Morphological changes in the experimental model of polycystic ovary syndrome and effects of vitamin D treatment.

AIM: This research was designed to develop an animal model by inducing polycystic ovary syndrome (PCOS) with dehydroepiandrosterone (DHEA) and observe reproductive and morphological changes after treatment with vitamin D. MATERIALS AND METHODS: Thirty pre-pubertal female Sprague-Dawley (SD) dams were recruited. The animals were distributed 10 each in control, PCOS and vitamin D-treated groups. In control group 0.2 ml of sesame oil was given. PCOS group was administered DHEA by the daily dose of 6 mg/kg for 30 days. In vitamin D-treated group, animals were injected 6 mg/kg/day DHEA daily and 120 ng 1, 25(OH) 2D3/100 g subcutaneously once a week. The occurrence of reproductive phenotypic PCOS was evaluated by estrous cycle, morphology and histological changes of ovary, uterus on light microscope. RESULTS: The results of this study showed significant weight gain, obesity, and estrous irregularity in PCOs group as compared to control and vitamin D-treated group. CONCLUSION: Administration of vitamin D (120 ng 1, 25(OH) 2D3/100) improved the cycle characteristics, reduced body weight and morphological features in PCOS induced animals. The results support the effect of vitamin D treatment for metabolic and reproductive characteristic features in PCOS females.

Endothelial function and carotid intima media thickness in obstructive sleep apnea without comorbidity.

PURPOSE: The objective of this study was to evaluate endothelial function and carotid intima media thickness (CIMT) in moderate to severe obstructive sleep apnea (OSA) without comorbidities. METHODS: It is an observational case control study in which endothelial function was assessed using flow-mediated dilatation (FMD) and peripheral arterial tonometry (PAT), and carotid artery ultrasound was used to measure CIMT in study group subjects that included 20 normotensive, non-diabetic, treatment naïve, and moderate to severe OSA patients, and 20 normotensive, non-diabetic, and non-OSA subjects served as a control group. Study was conducted in Polysomnography Laboratory, Department of Internal Medicine, All India Institute of Medical Sciences (AIIMS) Hospital, New Delhi. RESULTS: FMD was significantly lower in the moderate to severe OSA group compared to non-OSA group (mean ± SD, 8.3 ± 2.8 vs. 13.4 ± 4.1 %; p = 0.0001). Reactive hyperemia index (RHI) was also significantly lower in the OSA group (1.55 ± 0.27 vs. 2.01 ± 0.48, p = 0.0007). CIMT was observed to be significantly higher in the OSA group compared to the non-OSA group (0.54 ± 0.09 vs. 0.48 ± 0.08 mm; p = 0.049). In the OSA group, FMD, RHI, and CIMT did not show a significant correlation with OSA disease severity indices [apnea hypopnea index (AHI), oxygen desaturation index (ODI), and minimum O2 saturation]. CONCLUSION: Endothelial function in macrovascular and microvascular circulation is significantly impaired in moderate to severe OSA patients without comorbidities. These patients also show evidence of subclinical atherosclerosis, in the form of increased CIMT.

Evaluation of the Training Program to Train HIV Treatment Center Staff in Pakistan.

Introduction In Pakistan, HIV training programs, especially for health professionals working in HIV treatment centers, are limited. Consequently, there is little data about HIV awareness among physicians and allied health workers and how it may affect their care for people living with HIV (PLWH). Recently, the Global Fund to Fight AIDS, Tuberculosis, and Malaria (GFATM) grant Principal Recipient UNDP engaged an NGO experienced in HIV/AIDS training, on a competitive basis, to develop a training manual and conduct training of all categories of HIV treatment centers staff. The goal of this study was to assess the training program's influence on trainees' (both physicians and allied health staff) knowledge and abilities and describe its major lessons. Methodology This was a one-group pre-post test study, carried out between January 17 and February 22, 2023. The study was carried out in three phases. In the first phase, a team of experts developed an antiretroviral treatment (ART) training manual. In the second phase, 9- and three-day training workshops were conducted in six different cities of Pakistan, which were attended by physicians and allied health staff working in different HIV treatment centers across Pakistan. The workshops had plenary lectures, discussions, role plays, video cases, and case studies. In the third phase, a quiz, comprising multiple/best choice questions (MCQs/BCQs) and true and false questions, was administered before (pre) and after the workshop (post) to assess the impact of these training sessions in enhancing the level of HIV knowledge, especially related to ART. The workshop was attended by a total of 256 health workers from different cities in Pakistan. The participants had backgrounds in medical science, psychology, laboratory science, nursing, and computer science. Pre-and post-test responses were statistically analyzed to determine the impact of the training program on participant's knowledge. For this, the Shapiro-Wilk test was applied to test data normality, followed by the application of paired t-test or Wilcoxon Signed Rank Test for normally and non-normally distributed data, respectively. Finally, a chi-square test was applied to examine the significant (p<0.05) association between training workshops and improvement in the participant's level of understanding of HIV. In all statistical tests, p<0.05 was considered significant.  Results The results from our study showed that before the training session, both physicians and allied staff possessed limited knowledge about HIV-related domains. After the workshops, participants from all cities demonstrated a uniform enhancement of knowledge related to different HIV-related domains, evident from the improvement in post-test scores compared to pre-test scores (p<0.0001). The chi-square test showed a significant association between training workshops and improvement in the participant's level of understanding about HIV (p-values for BCQ, MCQ, and true and false: 0.001, 0.0047, and 0.0024, respectively). Conclusions Pre- and post-test evaluation provides an objective, data-driven method for measuring the impact of educational interventions in improving healthcare workers' awareness about HIV. The results emphasize the role of continuous workshops and training programs in enhancing the knowledge and understanding of healthcare and allied health workers regarding HIV.

Association of Interferon Lambda 3 and 4 Gene SNPs and Their Expression with COVID-19 Disease Severity: A Cross-Sectional Study.

Introduction: Expression and certain SNPs of interferon lambda 3 and 4 (IFNL3 and 4) have been associated with variable outcomes in COVID-19 patients in different regions, suggesting population-specific differences in the disease outcome. This study examined the association of INFL3 and INFL4 SNPs (rs12979860 and rs368234815, respectively) and nasopharyngeal expression with COVID-19 disease severity in Pakistani patients. Methods: For this study, 117 retrospectively collected nasopharyngeal swab samples were used from individuals with mild and severe COVID-19 disease. qPCR assays were used to determine the viral loads and mRNA expression of IFNL3 and 4 through the Ct and delta Ct methods, respectively. Due to funding limitations, only one SNP each in INFL3 and INFL4 (found to be most significant through literature search) was analyzed using tetra-arm PCR and RFLP-PCR strategies, respectively. The Mann-Whitney U-test was applied to evaluate the statistical differences in the expression of IFNL3/4 genes in the mild and severe groups, while for SNPs, a Chi-square test was employed. A multivariate Cox regression test was performed to assess the relationship of different variables with COVID-19 severity. Results: Comparative analysis of SNPs between mild and severe groups showed only the difference in SNP of the IFNL4 gene to be statistically significant (p = 0.001). Similarly, nasopharyngeal expression of IFNL3 and IFNL4 genes, respectively, was found to be 3.48-fold less and 3.48-fold higher in the severe group as compared to the mild group. Multivariate analysis revealed SNP in the IFNL4 gene and age to have a significant association with COVID-19 severity. Conclusion: Despite the small sample size, IFNL4 gene SNP and patient age were associated with COVID-19 severity. Age, IFNL3/IFNL4 mRNA expression in the nasopharyngeal milieu, and the presence of SNP in the IFNL4 (rs368234815) gene in COVID-19 patients may be biomarkers for infection severity and help improve SARS-CoV-2 infection management.

Genetic and antiretroviral drug resistance mutations analysis of reverse transcriptase and protease gene from Pakistani people living with HIV-1.

BACKGROUND: Antiretroviral therapy (ART) effectiveness is compromised by the emergence of HIV drug resistance mutations (DRM) and can lead to the failure of ART. Apart from intrinsic viral factors, non-compliance with drugs and/or the use of sub-optimum therapy can lead to the emergence of DRMs. In Pakistan HIV currently exists as a concentrated epidemic, however, ART coverage is very low, and drug adherence is poor. ART is selected assuming without baseline genotyping. Pakistan has recently seen a rise in treatment failures, but the country's actual burden of DRM is still unknown. In this study, we perform the genetic and drug resistance analysis of the pol gene from Pakistani HIV-positive ART-naïve and ART-experienced individuals. METHODS: In this study, HIV-1 pol was sequenced from 146 HIV-1 positive individuals, divided into ART-naïve (n = 37) and ART-experienced (n = 109). The sequences were also used to determine HIV-1 subtypes, the prevalence of DRM, and pol genetic variability. RESULTS: DRM analysis identified numerous DRMs against reverse transcriptase inhibitors in both ART-naïve and ART-experienced groups, including a few that are classified as rare. Additionally, the ART-experienced group showed mutations associated with resistance to protease inhibitors. Genetic analysis showed negative selection pressure in both groups, but a higher rate of evolution in the ART-naïve group. CONCLUSION: High prevalence of DRMs, especially against previous first-line treatment in ART- naïve and the accumulation of DRMs in ART-experienced groups is concerning and warrants that a more extensive DRM survey be carried out to inform first-line and second-line ART regimen recommendations.

Analysis of temporal changes in HIV-1 CRF01_AE gag genetic variability and CD8 T-cell epitope evolution.

Currently, little is known about the time-dependent evolution of human immunodeficiency virus-1 (HIV-1) circulating recombinant forms (CRF) 01_AE, a dominant recombinant form associated with HIV-1 epidemics worldwide. Since gag is a highly immunodominant HIV-1 protein, we performed a comparative analysis of the CRF01_AE gag protein's time-dependent changes and evolution. A total of 3105 HIV-1 CRF01_AE gag sequences representing 17 countries from the timeline 1990-2017 were obtained. The sequences' phylogenetic relationship and epidemic dynamics were analyzed through a Maximum Likelihood tree and Bayesian Skyline plot, respectively. Genomic variability was measured through Shannon entropy and time-dependent immunoevolution was analyzed using changes in proteasomal degradation pattern, cytotoxic T lymphocytes (CTL) epitopes, and Human leukocyte antigens (HLA) restriction profile. The most recent common ancestor of the HIV CRF01_AE epidemic was estimated to be 1974±1. A period of exponential growth in effective population size began in 1982, fluctuated, and then stabilized in 1999. Genetic variability (entropy) consistently increased, however, epitope variability remained comparable; the highest number of novel CTL epitopes were present in 1995-1999, which were lost over time. The spread of the HIV-1 CRF01_AE epidemic is predominant in countries within Asia. Population immunogenetic pressures in the region may have contributed to the initial changes and following adaptation/stabilization of epitope diversity within gag sequences.

Phylogenetic and drug- and vaccine-resistance profiles of Hepatitis B Virus among children with HIV co-infection in Pakistan.

INTRODUCTION: HIV-1 and hepatitis B virus (HBV) share common routes of transmission and therefore co-infection is common. In 2019, an HIV-1 outbreak that resulted in >1000 children being infected, predominantly through nosocomial transmission, occurred in Sindh, Pakistan. We conducted a phylogenetic and drug resistance analysis of the HBV Reverse Transcriptase (RT) gene in children with HIV-1 and HBV co-infection. METHODOLOGY: Blood samples were collected from 321 children with HIV who were recruited as part of a study to investigate the HIV-1 outbreak. All samples were tested for HBV surface antigen (HBsAg) using an ELISA assay, and positive samples were used to amplify and sequence the HBV RT gene. The phylogenetic relationship between sequences was analyzed, and drug- and vaccine- resistance mutations in the RT gene were explored. RESULTS: Of 321 samples, 23% (n = 75) were positive for HBsAg on ELISA. Phylogenetic analysis of the sequences revealed that 63.5% of HBV sequences were sub-genotype D1, while the rest were sub-genotype D2. Cluster analysis revealed grouping of sub-genotype D1 sequences exclusively with Pakistani sequences, while clustering of sub-genotypes D2 predominantly with global sequences. The 236Y mutation associated with resistance to tenofovir was observed in 2.8% of HBV sequences. Additionally, seven vaccine escape mutations were observed, the most common being 128 V. CONCLUSION: Our study suggests ongoing transmission of HBV D1 and D2 sub-genotypes in the HIV-1 co-infected population, likely nosocomially, given common routes of HVB and HIV-1 transmission. The prevalence of major HBV drug- and vaccine-resistant mutations remains low. Surveillance for further transmissions and the possible emergence of major drug- or vaccine-resistant variants is required.

Analysis of differential gene expression of pro-inflammatory cytokines in the nasopharyngeal milieu of mild & severe COVID-19 cases.

INTRODUCTION: A subset of individuals with COVID-19 can suffer from a severe form of the disease requiring breathing support for respiratory failure and even death due to disease complications. COVID-19 disease severity can be attributed to numerous factors, where several studies have associated changes in the expression of serum pro-inflammatory cytokines with disease severity. However, very few studies have associated the changes in expression of pro-inflammatory changes in the nasopharyngeal milieu with disease severity. Therefore, in the current study, we performed differential gene expression analysis of various pro-inflammatory cytokines in the nasopharyngeal milieu of mild & severe COVID-19 cases. MATERIAL AND METHOD: For this retrospective, cross-sectional study, a total of 118 nasopharyngeal swab samples, previously collected from mild and severe (based on the WHO criteria) COVID-19 patients were used. A real-time qPCR was performed to determine the viral loads and also evaluate the mRNA expression of eight cytokines (IL-1, IL-2, IL-4, IL-6, IL-10, IFN-γ, TGF-ß1, and TNF-α). Subsequently, an unpaired T-test was applied to compare the statistical difference in mean expression of viral loads and each cytokine between the mild and severe groups, while the Pearson correlation test was applied to establish a correlation between disease severity, viral load, and cytokines expression. Similarly, a multivariable logistic regression analysis was performed to assess the relationship between different variables from the data and disease severity. RESULTS: Out of 118 samples, 71 were mild, while 47 were severe. The mean viral load between the mild and severe groups was comparable (mild group: 27.07± 5.22; severe group: 26.37 ±7.89). The mRNA expression of cytokines IL-2, IL-6, IFN- γ, and TNF-α was significantly different in the two groups (p<0.05), where the Log2 normalized expression of IL-2, IL-6, IFN- γ, and TNF-α was found to be 2.2-, 16-, 2.3-, and 1.73-fold less in the severe group as compared to the mild group. Furthermore, we also observed a significant positive correlation between all the cytokines in the severe group. The multivariate analysis showed a significant relationship between age, IL-6, and disease severity. CONCLUSION: This decreased expression of certain cytokines (IL-2, IL-6, TNF-α, and IFN-γ) in the nasopharyngeal milieu may be considered early biomarkers for disease severity in COVID-19 patients.

Endometriosis, infertility and MicroRNA's: A review.

The review aims to explore circulating small non- coding regulatory Ribonucleic Acids (miRNA) as biomarkers of endometriosis; a reproductive age group disorder. miRNA are linked with genetic, epigenetic and angiogenic factors, hormones, cytokines, chemokines, oxidative stress (OS) markers, mediators of inflammation, hypoxia, angiogenesis and altered immune system contributing to the pathogenesis of endometriosis. Hormonal imbalance occurs by decreased levels of miRNAs -23a and miRNAs -23b and increase in miRNAs -:135a, 135b, 29c and 194-3p. Angiogenesis by vascular endothelial growth factor is attributed to increased miRNAs -126, miRNAs -210, miRNAs -21, miRNAs -199a-5p and miRNAs 20A. OS upregulates miRNAs -302a by increased levels of Tumor Necrosis factor (TNF)-α, TNF- ß and Interleukin -1ß. Upregulation of miRNAs -199a and miRNAs -16 promotes inflammation and cell proliferation in the endometriotic lesions. The gold standard to diagnose endometriosis is laparoscopy, yet miRNA can be validated as diagnostic tool for detection, progression and prevention of endometriosis in large, independent cohorts of women, with and without endometriosis.