Real-time Polymerized Chain Reaction Research Articles

Isolation and Molecular Detection of Marek's Disease Virus from Outbreak Cases in Chicken in South Western Ethiopia.

BackgroundMarek’s disease virus is a devastating infection, causing high morbidity and mortality in chickens in Ethiopia.MethodsThe current study was conducted from March to November, 2021 with the general objective of performing antemortem and postmortem, isolation, and molecular detection of Marek’s disease virus from outbreak cases in southwestern Ethiopia. Accordingly, based on outbreak information reported from the study sites namely, Bedelle, Yayo, and Bonga towns in southwestern Ethiopia, 50 sick chickens were sampled. The backyard and intensive farming systems of chickens were included in the sampling and priorities were given for chickens that showed clinical signs that are characteristics of Marek’s disease.ResultsBy clinical examinations, paralysis of legs and wings, gray eye, loss of weight, difficulty in breathing, and depression were recorded on all chickens sampled for this study and death of diseased chickens was observed. In addition, enlargement of the spleen and gross lesions of the liver and heart were recorded during postmortem examination. The death of infected chickens was observed in both vaccinated and non-vaccinated flocks. Out of 50 pooled feather follicle samples, Marek’s disease virus was isolated from 14/50 (28%) by cell culture method and out of six tissue samples, the virus was isolated from 5/6(83.30%). By Real time polymerization chain reaction technique, which was targeted to detect the Meq gene, Marek’s disease virus was detected from 18/50 feather follicles which accounts for 36% of sampled chickens.ConclusionIn general, current study showed that the circulating Marek’s disease virus in southwestern Ethiopia was caused by the oncogenic Gallid herpesvirus-2 (Serotype-1). Further research on molecular characterization of revolving virus in current and other regions is recommended for effective control of the disease through vaccination.

Comparison of liver biomarkers with N/L ratio, CRP, d-dimer in Covid 19 pneumonia and its effect on mortality

Coronavirus Disease-2019 (Covid-19) is the cause of a pandemic that has high mortality and global effects. The liver damage in Covid-19 cases in mechanical ventilation was investigated in this study. A total of 60 patients (the Study Group) who were diagnosed with Covid-19 pneumonia, and 65 individuals (the Control Group) were included prospectively in the study. The cases were divided into 3 groups as those who were intubated (severe), those who were not intubated (mild), and the healthy Control Group. The lung tomography results of those who were diagnosed with Covid-19 were examined in the study. The cases with positive RT-PCR (Real Time Polymerized Chain Reaction) test results were recorded from the system. The liver tests of the patients were compared with those of the Control Group. The two groups with and without intubation were also compared. The results were evaluated and analyzed statistically. When all the data were evaluated, it was found that LDH, GGT, AST, and aPTT levels were significantly higher in the mild and severe patient group compared to the Control Group, and the T. Protein and albumin levels were low (p<0.01). The N/L Ratio, and the CRP levels, which are the other acute phase reactants, were significantly higher (p<0.05). No statistically significant differences were detected when all parameters of the groups with and without intubation were compared (p>0.05). Increased D-dimer, GGT, D. Bil, LDH, NLR, and AST levels in Covid-19 patients in invasive mechanical ventilation are associated with mortality.

In Vitro Study Evaluating the Effect of Different Immunosuppressive Agents on Human Polyomavirus BK Replication

Human polyomavirus BK (BKPyV) is the etiologic agent of polyomavirus-associated nephropathy, a leading cause of kidney transplant dysfunction. Because of the lack of antiviral therapies, immunosuppression minimization is the recommended treatment. This strategy offers suboptimal outcomes and entails a significant risk of rejection. Our aim was to evaluate the effect of different immunosuppressive drugs (leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus) and their combinations in an in vitro model of BKPyV infection. Human renal tubular epithelial cells were infected with BKPyV and treated with leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus, administered alone or in some combination thereof. Viral replication was assessed every 24 hours (up to 72 hours) by BKPyV-specific quantitative real-time polymerized chain reaction for the VIRAL PROTEIN 1 sequence in cell supernatants and by western blot analysis targeting the viral protein 1 and the glyceraldehyde 3-phosphate dehydrogenase on total protein lysates. Results were described as viral copies/mL and compared between treatments at any prespecified time point of the study. The highest inhibitory effects were observed using leflunomide or everolimus plus mycophenolic acid (mean BKPyV replication log reduction 0.28). The antiviral effect of everolimus persisted when it was used in combination with tacrolimus (mean BKPyV replication log reduction 0.27). Our experience confirms that everolimus has anti-BKPyV properties and prompts future research to investigate possible mechanisms of action. It also provides a rational basis for targeted clinical trials evaluating alternative immunosuppressive modification strategies.

Comparison of amylase, lipase and d-dimer levels in the etiopathogenesis of mortality in Covid 19 pneumonia

Objective: Coronavirus Disease 2019 (Covid-19) is a pandemic cause affecting the whole world with high mortality. The effects of lipase, amylase and d-dimer levels on mortality were investigated in Covid-19 pneumonia in the present study. Method: A total of 131 patients diagnosed with pneumonia due to Covid-19 and 60 healthy control groups were included in the study prospectively. The cases were divided into 3 groups as the healthy control group, those who recovered, and those who died. Those who had pneumonia in the lung tomography among the cases diagnosed with Covid-19 were examined. The RT-PCR (Real-Time Polymerized Chain Reaction) test results were recorded from the system. The serum lipase, amylase, albumin levels, WBC (White Blood Cell), N/L (Neutrophil/Lymphocyte Ratio), and CRP (C-Reactive Protein) levels of the patients were compared with those of the Control Group. Results: When all the data were reviewed, the Lipase, Amylase, and d-dimer levels were found to be statistically significantly higher in the exitus group when compared to the control group, and T. protein and albumin levels were lower (p<0.01). Other acute phase reactants, WBC, N/L Ratio, and CRP levels were significantly higher (p<0.01). As a result of the comparison of the recovered and exitus group, no statistically significant changes were detected in all parameters (p>0.05). Conclusions: The elevation in serum amylase, lipase, and d-dimer levels may be significant in prognosis in pneumonia developing due to Covid-19.

Evaluation of patients with acute pancreatitis associated with SARS-CoV-2 (COVID-19); The importance of lipase/lymphocyte ratio in predicting mortality.

SARS-CoV-2 is the cause of a pandemic with high mortality. In the present study, the effects of the lipase/lymphocyte ratio on mortality were investigated in cases diagnosed with Covid-19 and acute pancreatitis. A total of 21 patients who were diagnosed with Covid-19 and acute pancreatitis, 34 patients who were not diagnosed with COVID-19 but diagnosed with acute pancreatitis, and 55 healthy control groups were divided into 3 groups and included in the study retrospectively. The patients who had positive RT-PCR (real‑time polymerized chain reaction) test results were included in the study. Complete blood count and biochemical values ​​of the patients were compared with those of the control group. When the data of the cases diagnosed with COVID-19 and acute pancreatitis were examined retrospectively, the amylase, lipase, lipase/lymphocyte ratio, and D-dimer levels were found to be significantly higher than in the control group (p < 0.01). In the ROC analysis, the amylase, lipase, and lipase/lymphocyte ratio had a high AUC (area under the curve) value (0.993 / 0.949 / 0.978, respectively). The lipase/lymphocyte ratio can be used in cases diagnosed with Covid-19 and acute pancreatitis to predict mortality (Tab. 3, Fig. 3, Ref. 23).

Routine wastewater-based monitoring of antibiotic resistance in two Finnish hospitals: focus on carbapenem resistance genes and genes associated with bacteria causing hospital-acquired infections

Wastewater-based monitoring represents a useful tool for antibiotic resistance surveillance. To investigate the prevalence and abundance of antibiotic resistance genes (ARGs) in hospital wastewater over time. Wastewater from two hospitals in Finland (HUS1 and HUS2) was monitored weekly for nine weeks (weeks 25-33) in summer 2020. A high-throughput real-time polymerization chain reaction (HT-qPCR) system was used to detect and quantify 216 ARGs and genes associated with mobile genetic elements (MGEs), integrons, and bacteria causing hospital-acquired infections (HAIs), as well as the 16S rRNA gene. Data from HT-qPCR were analysed and visualized using a novel digital platform, ResistApp. Eight carbapenem resistance genes (blaGES, blaKPC, blaVIM, blaNDM, blaCMY, blaMOX, blaOXA48, and blaOXA51) and three genes associated with bacteria causing HAIs (Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) were studied. There was a significantly higher number of ARGs at both hospitals in weeks 27-30 (174-191 genes) compared to other sampling weeks (151-171 genes). Our analyses also indicated that the two hospitals, which used different amounts of antibiotics, had significantly different resistance gene profiles. Carbapenem resistance genes were more prevalent and abundant in HUS1 than HUS2. Across both hospitals, blaGES and blaVIM were the most prevalent and abundant. There was also a strong positive association between blaKPC and K.pneumoniae in HUS1 wastewater. Routine wastewater-based monitoring using ResistApp can provide valuable information on the prevalence and abundance of ARGs in hospitals. This helps hospitals understand the spread of antibiotic resistance in hospitals and identify potential areas for intervention.

A Randomized Pilot Clinical and Microbiological Study Comparing Laser Microtextured Implants with and without Platform Switching

Minimising marginal bone loss around dental implants is of paramount importance. The success of methods such as platform switching (PS) and laser-micro-texturing (LM) are well documented. Whether or not a combination of these designs will further improve outcomes has not been studied previously. Hence, this prospective, randomized controlled single-centre pilot study compared the clinical and microbiological outcomes of implants with both PS and LM (test) to implants with only LM (control). A test and control implant were placed in thirteen patients totalling 26 implants. The primary investigated outcome was marginal bone level (MBL); secondary outcomes were peri-implant probing depths (PPD), bleeding on probing (BOP) and marginal tissue height (MTH). Additionally, the presence of five putative periodontal pathogens were assessed using real-time polymerized chain reaction. At 12 months the overall implant survival rate was 95.8%. MBL change was not found to be different between test and control at any time points, but a significant change was detected within the test implants at 6 months compared to baseline (p = 0.006). No differences were found in the secondary outcomes. Average PPD at 12 months was 2.68 ± 0.73 mm and 2.30 ± 0.46 mm and average change in MTH was 0.05 ± 0.72 mm and −0.24 ± 0.59 mm at tests and controls. No differences were reported in BOP frequency. Total periodontal pathogens count revealed no significant difference among control, test implants and adjacent tooth sites. Within the limitations of this study, it can be concluded that the addition of PS to LM implants does not significantly alter either short-term clinical outcomes or the vulnerability to pathogenic microflora colonization.

Evaluation of yellow fever surveillance in Chad, 2015-2020

The present study was conducted to determine the seroprevalence of yellow fever virus in patients with clinical signs of febrile jaundice giving suspicion of yellow fever and to evaluate diagnostic techniques for surveillance of yellow fever in the Republic from Chad. From January 2015 to July 2020, an observational study of virological markers was conducted in jaundice patients in Chad. Of the 1730 patients included in this study, a seroprevalence of 0.28% of yellow fever virus was determined. The distribution of pathogens responsible for diseases associated with yellow fever was: 49.47% (Plasmodium falciparum), 2% (hepatitis E virus), 4.62% (hepatitis C virus), and 29.00% (hepatitis virus B), respectively. Discrepancies in the results between the regional reference laboratories and the national laboratory of the Republic of Chad were observed. All genders and age groups were affected. Of the 1730 samples taken, 55.49% were female and 44.51% male (p = 0.01, a significant difference in favor of the female sex). The male/female sex ratio was 1.25. This study, the first, made it possible to determine the rate of the yellow fever virus in the absence of an outbreak in forest areas and with heavy rainfall and to evaluate the MAC-ELISA-CDC techniques used between the national laboratory of the Republic of Chad and those regional references. It was recommended that real-time polymerization chain reaction techniques be made available to national laboratories and reagents from the same manufacturing company in order to carry out effective monitoring of yellow fever. Key words: Evaluation, surveillance, seroprevalence, techniques, yellow fever.

Profile and Outcome of Children with COVID-19 Attending Bangabandhu Sheikh Mujib Medical University

<i>Background</i>: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is a global pandemic. Covid-19 has spread around Bangladesh and children are also being infected with comparatively fewer symptoms and lower case-fatality rates. <i>Objectives</i>: To assess the clinical profile, disease severity, presence of co-morbidities and outcome of pediatric COVID-19 patients. <i>Methods</i>: This prospective observational study was conducted in the fever clinic and the Department of Pediatrics, Bangabandhu Sheikh Mujib Medical University (BSMMU). Data were collected from the confirmed cases by using a structured questionnaire by face to face interview and also over telephone at 1^st visit and at 2^nd and 8^th week of disease course. Collected data were analyzed using appropriate statistical tests. <i>Result</i>: Among 2091 suspected COVID 19 cases attending fever clinic and department of Pediatrics, 462 (22.09%) were real time polymerized chain reaction (RT PCR) COVID 19 positive. Of them 377 (81.60%) responded to the questionnaire and subsequent follow up. Highest numbers of cases were found in June 2020. More than 60% of cases were within the age group of 10 to 18 years and 3.44% were infants. Male: female ratio was 1.04:1. Associated co-morbid conditions were present in 21.48% patients. Fever was the most common (72.67%) clinical feature followed by others. Incidences of severe and critical illness were more in older children. Most of the moderate cases and all of the severe and critical COVID cases were managed in the inpatient department including Pediatric intensive care unit. Among them, 100% required oxygen therapy, 72.41% needed oral or IV steroids, 8.6% and 6.9% needed intravenous immunoglobulin and biological agents (Tocilizumab) respectively. Seventeen patients (4.5%) recovered with some complications. <i>Conclusions</i>: Among suspected cases 22% were RT-PCR positive. Majorities were in the older age group and most of the moderate and all the severe and critical cases also belonged to older age group. Most of the cases (94.42%) recovered without complication and 4.5% recovered with some complications. Mortality was 1.06%.

Investigation of promoter methylation of MCPH1 gene in circulating cell-free DNA of brain tumor patients.

Despite advanced diagnostic and therapeutic techniques, many brain tumors are still diagnosed at high grades and, therefore finding novel molecular markers may assist in early detection and reducing brain tumors-related mortality rate. Owing to the previous reports on the importance of MCPH1 gene in tumorigenesis, the present study was aimed to study the promoter methylation of MCPH1 gene in paired circulating cell-free DNA (cfDNA) and tumor tissues of brain tumor patients. Fourteen fresh paired serum and tumor tissue samples in addition to 18 isolated serum samples were collected from patients affected by different grades of brain tumor. Genomic DNA and cfDNA was isolated from tissue and serum samples using QIAamp DNA Mini Kit Norgen Bioteck Kit, respectively. Methylation DNA immunoprecipitation Real-time polymerization chain reaction (MeDIP-Real-time PCR) was performed on isolated DNA samples using EpiQuik MeDIP Ultra Kit and specific primer pairs. cfDNA quantity was determined through Real-time PCR analysis using specific primer pairs designed for GAPDH gene. MCPH1 was methylated in 54% of cfDNA samples which was significantly associated with tumor grade, as well (P-value = 0.02). The methylation rate of MCPH1 was found as 78% in the tissue samples which was meaningfully associated with tumor grade (P-value = 0.03). Moreover, methylation of the MCPH1 gene was consistent in 57% of the same cfDNA and tissue samples. Methylation of MCPH1 gene in neither tumor tissues nor cfDNA was not correlated with age and sex of the patients. Due to the conformity of methylation of MCPH1 gene in cfDNA and tissue samples in more than half of the enrolled patients, especially in higher grades of tumors, it seems that MCPH1 promoter methylation could be a potential epimarker in not only detection of brain tumors but also in response to chemo- and radiotherapy which warranted further assessment.

The Proportion of Bifidobacterium and Escherichia coli in Colon of Children with Recurrent Urinary Tract Infection.

Recurrent urinary tract infection (UTI) in children is a well-known risk factor of chronic kidney disease. Periurethral area is normally inhabited by non-pathogenic flora, such as Bifidobacterium sp., and pathogenic flora from gastrointestinal tract, such as Escherichia coli (E. coli), which can cause UTI. Dysbiosis between pathogenic and non-pathogenic bacteria leads to infections, but studies regarding dysbiosis and recurrent UTI have not yet been documented. To estimate the proportional differences between gastrointestinal E. coli and Bifidobacterium sp. in children with recurrent UTI, a cross-sectional study was conducted in children from age six months to <18 years old diagnosed with recurrent UTI in Dr. Cipto Mangunkusumo Hospital. Healthy children matched in gender and age were recruited as control group. Stool samples were obtained from all the children in the two groups. Stool DNA was extracted using real-time polymerized chain reaction method to count E. coli and Bifidobacterium sp. proportion. Children with recurrent UTI had significantly higher proportion of E. coli compared to control group (10.97 vs. 4.74; P = 0.014) and lower proportion of Bifidobacterium sp. (6.54 vs. 9.33; P = 0.594). In children with recurrent UTI group, E. coli proportion was found higher than Bifidobacterium sp. although not statistically significant (10.97 vs. 6.54; P = 0.819). In healthy controls, Bifidobacterium sp. proportion was significantly higher than E. coli (4.74 vs. 9.33; P = 0.021). The total amount of E. coli (996,004 vs. 1,099,271; P = 0.798) and Bifidobacterium sp. (835,921 vs. 1,196,991; P = 0.711) were higher in secondary UTI compared to the simple UTI. Proportion of E. coli is higher in children with recurrent UTI than in healthy children. The proportion of E. coli is higher than Bifidobacterium sp in the colon of children with recurrent UTI.

Sirtuin 3 Therapy Attenuates Aging Expression, Oxidative Stress Parameters, and Neointimal Hyperplasia Formation in Vein Grafts

Vein graft (VG) failure due to neointimal hyperplasia remains an important and unresolved problem in cardiovascular surgery. Sirtuin3 (SIRT3) is associated with oxidative stress and lifespan. We aimed to measure SIRT3 expression in the veins of humans and rats during aging, explore the inhibitory effects of SIRT3 on vascular smooth muscle cell (VSMC) proliferation and neointimal hyperplasia in VGs, and investigate the underlying mechanisms. SIRT3 mRNA and protein levels in saphenous veins of young and older humans and in veins of young and old rats were measured by quantitative real-time polymerized chain reaction (PCR) and Western blot analysis. Young and old male rats were randomized to the control (control), graft (graft), adenovirus-encoding green fluorescent protein (Ad-GFP), and adenovirus encoding SIRT3 (Ad-SIRT3) groups. At 7days after operation, the mRNA and protein levels of SIRT3 and endothelial nitric oxide synthase (eNOS) were measured by quantitative real-time PCR and Western blot analysis. The mRNA levels and enzyme activity of manganese superoxide dismutase (MnSOD) and catalase (CAT) were measured by quantitative real-time PCR and enzymatic activity assay kits, and total nitric oxide (NO) levels were measured by biochemical assay kits. Histomorphometric analysis of VGs and immunohistochemical staining for proliferative activity were performed at 4weeks after operation. The hemodynamic parameters of the VGs were also measured by ultrasonic examination. SIRT3 mRNA and protein levels were lower in older human and rat veins than in younger human and rat veins. Ad-SIRT3 treatment significantly increased the expression and concentration of SIRT3, MnSOD, CAT, eNOS, and NO in VGs at 7days after operation. Ad-SIRT3 gene transfer reduced the neointimal thickness and neointimal area/media area ratio in the VGs of the Ad-SIRT3 groups compared with the graft and Ad-GFP groups, especially in old rats. Proliferative activity was lower in the Ad-SIRT3 groups than in the other groups. The hemodynamic parameters of VGs were obviously improved in the Ad-SIRT3 groups. SIRT3 expression decreases in the veins of humans and rats during aging. Furthermore, SIRT3 overexpression can significantly reduce VSMC proliferation and neointimal hyperplasia in VGs. Local intravenous delivery of adenovirus encoding SIRT3 may be a promising gene therapy for preventing VG failure.

A Double-Blind Randomized Trial Comparing Implants with Laser-Microtextured and Machined Collar Surfaces: Microbiologic and Clinical Results.

The purpose of this study was to compare the clinical outcome of and determine the differences in periodonto-pathogenic microbiota around two types of implant collar surfaces: laser-microtextured (test) vs machined (control). Seventeen patients (11 periodontally healthy, and 6 periodontally compromised) were selected to receive the two different implants, placed randomly, in two edentulous sites. Six months following the surgical placement of the dental implants, subgingival plaque samples were collected using paper points from the peri-implant sulcus and from the sulcus of an adjacent tooth. The presence of five putative periodontal pathogens, namely, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythensis, was assessed using real-time polymerized chain reaction (RT-PCR). Peri-implant parameters and intraoral radiographs were recorded up to 1 year after abutment connection. In the main population, and in the periodontally compromised subgroup, the total number of periodontal pathogens around test implant sites was lower than control implant sites and adjacent tooth sites, with a statistically significant difference (P < .05). In periodontally healthy patients, the mean probing pocket depth for the test implant was 1.31 ± 0.51 mm, compared with 2.66 ± 0.83 mm for the control implant, while in periodontally compromised patients, it was 1.61 ± 0.58 mm for the test implant, compared with a mean value of 2.84 ± 1.0 mm for the control implant. Implants with a laser-microtextured collar surface are not more vulnerable to pathogenic microflora colonization than implants with a machined collar surface. In both of the subgroups of patients (periodontally healthy and periodontally compromised), implants with a laser-microtextured collar surface have a better clinical outcome at 1 year of loading, compared with implants with a machined collar surface.

Binding and detoxification of chlorpyrifos by lactic acid bacteria on rice straw silage fermentation

ABSTRACTThis investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3–42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography–mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.

Incorporation of chitosan nanoparticles into silk fibroin-based porous scaffolds: Chondrogenic differentiation of stem cells

ABSTRACTA silk fibroin-chondroitin sulfate-sodium alginate (SF-CHS-SA) porous scaffold containing chitosan nanoparticles (NPs) was investigated. The proliferation of adipose-derived stem cells (ASCs) was studied by SEM, fluorescent microscopy, alcian blue staining, dimethylmethylene blue assay, and real-time polymerization chain reaction. The results showed that incorporation of NPs into the scaffold improved compressive modulus (5.6 ± 0.15 MPa). The amount of glycosaminoglycan expression of the ASCs was reached to 8.9 ± 0.3 µg/mL. The gene expressions of aggrecan, collagen II, and SOX9 of the ASCs were significantly improved. This study revealed that the prepared scaffold can be used as a substrate for cartilage tissue engineering.

Protective Effect of Rutin Against H2O2-Induced Oxidative Stress and Apoptosis in Human Lens Epithelial Cells

ABSTRACT Purpose: The aim of this study was to evaluate the effect of rutin on oxidative stress and apoptosis induced by H2O2 in human lens epithelial (HLE) cells and the associated mechanisms involved. Methods: Cell viability was assessed by 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and cell apoptosis was determined by flow cytometry, TUNEL assay and DNA fragmentation assay after 24 h treatment of 100 μM H2O2 with or without rutin pretreatment at various concentrations. The level of reactive oxygen species (ROS) was examined using 2′,7′-dichlorodihydrofluorescein diacetate by flow cytometry. The activity of superoxide dismutase (SOD) was measured by xanthinoxidase method and the contents of glutathione (GSH) and malondialdehyde (MDA) were quantified by enzyme-linked immunosorbent assay. The expression change of Bcl-2, Bax and caspase-3 at mRNA and protein levels were detected by real-time polymerized chain reaction (RT-PCR) and Western-blot analysis, respectively. Activation and translocation of nuclear factor-kappaB (NF-кB/p65) were examined by Western blot and immunocytochemistry. Results: Rutin pretreatment protected HLE cells from H2O2-induced cell viability decrease and apoptosis. In addition, in the presence of rutin, H2O2-induced intracellular excessive ROS and MDA were attenuated, whereas intracellular SOD and GSH depletion were prevented. Moreover, rutin also inhibited the up-regulation of caspase-3 and Bax expression and rescued down-regulation of Bcl-2 expression. Lastly, rutin blocked the activation and translocation of NF-кB/p65 induced by H2O2. Conclusions: Our results demonstrated that rutin effectively protects HLE cells from H2O2-induced oxidative stress and apoptosis in a dose-dependent manner. The involved mechanisms may be related to the regulation of ROS production, the inhabitation of lipid peroxidation, the protection of intracellular antioxidant system and its modulation of Bcl-2/Bax family and NF-кB/p65 signaling pathway.

Prevalence and significance of hepatitis B core antibodies among hepatitis B surface antigen-negative Egyptian polytransfused adult patients

Background/aims Blood transfusion is a well-established line of therapy associated with risk of infection transmission. Occult hepatitis B virus (HBV) infection is defined as the presence of HBV-DNA in the serum and/or the liver in the absence of hepatitis B surface antigen (HBsAg). Combined screening of HBsAg and hepatitis B core antibody (anti-HBc) can virtually eradicate blood-transmitted HBV. The aim of this study was to evaluate the presence of anti-HBc among Egyptian polytransfused adult patients and to determine the presence or absence of HBV-DNA in the serum samples of HBsAg-negative, anti-HBc-positive polytransfused adult patients using the polymerized chain reaction (PCR) method to assess the magnitude of occult HBV infection in these patients. Patients and methods This cross-sectional study included 79 polytransfused patients negative for HBsAg, HBsAb, and anti-hepatitis C virus. Patients were investigated for anti-HBc and samples of anti-HBc-positive patients were tested for HBV-DNA using real-time PCR. Results Among the 79 HBsAg-negative sera, anti-HBc was detected in 12 of 79 (15.19%) cases. All anti-HBc-positive sera were anti-HBs-negative. HBV-DNA was detected in five of 12 (41.67%) cases. Occult HBV infection was present in 6.33% of patients. Conclusion The overall prevalence of occult HBV in adult Egyptian polytransfused patients on regular blood transfusion is 6.33%.

Expression of Nodal on Bronchial Epithelial Cells Influenced by Lung Microbes Through DNA Methylation Modulates the Differentiation of T-Helper Cells

Background/Aims: The previous study in our lab showed that Nodal molecule on bronchial epithelial cells (BECs) was modulated by all kinds of lung microbes. The present study was designed to determine the effects of Nodal on proliferation of BECs and BECs-induced differentiation of T-helper (Th) cells. The epigenetic mechanisms of Nodal expression following treatments of different lung microbes were also identified. Methods: Real-time polymerization chain reaction (PCR) and western blot were used to determine the expression of Nodal. Flow cytometry was used to observe the effects of proliferation of BECs and subsequent BECs-induced differentiation of Th cells. Methylation levels of CpG islands in Nodal promoters were also analyzed by time of flight mass spectrometry. Results: The results showed that Nodal promoted proliferation of BECs and BECs-induced differentiation of Th cell from Th1 to Th2 and Th17. Nodal promoter showed a hyper-methylation in normal BECs. Through methylation modification in the promoter, P. aeruginosa or A.baumanni inhibited the expression of Nodal while RSV promoted the expression of Nodal. Conclusions: Our data showed that Nodal promoted Th2 and Th17 differentiation and inhibited Th1 differentiation which may cause imbalance of airway microenvironment. P. aeruginosa or A.baumanni may be hopeful for the treatment of airway hyperresponsveness by inhibition Nodal expression through DNA methylation modification in the promoter.

Alterations of maternal serum and placental follistatin‐like 3 and myostatin in pre‐eclampsia

To clarify the alterations of myostatin, a member of the transforming growth factor-β superfamily, and follistatin-like 3 (FSTL3), a binding protein for myostatin, in pre-eclamptic women. Samples of blood and placenta were collected from 40 pre-eclamptic women and 40 controls. The serum level and placental expression of FSTL3 and myostatin were determined with enzyme-linked immunosorbent assay, real-time polymerized chain reaction and western blotting. The serum levels of myostatin and FSTL3 were significantly higher in pre-eclamptic women than in the controls (P < 0.001 for both). Placental expression of myostatin and FSTL3 were also significantly increased in the pre-eclamptic placenta compared with that of the controls (P < 0.001 for both); however, there were no significant differences in myostatin or FSTL3 in either the maternal serum or the placenta in women with mild or severe pre-eclampsia (P > 0.05 for both). The serum levels and placental expression of myostatin and FSTL3 are elevated in pre-eclampsia, suggesting the role of myostatin and its binding protein in pre-eclampsia.

Losartan Decreases Cardiac Muscle Fibrosis and Improves Cardiac Function in Dystrophin-Deficient Mdx Mice

Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmd(mdx)/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice.