In brief, 1 × 104 BCap37 or MDA-MB-231 target cells were seeded in triplicate at NK cell-to-target cell (E:T) ratios of 10:1, 5:1 and 2.5:1. An anti-NKG2D Ab (50 mg/ml, Novus, Littleton, USA, clone 149810) or control mAb (50 mg/ml, Novus) was added to experiments 1 h before co-culture. After 4 h of incubation, the supernatant was removed for analysis. The following formulas were used to calculate spontaneous and specific cytotoxicity: % specific release=(experimental release−effector spontaneous release−target spontaneous release)/(target maximum release−target spontaneous release) × 100%.
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study
| 100.0 |
A short-term lung clearance assay was performed as previously described,23, 30 with some modifications to evaluate the NKG2DL-mediated immune effect in vivo. HeLa cells, which were not effectively recognized and killed by mouse NK cells, were used as internal negative controls. In brief, 36 male C57BL/6 mice were divided into three groups and were intraperitoneally injected with an IgG isotype control (300 μg per mouse; Novus, Clone 20116, MAB004), an anti-mouse NKG2D mAb (300 μg per mouse; Novus, Clone 191004, MAB1547) or an anti-NK1.1 Ab (300 μg per mouse; Biolegend, Clone PK136, 108712, San Diego, CA, USA), respectively. Twenty-four hours later, HeLa cells were labeled with PKH26 (Invitrogen, MINI26-KT, Carlsbad, CA, USA), and various BCap37 cells (transfected with miR-20a mimic, miR-20a inhibitor, or control miRNAs) were labeled with CFSE (Invitrogen, C34554). Then, the stained BCap37 cells were mixed with the stained HeLa cells of each population at a density of 5 × 106 cells in 1 ml PBS. A 0.4-ml aliquot of the cells was injected into the tail vein. Five hours later, the lungs were harvested to prepare single-cell suspensions, and flow cytometry analysis was performed. The relative ratio of tested cells to HeLa cells was calculated as the FI ratio of the tested cells to HeLa cells. Another human BC cell line (MDA-MB-231) was treated and analyzed as BCap37 cells.
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study
| 100.0 |
Detailed methods are described in the Supplementary Materials, including immunohistochemical staining (IHC), transient transfection and drug treatment, clinical sample collection and NK cell isolation, RNA extraction and real-time quantitative PCR analysis, ligand expression analysis, Annexin V and propidium iodide staining analysis, flow cytometry analysis of cell cycle, western blotting assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, vector constructs, statistical analysis and ethics statement.
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other
| 99.9 |
Despite the abundance of studies on unplanned pregnancy and the fact that the high incidence remains a major public health concern worldwide , the dominant focus tends to be on the vulnerabilities of young women of low socio-economic status and those with little or no formal education [2, 3]. Even so, the association between perceived unplanned pregnancy-risks and the use of emergency contraception remains unclear, as studies on this subject tend to focus mainly on EC effectiveness as well as knowledge, attitude and practices of EC. This article draws on the findings of a survey conducted in two Nigerian universities to shed light on the sexual behaviour of female undergraduate students and the dynamics of emergency contraceptive use among this cohort of students.
|
study
| 99.7 |
In 2012, approximately 213 million pregnancies occurred worldwide; over 40% of which were unplanned . Studies have also shown that one of the major reasons women seek abortion is to deal with the problem of unplanned pregnancy, 50% of them seek unsafe methods to get rid of the pregnancies . Globally, an estimated 22 million unsafe abortions occur each year, and nearly all of these occurring in developing countries . In Africa, 50% of all abortion-related mortality occur among the youth .
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other
| 99.8 |
Unintended pregnancies and unsafe abortion are among the reproductive health challenges women face in Sub-Saharan Africa [3, 6–9]. Despite the numerous intervention programmes to create awareness and encourage women in Sub-Saharan Africa to make use of contraceptives, the level of use of both traditional and modern methods remains low (29%) compared to countries like Norway, United Kingdom and Malta, which have the highest rates (above 80%) of contraceptive usage . Even though there are variations in contraceptive use by age, socioeconomic status and location, available evidence suggests that contraceptive practice remains low in Sub-Saharan Africa irrespective of the context . The consensus on the underlying reasons why the practice of contraception remains low in sub-Saharan Africa are health risks/side effects, opposition by the woman and/or partner, lack of resources, lack of awareness, lack of personal vulnerability and gender power issues (dislike of condoms) .
|
review
| 99.8 |
Emergency contraception is a method that can prevent pregnancy if used correctly a few days after unprotected sexual intercourse or due to contraceptive failure [12–16]. However, EC has not been demonstrated to lead to a population-level reduction in unintended pregnancy and induced abortion [12, 14]. This is primarily because the incidence of unprotected sexual intercourse is very high. Emergency Contraceptive Pills (ECPs) are only moderately effective, and ECPs are often not used . Increased access to emergency contraception has been reported to enhance usage but has not been shown to reduce unintended pregnancy rates . Over-the-counter access and advance supply of EC indeed make access easier for women who wish to use EC . Nevertheless, the use of EC remains considerably low in both less developed and more developed countries partly because many women are yet to embrace EC, misinformation about EC and negative perception of EC side effects [19–22].
|
review
| 99.9 |
There is little information in the literature about just how knowledgeable young educated women are vis-à-vis the use of EC, and above all, what EC methods they use, if at all they do. Besides, because of their privileged position in the social structure (that is, being educated), the impression created by the dominant discourse is that they would not seek recourse to unproven and unsafe contraceptive methods. The present article seeks to fill these knowledge gaps by focusing on middle-class female youths, specifically university students.
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other
| 99.7 |
A cross-sectional study was conducted among unmarried female students in Ekiti state University (government owned) and Afe Babalola University in Ekiti State (privately owned), South Western Nigeria, between February and May 2012. The total population of female students in Ekiti State University and Afe Babalola University was 5840 and 880 respectively. The population of female students in Afe Babalola University was equivalent to 15% of the population of female students in Ekiti State University, hence, 15% of the sample size (63 respondents) were drawn from Afe Babalola University and the remaining 357 participants were selected from Ekiti State University.
|
study
| 100.0 |
For inclusiveness, respondents were stratified into year of study and faculty of study. A random sample of eligible participants corresponding to the sizes of the strata was recruited. However, the data analysis was based on a sample size of 370, as 50 questionnaires were returned with incomplete responses.
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study
| 99.94 |
The University of Ibadan Social Science and Humanities ethical committee approved the study protocol. The inclusion of participants was voluntary and informed consent was obtained from each participant. Participants were guaranteed confidentiality and anonymity.
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other
| 99.94 |
A semi-structured questionnaire was piloted with 20 participants, who were not included in the main study. Feedback from the participants was used to improve the questionnaire. The questionnaire consisted of two parts. Close-ended and open-ended questions allowed the capturing of structured responses and the probing and capturing of narratives about specific sexual behaviour. Data on socio-demographic variables such as age, type of home and school residence, religious practices, year and faculty of study, and ethnicity were obtained. Questions probing sexual activity focused on recent use of contraceptives.
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study
| 100.0 |
A number of questionnaire items probed risk perceptions relating to unplanned pregnancy, actual and/or preferred actions in the event of unplanned pregnancy, awareness about emergency contraception, and knowledge of the efficacy of (and health risks associated with) various clinical and local EC methods, as well as actual use of EC methods.
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study
| 52.1 |
Data were coded and entered into Statistical Package for Social Sciences (SPSS version 19, Chicago, IL, USA). Frequency and proportions were reported for categorical variables. Bivariate analysis (Chi Square test) was applied to examine the association between the use of EC and age, year of study, risk of unplanned pregnancy, knowledge of timing of use of EC, and perceived side effects. A p-value of 0.05 was considered as statistically significant.
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study
| 99.94 |
The socio-demographic characteristics of the participants are presented in Table 1. Ninety-two percent of the respondents were aged 24 years and below; 71% reside off campus.Table 1Demographic characteristics of the participantsCharacteristicsFrequencyPercentAge ≤ 199425.4 20–2318048.6 ≥ 245715.4Faculty Social science6918.6 Arts & humanities5214.1 Engineering225.9 Management Sciences6818.4 Education6517.6 Law236.2 Science4311.2 Agricultural Sciences287.6Year of study First6818.4 Second7720.8 Third9826.5 Fourth9926.8 Fifth287.6Place of residence in school University residence10829.2 Off Campus26270.8Home residence Rural11230.3 Urban25869.7Ethnic Group Yoruba32186.8 Igbo349.2 Hausa/Fulani61.6 Edo/ijaw92.4
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study
| 99.94 |
About half of the respondents (47.6%) engaged in sexual intercourse in the year preceding the survey, even though over 10% of the respondents did not respond to this question. Of the 176 respondents that had engaged in sexual intercourse in the year preceding the study, 38.6% reported the use of condom, 26.1% after sex contraception, the remaining did not use any form of contraception. Inferring from the result, there is high rate of unprotected sexual intercourse (61.4%) among the participants. Likewise, there is increasing trend of sexual activity with increasing age and year of study (Figs. 1 and 2).Fig. 1Relationship of sexual activity by age of students (p = 0.001) Fig. 2Relationship of sexual activity by Students’ year of study (p = 0.001)
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study
| 100.0 |
When asked if they are aware of any methods of preventing pregnancy after sex, the majority (63.1%) of the respondents stated that they were aware of drugs that could prevent pregnancy after unprotected sexual intercourse. However, when asked to name the methods of preventing pregnancy after sex, 22.6% of those who claimed they knew of a method of preventing pregnancy after named incorrect methods. Other non-EC drugs reported by the participants include: menstrogen, gynacocied, antibiotics, cytotec, Andrews liver salt, MNB 760, Alabukun, salt and water, alcohol, lime, potash, and yoyo bitters. The majority of the respondents indicated knowledge of more than one method. Younger students were more likely to consider non-EC pills as EC methods.
|
study
| 99.94 |
Pearson chi-square statistics was used to examine the relationship between demographic variables and awareness of EC. The results show that age and level of study were significantly associated with awareness of EC among the respondents (p < 0.026). The level of awareness of EC increases with increase in level of study and age of the respondents (Table 2). The level of awareness varies from 47% in first year to 74.1% in the fifth year. Older students (87.5%) were more likely to be aware of EC compared with younger students (48.4%).Table 2Bivariate analysis showing awareness of emergency contraception by background characteristicsVariablesEver heard of pills that can prevent pregnancy after sex P-valueName the pills P-valuePostinorNon EC pillsI cannot rememberAll214 (63.1)158 (77.5)35 (17.2)11 (5.4)Age ≤ 1947 (49.5)0.0035 (74.5)10 (21.3)2 (4.3)0.10 20–23123 (63.4)84 (73.7)22 (19.3)8 (7.0) ≥ 2443 (89.6)39 (92.9)3 (7.1)0 (0.0)Year of study First31 (47.0)0.0418 (60.0)10 (33.3)2 (6.7)0.12 Second47 (64.4)37 (82.2)5 (11.1)3 (6.7) Third57 (65.5)47 (85.5)6 (10.9)2 (3.6) Fourth59 (68.6)44 (80.0)9 (16.4)2 (3.6) Fifth20 (74.1)12 (63.2)5 (26.3)2 (10.5)Place of residence in school University residence58 (58.6)0.1639 (69.6)11 (19.6)6 (10.7)0.09 Off campus residence156 (65.0)119 (80.4)24 (16.2)5 (3.4)Place of residence outside school Rural62 (62.0)0.4441 (71.9)13 (22.8)3 (5.3)0.41 Urban152 (63.6)117 (79.6)22 (15.0)8 (5.4)
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study
| 100.0 |
Table 3 shows that the majority of the respondents believed that EC should be taken within 24 h after sexual intercourse. Only 5% reported that the EC pills could still be used up to three days after sexual intercourse. Accurate knowledge about timing of EC use increases with increase in age and year of study. Knowledge of side effects (Table 3) showed that fewer respondents (28%) were familiar with the World Health Organization (WHO) documented side effects of EC, which include; menstrual irregularity, nausea, bleeding, and body pain. Perceived risks associated with the use of EC as reported by the participants include; irreparable damage to the womb leading to infertility (53%). Knowledge of EC side effects slightly increases with age and year of study. Contrastingly, perception of EC’s side effects improves with increasing age and year of study. About 18% of them did not know the side effects of EC.Table 3Bivariate analysis indicating perception of emergency contraception timing and side effectsQuestions and responsesAllAge P-value≤1920–23≥24Timing of use Up to 24 h of sex140 (77.8)28 (70.0)79 (79.0)33 (82.5)0.81 72 h after sex9 (5.0)4 (10.0)3 (3.0)2 (5.0) Before Sex10 (5.6)3 (7.5)5 (5.0)2 (5.0) A week after sex9 (5.0)2 (5.0)6 (6.0)1 (2.5) Don’t Know12 (6.7)3 (7.5)7 (7.0)2 (5.0)Perception of Side Effects WHO proven side effects of ECa 50 (27.9)10 (22.7)29 (29.6)11 (30.6)0.85 Unproven side effects of ECb 95 (53.1)25 (56.8)50 (51.0)19 (52.8) Don’t Know32 (17.9)9 (20.5)17 (17.3)6 (16.7) No side effect2 (1.1)0 (0.0)2 (2.0)0 (0.0) aNausea, Irregular menstrual cycle, Bleeding bDamage to the womb, Future infertility
|
study
| 99.94 |
Many of the young women expressed fears regarding the use of pills to prevent pregnancy after sex. A recurring theme in their responses was that ECPs could have consequences on women’s fertility. These sentiments were echoed by a number of respondents:It can cause infertility in the future (18-year-old first year student) It can damage the womb (19-year-old second year)
|
other
| 99.9 |
This result is in sharp contrast to the correlation result that shows that perceived side effects of EC (P > 0.05) was not significantly correlated with the use of EC. Although some respondents who believe EC could negatively impact women’s fertility do not use EC, the cross tabulation shows that some of them actually do use EC. Their narratives reveal an interesting pattern about their perceptions of the side effects of emergency contraception. To some, it is only ‘too much use’ of EC that could damage the womb, although what they regard as ‘too much’ could vary. Going by their narratives, there is a clear indication that some would use EC but avoid ‘too much’ use. A fifth-year agricultural science student at the Ekiti state university explained how this could happen:Too much of postinor2 (Levonorgestrel) will weaken the wall of the womb and damage the uterus. This will make someone experience miscarriage in the future (24-year-old student)
|
study
| 99.56 |
The overall level of use of after sex contraception among the participants that responded (n = 330) is 27.4%. Forty participants did not respond to this question. However, when asked to specify the pills used in preventing unplanned pregnancy, only 17.6% (65) reported having used Levonorgestrel (postinor), while 6.8% (n = 25) had used non-EC pills such as menstrogen, gynacocied and Cytotec. Of the participants that engaged in sexual intercourse (n = 176) in the year preceding the survey, only 36.9% had ever used Levonorgestrel. The proportion is slightly higher for those that reported non-use of condom or unprotected sex (40%) in the most recent sexual encounter. The proportion of EC users increased with age and year of study. The use of non-EC appears to be more common among younger students.
|
study
| 99.94 |
Pearson chi-square statistics was used to examine the relationship between demographic variables and use of EC. The results show that awareness of EC (p < 0.001), knowledge of timing (p < 0.001), perceived risk of unplanned pregnancy (p < 0.001), and level of study (p < 0.001), were significantly associated with the use of EC (Table 4). Perceived side effect of EC and age (P > 0.05) were not significantly associated with the use of EC.Table 4Pearson chi-square results showing factors associated with the use of emergency contraceptionVariablesEver used pills after sex to prevent unplanned pregnancy P-valueYesNoAll90 (27.4)238 (72.6)Age ≤ 1918 (20.5)70 (79.5)0.13 20–2354 (28.4)136 (71.6) ≥ 2418 (36.0)32 (64.0)Year of study First7 (11.3)55 (88.7)0.03 Second19 (27.9)49 (72.1) Third30 (33.3)60 (66.7) Fourth26 (30.6)59 (69.4) Fifth8 (32.0)17 (68.0)Place of residence in school University residence21 (21.2)78 (78.8)0.08 Oppidans69 (29.9)162 (70.1)Timing of use Up to 24 h of sex66 (47.8)72 (52.2)0.10 72 h after sex3 (50.0)3 (50.0) Before Sex1 (10.0)9 (90.0) A week after sex2 (22.2)7 (77.8) Don’t Know1 (8.3)11 (91.7)Perception of Side Effects WHO proven side effects of ECa 23 (46.0)27 (54.0)0.52 Unproven side effects of ECb 42 (45.2)51 (54.8) Don’t Know12 (37.5)20 (62.5) No side effect0 (0.0)2 (100.0)Name of pills ever used Postinor62 (48.7)78 (51.3)0.001 Non EC pills9 (25.7)26 (74.3) Cannot remember0 (0.0)11 (100.0)Aware of EC86 (42.0)119 (58.0)0.00Perceived pregnancy risk55 (61.1)35 (38.9)0.00Ever engaged in sexual intercourse90 (51.1)82 (48.9)0.00 aNausea, Irregular menstrual cycle, Bleeding bDamage to the womb, Future infertility
|
study
| 100.0 |
Of the 94 respondents with perceived risk of unplanned pregnancy in the past year, 28 used Levonorgestrel, 19 use non-EC pills, and the remaining did not use any EC. However, three of them got pregnant and had abortion. Pearson chi-square was used to examine the relationship between action taken to combat perceived risk of unplanned pregnancy and demographic characteristics. The results show that the risk of unplanned pregnancy increases with increasing age and year of study, likewise, the use of EC pills (Table 5). Older female students were more likely to seek abortion. The use of non-EC pills was not significantly influenced by age and year of study.Table 5Bivariate analysis showing unplanned pregnancy risk and action taken by age and level of StudyQuestions and responsesLevel of study (in years)Age1 n = 662 n = 713 n = 864 n = 875 n = 22≤19 n = 9420–23 n = 187≥24 n = 49Ever at risk of unplanned pregnancy in the last one year13(19.7)18(25.4)24(29.1)28(32.2)10(45.5)20(21.3)54(28.9)20(40.8)Action Taken Use Levonorgestrel3(25.0)6(33.3)10(40.0)5(17.9)4(36.4)5(26.3)18(33.3)5(23.8) Use other non-EC Pills3(25.0)6(33.3)3(12.0)6(21.4)2(18.2)5(26.3)11(20.4)(19.0) Did Pregnancy Check up1(8.3)2(11.1)2(8.0)1(3.6)1(9.1)2(10.5)4(7.4)1(4.8) Sort abortion later0(0.0)0(0.0)2(8.0)0(0.0)1(9.1)0(0.0)2(3.7)1(4.8) Did Nothing5(41.7)4(22.2)8(32.0)16(57.1)3(27.3)7(36.8)19(35.2)10(47.6)
|
study
| 100.0 |
The results of this study indicate that the practice of unprotected sex is common among university students and awareness of methods of preventing unplanned pregnancy is far from universal. Contrary to the dominant discourse, this study reveals that middle-class youths, specifically university students, are prone to unplanned pregnancy, use of unsafe EC methods and unsafe abortion. Indeed, even though many of the respondents were aware of EC, the study found that this awareness did not translate to EC use. Many respondents had poor knowledge of EC methods, lacked accurate knowledge of timing of the use of EC and held erroneous perceptions about the side effects of emergency contraception.
|
study
| 99.94 |
Emergency contraception remained underutilised among the respondents despite their self-reported perceived risks of unplanned pregnancy. It is important to note that emergency contraceptive pills are available over the counter in Nigeria. The low rate of utilisation could be due to stigma attached to premarital sex in Nigeria. Purchasing EC will automatically portray a young woman as having engaged in premarital sex, which is disapproved culturally. Our findings also confirms the assertion of Trussell and Cleland that emergency contraception remains underutilised and has not been demonstrated to lead to a population-level reduction in unintended pregnancies.
|
study
| 64.94 |
The finding that perceived pregnancy-risk was associated with the use of EC is unsurprising. For a young woman to successfully use EC, she had to perceive the risk of potential unplanned pregnancy. However, the finding that many students with perceived risk of unplanned pregnancy did nothing or used non-EC pills is worrisome and suggests the need for awareness campaign about EC in higher institutions. Perhaps the three students that reported abortion could indeed have prevented the pregnancies. Of course, even more of them might have become pregnant and sought recourse to abortion.
|
other
| 99.9 |
Some of the barriers affecting the use of EC reported in this study include; lack of knowledge of EC, the use of non-EC pills as EC, inaccurate knowledge of timing of use, use of local concoction as EC, and misperceptions of side effects of EC. The findings that 48% of women do not want to use EC due to perceived side effects by Tajure in Ethiopia further corroborates the present study’s finding about barriers to EC utilisation. Wesley and Glasier highlighted the mixed information about EC from the media as contributing to misperceptions in the population. However, negative perception of EC side effects was not significantly associated with the use of EC in our study. This is unsurprising because desperation to avoid unplanned pregnancy may push young women to use EC irrespective of their perception of EC side effects.
|
study
| 99.94 |
This paper challenges the common assumption that the use of unproven and even dangerous EC methods is mostly to be found among poor and the uneducated women. The level of ignorance among educated young women clearly shows that young women generally, irrespective of their level of education, should be targeted for intervention. Many of the medications reported by the participants for prevention of pregnancy following unprotected sexual intercourse do not have proven efficacy. Hence, the risk of avoidable drug-induced adverse effects and unplanned pregnancy may compromise the health of young university students. The use of medications such as menstrogen, gynacocied, antibiotics, Cytotec, Andrews Liver Salt, MNB 760, and “Alabukun” as emergency contraception would definitely have some health implications. Equally worrying is the use of concoctions such as salt and water, alcohol, lime, potash, and ‘yoyo bitters’ as emergency contraception. Future studies are needed to demonstrate the efficacy and health implications of the use of these medications and concoctions.
|
review
| 98.9 |
A causal association between the barriers and the low utilisation of emergency contraception cannot be established due to the cross-sectional nature of the study. The authors cannot exclude information bias on the sexual behaviours of the participants due to the self-reporting utilised for data collection. Hence, the rate of unprotected sexual intercourse, unplanned pregnancies and abortions may have been under-reported among the participants.
|
study
| 99.94 |
Contrary to the dominant discourse, this study reveals that middle class educated youths, specifically university students, engage in high-risk sexual behaviour, and also do seek recourse to unproven and unsafe contraceptive methods. Poor knowledge of EC methods and timing of use, misperceptions about side effects and the use of unproven non-EC methods are probable barriers to the use of EC among this cohort. Hence, appropriate educational programme addressing the barriers and dispelling the myths surrounding EC are urgently needed not only among poorly educated youths from low socioeconomic status, but also among young educated middle-class youths.
|
other
| 95.0 |
Lamotrigine (LTG) is a phenyltriazine derivative which is used in the treatment of epilepsy and bipolar disorder. It is one of the aromatic antiepileptic drugs (AEDs) which together are the most common cause of cutaneous adverse reactions (CADR) (Arif et al., 2007). The current widely used AEDs include carbamazepine (CBZ), oxcarbazepine (OXC), phenytoin (PHT), and phenobarbital (PB) (Maggs et al., 2000; Arif et al., 2007; Chung et al., 2010). CADR manifestations range from mild maculopapular exanthema (MPE) to severe cutaneous adverse reactions (SCAR), including Stevens–Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reactions with eosinophilia and systemic symptoms (DRESS). The mortality rates are 1–5% of patients with AED-induced SJS and up to 30% in AED-induced TEN (Roujeau and Stern, 1994; Harr and French, 2010; Yang et al., 2011). The incidences of SJS and TEN range between 1 and 10 cases per 10,000 patients (Mockenhaupt et al., 2005).
|
review
| 99.9 |
Pharmacogenetic studies have identified genetic associations between the human leukocyte antigen (HLA) allele and AEDs-induced CADR. According to specific AEDs medication, AEDs-induced SJS/TEN has been associated with specific HLA alleles in various populations, namely HLA-B∗15:02 and CBZ in the Han Chinese and Thai populations (Chung et al., 2004; Lim et al., 2008; Locharernkul et al., 2008; Tassaneeyakul et al., 2010) but not in the Japanese (Kaniwa et al., 2008) and European population (Alfirevic et al., 2006), HLA-B∗15:02 and OXC in Chinese and Thai populations (Chen et al., 2017). LTG is the most common AED used in Thailand. It has a similar chemical structure to CBZ, and high cross-reactivity rates of skin reaction from the group of AEDs in Han Chinese epilepsy patients have been reported (Greenwood, 2000; Maggs et al., 2000; Wang et al., 2010). LTG-induced SJS/TEN has been associated with HLA-B∗44:03 in Korean patients but no association was found with HLA-B∗15:02 (An et al., 2010; Hung et al., 2010; Shi et al., 2011; Park et al., 2015). In addition, HLA-A∗30:01 and HLA-B∗13:02 have been associated with a higher risk of LTG-induced MPE in Han Chinese (Li et al., 2013). However, there are no specific HLA alleles associated with LTG-induced CADR and an association between LTG-induced CADR and HLA alleles in Thailand has not been identified. Therefore, we aimed to examine the association between HLA-A and HLA-B and LTG-induced CADR in the Thai population.
|
study
| 99.94 |
A case–control study was performed at the Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center (SDMC), Ramathibodi Hospital, Thailand. Fifteen LTG-induced CADR (4 cases of SJS, 1 case of DRESS, and 10 cases of MPE) were recruited from the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Manarom Hospital, and Srinagarind Hospital between 2011 and 2015. All patients who developed CADR, as SJS, MPE, or DRESS, within 2 months after initiating LTG treatment were recruited for the study. The dermatological diagnosis was made by a dermatologist or allergist who reviewed photographs, pathological slides, clinical morphology, and medical records. MPE was defined as the presence of erythematous macules and papules without mucosal involvement, and in which the skin rash resolved after the drug was discontinued. The RegiSCAR criteria were used to establish SJS and DRESS. SJS was defined as skin detachment of BSA <10%, and TEN as skin detachment of BSA >30%. DRESS was defined as presenting with fever, maculopapular rash with internal organ involvement, and hematologic abnormalities. Patients who had been taking LTG for more than 6 months without evidence of cutaneous adverse effects were recruited as LTG-tolerant controls. In addition, the general population who had not taken LTG and had no history of drug-induced CADR were included in this study. Both case and control subjects were independently recruited with no family relationship. Data for this general control group were obtained from 369 and 986 subjects undergoing HLA-A and -B genotyping, respectively.
|
study
| 100.0 |
Genomic DNA samples were extracted from EDTA blood using a MagNA pure compact Nucleic Acid Isolation Kit on a MagNApure Compact machine. HLA alleles were genotyped using polymerase chain reaction-sequence-specific oligonucleotides (PCR-SSOs) according to the manufacturer’s protocol. In brief, diluted DNA samples were amplified by PCR using a GeneAmp®PCR System 9700 (Applied Biosystems, Waltham, MA, United States). The PCR products were then hybridized against a panel of oligonucleotide probes on coated polystyrene microspheres that had sequences complementary to stretches of polymorphisms within the target HLA-A, B alleles (a LABType®SSO, One Lambda Inc. Kit). The amplicon–probe complex was visualized using a colorimetric reaction and fluorescence detection technology (Luminex®IS 100). Data analysis for the HLA assays was performed with the software package HLA fusion 2.0.
|
study
| 99.94 |
Statistical analyses were performed using SPSS for Windows (version 16.0; SPSS, Chicago, IL, United States). Means and standard deviations were calculated for continuous variables. Dosages of LTG intake were described as median and interquartile range (IQR). To detect differences in the clinical characteristics between the case and control groups, an independent t-test was used for continuous variables. Chi-square test and Fisher’s exact test were used to describe the differences in frequencies of the HLA-A, B alleles between the groups. Haplotype association analysis was carried out using the “haplo.stats” package. The level of statistical significance was set at p < 0.05 (two-sided).
|
study
| 100.0 |
DNA samples from 15 LTG-induced CADR patients (10 cases with MPE, 4 cases with SJS, and 1 case with DRESS) and 50 LTG-tolerant controls and the general population group were genotyped. The mean age of the LTG-induced CADR patients was 35.2 ± 22.1 and 73.3% were female. The median LTG dosage was 50 mg per day. There were no significant differences in gender, age, dosage of LTG, and concomitant use of valproic acid between the LTG-induced CADR patients and the LTG-tolerant patients (Table 1).
|
study
| 100.0 |
The HLA-A and HLA-B genotypes of the LTG-induced CADR patients are shown in Table 2. The HLA genotypes for the treatment tolerant controls and the comparison of the HLA allele found in the CADR patients and control groups (LTG-tolerant controls and general population) are shown in Supplementary Tables 1, 2. The HLA alleles that showed a significant association when compared with the tolerant controls and general population are presented in Table 3. We found the HLA-B∗15:02 allele in 40.0% of patients who developed CADR and in 12.0% of the tolerant patients. The proportion of patients carrying the HLA-B∗15:02 allele was significantly higher in LTG-induced CADR cases than in both the treatment controls and general population groups with odds ratios (OR) of 4.89, 95% CI = 1.28–18.66, P-value = 0.014 and OR = 3.63, 95% CI = 1.27–10.34, P-value = 0.027, respectively. In addition, we also found a significant association between LTG-induced CADR patients and both HLA-B∗35:08 and HLA-B∗39:01 when compared with the general population with OR = 70.36, 95% CI = 4.19–1182.21, P-value = 0.030 and OR = 10.68, 95% CI = 2.20–51.83, P-value = 0.022, respectively.
|
study
| 100.0 |
In subgroup analysis of LTG-induced CADR, a significant association between LTG-induced MPE and HLA-B∗15:02 was found when compared with the tolerant controls and general population (OR = 7.33, 95% CI = 1.63–33.02, P-value = 0.005 and OR = 5.44, 95% CI = 1.56–19.03, P-value = 0.003, respectively). Interestingly, a significant association between LTG-induced MPE and HLA-B∗44:03 was found when compared with both control groups (OR = 10.29, 95% CI = 1.45–72.81, P-value = 0.029 and OR = 4.73, 95% CI = 1.20–18.62, P-value = 0.046, respectively), whereas HLA-B∗35:08 was significantly associated only with the general population (OR = 109.44, 95% CI = 6.34–1889.11, P-value = 0.020) (Table 4); nevertheless, no significant associations were found in LTG-induced SCAR.
|
study
| 100.0 |
Compared with the HLA-B allele, HLA-A∗02:07 was present in 33.3% of LTG-induced CADR patients and showed significantly higher frequencies than both the treatment control and general population groups with OR = 7.83, 95% CI = 1.60–38.25, P-value = 0.013 and OR = 3.27, 95% CI = 1.07–9.96, P-value = 0.029, respectively; in addition, HLA-A∗33:03 also had a significantly higher frequency than in the general population (OR = 3.16, 95% CI = 1.11–8.98, P-value = 0.023). Moreover, we found a significant association of LTG-induced MPE with HLA-A∗33:03 compared with the tolerant controls group (OR = 8.27, 95% CI = 1.83–37.41, P-value = 0.005) and general population group (OR = 8.43, 95% CI = 2.13–33.34, P-value = 0.002) as shown in Table 4.
|
study
| 100.0 |
In the present study, we found the significant association between LTG-induced CADR and HLA-A∗02:07 and HLA-B∗15:02 when compared with both tolerant and general population controls. In addition, the HLA-A∗33:03 allele was present at a significantly higher rate in LTG-induced CADR patients than in the general population controls. These results suggest that HLA-B∗15:02, alone, might not be the only risk factor for LTG-induced CADR, but HLA-A∗02:07 and HLA-A∗33:03 may also be risk factors for LTG-induced CADR. The subgroup analysis revealed that the proportion of patients carrying the HLA-B∗15:02 allele was significantly higher in LTG-induced MPE cases than in both the tolerant control and general population groups, which is very different from previous studies in which HLA-B∗15:02 was not found to be associated with LTG-induced MPE (An et al., 2010; Shi et al., 2011). We demonstrated that HLA-A∗33:03 may be a risk factors for LTG-induced MPE, which again is different from the findings of a previous study in the Han Chinese population which found that patients carrying HLA-A∗33:03 had a lower risk for LTG-induced MPE (Li et al., 2013). The same study also found that patients carrying either of the HLA-A∗30:01 or HLA-B∗13:02 alleles had increased risk for LTG-induced MPE (Li et al., 2013). In this study, we did not find a significant association between HLA-A∗30:01 or HLA-B∗13:02 and LTG-induced MPE. One study found the HLA-A∗30:01 allele in 1 of 10 LTG-induced MPE patients, while HLA-B∗13:02 was absent in the same 10 LTG-induced MPE patients, which the authors suggested could be because of the low frequency of HLA-B∗13:02 (1.4%) in the Thai population (Puangpetch et al., 2015).
|
study
| 99.94 |
Conversely, no significant association between LTG-induced SCAR and HLA-B∗15:02 was found when compared with the two control groups, nor did we find significant differences in the other HLA alleles between the LTG-induced SCAR group and the two control groups. Earlier studies on CBZ-induced SJS/TEN and the HLA-B∗15:02 allele reported no associations with the MPE group (Chung et al., 2004; Hung et al., 2006). The chemical structure of LTG includes aromatic rings similar to CBZ and shared a common risk allele causing SJS/TEN which similarities with other aromatic AEDs, namely PHT and OXC (Hung et al., 2010). Previous studies have found an association between LTG-induced SJS/TEN and HLA-B∗15:02 in Han Chinese (An et al., 2010; Hung et al., 2010), but other studies have found no association in Japanese patients (Kaniwa et al., 2008) or European population (Alfirevic et al., 2006), due to the fact that the HLA-B∗15:02 allele is rare in the Japanese population (0.1%) and people of European descent (0%), according to data from Lee et al. (2010). However, data from all of these studies were limited due to the small sample sizes of LTG-induced SJS patients; therefore, association studies between the HLA genotype and LTG-induced SJS could not be performed.
|
study
| 99.9 |
In this study, we report for the first time a significant association between HLA-B∗35:08 and LTG-induced CADR or MPE, although this allele has been reported in only one case of LTG-induced MPE and once in the general population, as a result of this allele being very rare in the Thai population (less than 1%, data from Puangpetch et al., 2015). The interpretation of data from studies with a small sample size can only be tentative, and further investigations with larger sample sizes are needed. Similarly to HLA-B∗35:08, the association of LTG-induced MPE and HLA-B∗44:03 alleles was firstly reported in the Thai population. One recent study from Korea found that HLA-B∗44:03 was associated with LTG-induced SJS/TEN (OR: 12.75, 95% CI: 1.03–157.14, and P-value = 0.053) (Park et al., 2016).
|
study
| 100.0 |
One study found that LTG-induced CADR in the Japanese population was associated with HLA class II alleles, including HLA-DRB1∗04:05, HLA-DQB1∗04:01, and HLA-DQA1∗03:03 (Ito et al., 2015), but HLA class II genotyping was not performed in this current study. However, it would be interesting to investigate the association of HLA class II and LTG-induced CADR in each population and with a large number of patients to better understand any association. Nevertheless, previous studies identified age and concomitant use of LTG and valproic acid as risk factors for LTG-induced CADR (Cheung et al., 2013; Egunsola et al., 2015). However, in our study we did not find that age and concomitant therapy with valproic acid were risk factors for LTG-induced CADR.
|
study
| 99.94 |
Apart from the HLA alleles, drug-metabolizing enzymes may be a risk factor for developing CADR. LTG is primarily metabolized by uridine diphosphate glucuronosyltransferases (UGT), including UGT1A4 and UGT2B7 (Perucca, 2006). 2-N-Glucuronide conjugates are the major inactive metabolite of LTG and elimination from the body by any enzyme variant either than one of these UGT enzymes will affect the risk of cutaneous adverse drug reactions (Rowland et al., 2006). A recent study on drug metabolizing enzymes found that the cytochrome P4502C9 (CYP2C9) influenced PHT-induced SCAR in the Thai population (Tassaneeyakul et al., 2016). Further association studies are required to determine the association between the glucuronidation metabolic pathway and LTG-induced CADR.
|
study
| 99.94 |
We found a statistically significant association of the HLA-A∗02:07 and HLA-B∗15:02 alleles with LTG-induced CADR in the Thai population. Therefore, these two alleles might be potential risk markers for LTG-induced CADR in Thailand. To confirm these findings, further large-scale studies are required.
|
study
| 100.0 |
CS, WT, and NK designed the research study. TR, TT, and JK diagnosed and recruited the subjects. VT, AP, and TD collected the clinical data; NK, TJ, SS, UI, and AV performed genotyping and evaluated the results. JP and PS analyzed the data. CS, JP, and PS wrote the manuscript. CS and AP reviewed and edited the manuscript.
|
other
| 99.94 |
The objective of root canal treatment is to prepare a clean, microbe- and debris-free canal for obturation.1 In addition to a proper root canal preparation and disinfection, an effective coronal and apical sealing ensures a long-term and successful endodontic treatment.2 A fluid tight seal for the entire root canal system is proposed as it prevents percolation of fluid from inflamed periapical tissues into inappropriately obturated canals.1 Though instruments are important in the removal of the infected dentin from the main canal, irrigants are crucial in areas inaccesible to instruments, such as lateral and accessory canals as well as fins and webs all over the canal.3Root canal irrigants serve multiple purposes during chemo-mechanical procedures as antimicrobial agents, dissolve organic tissue remnants, lubricate the dentinal walls and aid in flushing out debris, simultaneously assisting in the removal of the smear layer.4 Increased adhesion and sealing capacity is achieved after elimination of the smear layer.5
|
review
| 77.75 |
Sodium hypochlorite (NaOCl) is one of the most commonly used irrigants at a concentration ranging from 0.5% to 6%. It is well known for its antibacterial properties and tissue dissolving activity. Since, it has no effect on the inorganic part of the smear layer, Goldman in 1982 recommended that 17% ethylenediaminetetraacetic acid (EDTA) be used after NaOCl irrigation for complete removal of the smear layer.3
|
other
| 99.56 |
Haapasalo developed a new irrigant, QMix,6 which has substantial antimicrobial properties and is also effective in eliminating the smear layer.7It is a mixture of 2% chlorhexidine (a bisbiguanide, antimicrobial agent), 17% EDTA (a polyaminocarboxylic acid calcium-chelating agent) and cetrimide in distilled water with acceptable additional salt.6
|
other
| 99.9 |
Recently, a MTA-based sealer (MTA Fillapex) has been proposed as an endodontic sealer, which consists of MTA, salicylate resin, natural resin, bismuth oxide and silica. This material has attracted the researchers’ attention due to its excellent biocompatibility, bioactivity and osteoconductivity.8A study showed that this sealer has suitable physiochemical properties such as good radiopacity, flow and alkaline pH.8 Ferreira et al concluded that MTA-based sealers reduce the leakage into the root canal walls over time.9
|
study
| 99.5 |
Many characteristics such as adhesion to the tooth structure, long working time, ease of manipulation and good sealing ability of epoxy resin-based sealers made them the most commonly used sealers.10 A mixture of epoxy resins, amines, calcium phosphate, zirconium oxide, ethylene glycol salicylate, bismuth sub-carbonate and calcium oxide is incorporated into the recently introduced epoxy resin-based root canal sealer, Adseal.11 There are very few reports in the literature about its physical properties except for the reported radiopacity value.10
|
study
| 99.75 |
Various methods such as dye penetration, bacterial penetration, radio-labeled tracer penetration, dissolution of hard tissue, clearing of teeth, spectrometry of radioisotopes, electrochemical methods and gas chromatography have been used for evaluating the apical sealing properties of root canal sealers.12,13 According to Goldman et al14 (1989), the results of tracer penetration are hindered by the presence of entrapped air and are probably not very reliable.14 However, because of its sensitivity and amenity, the most common measurement used is dye penetration method.12 The longitudinal sectioning method facilitates examination of the exposed filling material and any dye penetration into the material at the interface of the dentinal wall on one side.15 The depth of dye penetration represents the gap between the root filling and canal walls.12
|
study
| 94.5 |
The aim of this study was to evaluate the effect of two different root canal irrigation solutions, i.e. 5.25% sodium hypochlorite followed by 17% EDTA and QMix on the apical sealing ability of two different root canal sealers, i.e. MTA Fillapex and Adseal.
|
study
| 99.94 |
Forty-six single-rooted human mandibular premolar teeth extracted for orthodontic or periodontal reasons were selected for this study. Radiographs were taken to make sure of single canals and the teeth were examined under a stereomicroscope for any cracks, caries, external or internal resorption and canal calcifications. As for using extracted teeth, there were no special considerations from institutional Research Ethics Committee.
|
study
| 99.94 |
Before initiating the study, the coronal portion of the teeth were cut away near the cementoenamel junction using a diamond disc and a high-speed handpiece with water coolant, perpendicular to the long axis to achieve a length of 14 mm in all the samples. The teeth were then divided into four equal experimental groups (n=10). Three teeth were kept as positive and three as negative controls.
|
study
| 100.0 |
The length of each canal was visually established by placing a #15 file (Dentsply-Maillefer, Ballaigues, Switzerland) into each tooth until the tip of the file was visible at the apex. The stopper was placed at cementoenamel junction which was the reference point. The working length was determined by subtracting 1 mm from the previous length. The canals were prepared up to #45 K-file using the step-back technique and the shaping of middle and coronal thirds was carried out by #3 and #4 Gates-Glidden drills (Dentsply-Maillefer, Switzerland).
|
study
| 99.94 |
After every change of endodontic file or Gates-Glidden drill, each canal was irrigated with 3 mL of irrigation solution. Two different endodontic irrigants were used. The teeth in groups 1 and 2 were irrigated with 5.25% NaOCl (Dentpro, Ammdent, Chandigarh, India) followed by 17% EDTA (Prevest DenPro, Jammu, India) and the teeth in groups 3 and 4 were irrigated with QMix (Dentsply, Tulsa Dental Specialties, Tulsa, OK). Both control groups (groups 5 and 6) were irrigated with sterile saline solution. Finally, the root canals were flushed with 2 mL of saline solution.
|
study
| 99.94 |
The apical patency was checked again by passing a #10 file through the apical foramen. Paper points were used to dry the root canals and standardized gutta-percha points (Dentsply-Maillefer, Switzerland) with proper tug-back at the working length were selected as master gutta-percha points. In groups 1 and 3 MTA Fillapex sealer (Angelus, Londrina, Parana) was used. Adseal sealer (Meta Biomed, Cheongju, South Korea) was used in groups 2 and 4. The obturation was completed with cold lateral condensation technique in all the experimental groups. The teeth were filled with gutta-percha without sealer in the positive control group and kept empty in the negative control group.
|
study
| 100.0 |
After obturation, the coronal access cavities of all the teeth were sealed with glass-ionomer cement (GC Fuji IX GP). The obturation of each tooth was assessed by radiographs. It was considered adequate if consistent gutta-percha without voids could be observed.
|
study
| 99.8 |
All the samples were stored in saline solution at 37°C for 48 hours to accomplish complete setting of the sealer. After 2 days, all the study specimens were thoroughly dried. The roots of all the experimental groups and the positive control group were coated with two coats of nail varnish except for the apical 2 mm. The entire root surface and apical foramen of the negative control specimens were completely covered with two coats of the nail varnish. The specimens were allowed to dry completely, then placed in 2% methylene blue dye solution (Central Drug House, New Delhi, India) and centrifuged at 3,000 rpm for 5 minutes. Afterwards the teeth were rinsed under tap water to remove the dye on external tooth surfaces.
|
study
| 100.0 |
The roots were longitudinally grooved with a diamond disc and then split into two halves with a chisel by levering with a plaster knife, making sure that the root canal filling was not penetrated. The dye penetration was measured from the apical to the coronal part of the root canal under a stereomicroscope (Leica Fluorescent Microscope, Wetzlar, Germany) with an ocular micrometer.
|
other
| 78.5 |
SPSS 14 was used for analyzing the results. Kruskal-Wallis test was used to compare the mean dye penetration in the six groups. A P-value < 0.05 was considered to be significant. Pairwise comparisons of dye penetration in various groups were carried out by Mann-Whitney U test.
|
study
| 99.9 |
Table 1 shows means and standard deviations of various groups and comparison of mean scores by Kruskal-Wallis test. Pairwise comparisons of six groups were carried out with Mann-Whitney U test. Group 3 exhibited maximum amount of apical leakage (3.7 ± 0.3 mm) whereas group 2 showed the least amount of apical leakage (2.1 ± 0.4 mm) among all the experimental groups. The P-value for each group was < 0.001, which shows that there were highly significant differences between the groups.
|
study
| 100.0 |
The apical sealing ability of sealers was affected by the irrigation solutions. Higher apical leakage values were observed with QMix as compared to 5.25% NaOCl, followed by 17% EDTA solution. However, the least amount of apical leakage was detected with 5.25% sodium hypochlorite, followed by 17% EDTA and Adseal combination. Figures 1,2and3 represent stereomicroscopic images of dye leakage in various groups.
|
study
| 100.0 |
The study showed that the apical sealing ability of Adseal was better than that of MTA Fillapex when NaOCl was used followed by 17% EDTA as irrigation solutions. Also, the apical sealing ability of both Adseal and MTA Fillapex decreased when QMix was used as an irrigation solution.
|
study
| 100.0 |
The smear layer consists of an organic portion (coagulated proteins, necrotic and non-necrotic pulpal tissue, odontoblastic processes, saliva, blood cells, and microorganisms) and an inorganic portion (minerals from the dentinal structure).5 Elimination of the smear layer from the root canal system results in cleaner and unconcealed dentinal tubules, promoting a better apical sealing with the filling material by allowing easier infiltration of the dentinal tubules.5
|
other
| 95.3 |
NaOCl is the most commonly used irrigant; however, its action does not affect inorganic materials. EDTA complements the response of sodium hypochlorite by chelating calcium ions in dentin and making instrumentation of the root canal easier.16 QMix has been found to be effective in removing the smear layer and also has substantial antimicrobial properties.7 Dai et al (2011)17 and Stojicic et al (2012)3 reported that QMix was as effective as 17% EDTA in smear layer removal. This was based on the number of fully opened dentinal tubules.
|
study
| 99.94 |
Endodontic failure is mainly due to the residual microbial organisms remaining after chemo-mechanical preparation. Root canal filling ideally should entomb such residual microorganisms denying nutrient supply to them as well as preventing microbial access towards the periradicular tissues.18
|
other
| 99.9 |
In this study, dye penetration method was used to evaluate the apical sealing property of root canal obturating materials because of its sensitivity, simplicity and convenience. The depth of dye penetration apically or coronally represents the gap between the root filling and the canal walls.12
|
study
| 99.94 |
Hasheminia et al19 showed that the sealing ability of Adseal was comparable to that of AH Plus and other epoxy resin-based sealers.19 Studies by Sonmez et al20 and Ferreira et al9 showed that MTA Fillapex resulted in more microleakage than AH Plus and Topseal, respectively, when irrigating the canals with NaOCl and EDTA. The results of the current study were found to be consistent with these.
|
study
| 99.94 |
The better sealing ability of Adseal can be explained by the fact that epoxy resin sealer is considered contraction-free during setting, reactions which is responsible for its appropriate interfacial adaptation.10 Epoxy resins forms an intimate contact with dentin, remains micromechanically retained, reinforces the tooth structure and prevents re-contamination.21 Although it is believed that MTA Fillapex provides a good seal due to the expansion during setting, there is limited research about its physicochemical properties.9
|
study
| 99.94 |
MTA Fillapex showed more dye leakage (3.7±0.3 mm) than Adseal (3.4±0.3 mm) when QMix was used as an irrigation solution. The effect of QMix on apical sealing ability of root canal filling materials has not been reported yet; however, studies have shown that EDTA negatively interferes with the hydration of MTA.22
|
study
| 100.0 |
In the present study, dye leakage values were higher for groups irrigated with QMix (3.7±0.3 mm) than those irrigated with NaOCl and EDTA (2.9±0.3 mm) where MTA Fillapex was used as sealer. Similarly when Adseal was used as root canal sealer, dye leakage values for groups irrigated with QMix (3.4±0.3 mm) were higher than the groups irrigated with NaOCl and EDTA (2.1±0.4 mm).
|
study
| 100.0 |
This outcome might be attributed to the fact that QMix solutions cannot dissolve organic tissues in the root canal system. Hence, the remnants of these organic materials might interfere with the bonding of the sealer to the root canal walls when QMix is used as a root canal irrigant.23
|
study
| 98.5 |
Within the limitations of this ex vivo study it can be concluded that the apical sealing ability of both MTA Fillapex and Adseal decreased when QMix was used as an irrigation solution. NaOCl followed by 17% EDTA yielded better results than QMix. In addition, Adseal showed better sealing ability than MTA Fillapex. So to conclude, none of the groups showed complete fluid-tight seal.
|
study
| 100.0 |
RS was responsible for the study design, data collection and compiling the manuscript. SP and DA were responsible for designing the research protocol and manuscript writing and reviewing. AS was responsible for collecting reviews for the study. AM was responsible for designing the methodology and performing the analysis and data compilation. RMS was responsible for reviewing and editing the manuscript. All the authors have read and approved the final manuscript.
|
other
| 99.94 |
One of the cardinal features of ageing is brain ageing, manifest in a wide spectrum of behavioural deficits including anxiety and impaired cognitive function. Changes in brain structural connectivity, decrease in neurogenesis, lipids peroxidation, oxidative stress, mitochondrial dysfunction, decline in neurotransmitters levels and beta amyloid (Aβ) overproduction have all been suggested to be major mediators of brain ageing and age-related neurologic disorders [3–5].
|
review
| 99.9 |
Animal models can be extremely valuable tools for studying biological mechanisms, for testing hypotheses generated from clinical research, and for testing the efficacy of candidate interventions. Such models should have demonstrable reliability, predictive validity, construct validity and relevance if findings from such studies are to translate from bench to bedside .
|
other
| 99.9 |
In the last 10 years, ageing research using animals has gained an increasing attention with the availability of drug-induced animal models which can be used to study accelerated ageing. D-galactose-injected rodent models recapitulate many features of brain ageing and have been extensively applied to study the mechanisms of brain ageing [3, 7, 8]. Following administration, d-galactose reacts with … to form advanced glycation end-products (AGEs) and cause oxidative stress. This in turn can lead to increased malondialdehyde (MDA) levels and decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities [9–11]. Its administration in rodents also has been reported to cause neurobehavioral changes including cognition and motor impairment; reduced neurogenesis; neurodegeneration; and caspase-dependent apoptosis and mitochondrial dysfunction .
|
review
| 99.9 |
However, findings from different laboratories, often from small studies, are inconsistent. Systematic reviews and meta-analysis are techniques to provide an unbiased and transparent summary of existing research [13, 14]. They can be helpful in the design of clinical trials [15, 16] and in understanding discrepancies between the results of preclinical and clinical trials . Here, we report a systematic review and meta-analysis to appraise d-galactose-induced brain ageing as a prevalent ageing model in rodent.
|
review
| 99.9 |
We electronically searched two databases (MEDLINE via PubMed and SCOPUS) for studies that used d-galactose as a brain-aging-inducing agent in rodent, using the keywords “Brain”, “aging”, “d-galactose” and “Rodent” as follows: [(brain)] AND [(aging) OR (sensense) OR (geriatric) OR (gerontic)] AND [(rodent) OR (rat) OR (mice) OR (mouse) OR (rattus) or (mus)] AND [d-galactose]. Two investigators used the SyRF platform (app.syrf.org.uk) independently to screen title, abstract and where necessary full text, judging the work against the inclusion and exclusion criteria. Where there are disagreements, the SyRF platform automatically serves the citation to a third investigator for adjudication. There was no date and language restriction in our search and the study was restricted to “other (i.e. non-human) animals”.
|
review
| 99.75 |
We included all rodent (e.g. mouse and rat) studies reported in full-text publications. Which used d-galactose to induce features of ageing We included any route of administration, dose, and dose timing and frequency. The primary outcome measure was cognition-related neurobehavioral outcome and secondary outcome measures were changes in the abundance of biochemical markers MDA, GSH-px, SOD, protein carbonyl (PC), Caspase-3, Bcl-2, Bax and AChE (acetylcholine esterase).
|
review
| 93.06 |
We extracted the author, publication year and type, animal characteristics (including species, strain, and sex, weight and age range or categorical age) and supplier, d-galactose model details (including route, dose and frequency of injection and duration of exposure), study quality evaluation, and the reporting of measures to reduce the risk of bias (listed below). We also recorded data for the nature of the outcome reported (neurobehavioural or neurochemical), the number of d-galactose groups served by the control group, and the number of animals per group, mean outcome and SD or SEM. We did not record the time of outcome assessment.
|
review
| 90.3 |
When a single publication reported more than one experiment, the data were evaluated as independent experiments. Where neurobehavioral or neurochemical outcomes were reported more than once in the same cohort of animals we recorded only data for the last assessment time. For graphically-presented data, we measured values from graphs using Universal Desktop Ruler, version 2.9 or contacted the manuscript authors for more information.
|
other
| 99.5 |
The internal validity of the enrolled studies (e.g. selection, performance, detection and attrition bias) and other study quality measures (e.g. reporting quality, power) were assessed using a modified version of the CAMARADES' study quality checklist which comprised: publication in peer-reviewed journal, randomization to treatment or control, allocation concealment, blinded assessment of outcome, statement of inclusion and exclusion of animals from the study, sample-size calculation, statement of compliance with regulatory requirements and statement regarding possible conflict of interest.
|
study
| 99.75 |
We expected substantial heterogeneity between studies so used a random effects model. The primary outcome was the overall effect of d-galactose on neurobehavioral outcome. Secondary outcomes were the effect of d-galactose on 8 biochemical outcomes, with a Holm-Bonferroni adjusted critical value for p of 0.006. Stratifications were considered in two domains, with 8 aspects of study design and 8 aspects of study quality, each domain tested at p<0.05 overall for neurobehaviour, p<0.006 for each biochemical outcome, giving critical values for p across 8 tests of 0.006 and 0.0008 respectively. For study quality items we also calculated an effect size as the change in effect observed in studies at high risk of bias, along with their 99.5% confidence intervals. Since we used the statistically more conservative standardized mean difference (SMD) we assessed the significance of differences between n groups by partitioning heterogeneity and by using Chi-square test with n-1 degrees of freedom (11). For continuous variables, we divided these into quartiles for partitioning of heterogeneity (STATA, version 10). Due to the limitations of using funnel plotting , Egger’s regression and trim and fill in the assessment of SMD publication bias (Wever et al, manuscript under consideration), these tests were not applied to assess publication bias in this literature.
|
study
| 99.94 |
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