Prevalence of occult hepatitis C virus infection in beta-thalassemia major patients in Ahvaz, Iran

Prevalence of occult hepatitis C virus infection in beta-thalassemia major patients in Ahvaz, Iran
Occult hepatitis C virus infection (OCI) is defined by the presence of HCV RNA in peripheral blood mononuclear cells (PBMCs) and liver tissue cells despite the absence of HCV RNA in plasma. Currently, OCI is classified into two types: seropositive OCI (anti-HCV positive and serum HCV RNA negative) and seronegative OCI (anti-HCV and serum HCV RNA negative).
Beta-thalassemia is described as a blood disorder that decreases the synthesis of hemoglobin. Repeated blood transfusion is the standard treatment for patients with beta-thalassemia major (BTM), and this increases the risk of exposure to infectious agents.
The aim of this study was to investigate the prevalence of OCI among BTM patients. Plasma and PBMCs were collected from 90 BTM patients who were referred to Shafa Hospital in the city of Ahvaz and were screened for HCV antibody using a commercial ELISA kit as the first step. Next, nested RT-PCR was performed on extracts of plasma and PBMCs.
HCV RNA from positive PBMCs was sequenced, the sequences were aligned, and a phylogenetic tree was constructed to determine their relationship to reference sequences retrieved from the GenBank database. Seventy-nine out of 90 patients (87.8%) were negative for HCV Ab (seronegative), while 11 patients (12.2%) were seropositive.
HCV RNA was found in PBMCs of four patients (66.7%) who were negative for HCV Ab (seronegative) and two patients (33.3%) who were positive for HCV Ab (seropositive). HCV RNA was not detected in plasma samples from these six patients. Six out of 90 BTM patients (6.7%) had OCI. HCV genotyping revealed that all six patients were infected with HCV subtype 3a.
We found a high frequency of OCI in BTM patients, which warrants more attention, considering the importance of this infection. Further studies are needed to determine the actual prevalence of OCI in BTM patients in Iran.

lncRNA MEG3 Downregulation Relieves Intracerebral Hemorrhage by Inhibiting Oxidative Stress and Inflammation in an miR-181b-Dependent Manner

BACKGROUND This study was designed to illustrate the effects and latent mechanism of lncRNA maternally expressed gene 3 (MEG3) on intracerebral hemorrhage (ICH)-induced brain injury. MATERIAL AND METHODS An ICH rat model was generated to determine the role of lncRNA MEG3 in ICH.
The interaction between lncRNA MEG3 and microRNA (miR)-181b were confirmed by Starbase and dual-luciferase reporter assay. One hour (h) or 3 days after ICH stimulation, rat neurological injury was evaluated by modified Neurological Severity Score (mNSS). Brain water content and cell apoptosis were assessed using brain edema assessment and flow cytometry (FCM), respectively.
Caspase3 activity was also determined. Enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the levels of pro-inflammatory cytokines. Moreover, the representative biomarkers of oxidative stress were evidenced using detection kits. RESULTS The lncRNA MEG3 level in ICH rat brain tissues was higher than that in the sham group. miR-181b was a direct target of lncRNA MEG3 and it was downregulated in brain tissues of ICH rats.
Notably, we found that neurobehavioral scores, brain water content, and neuronal apoptosis were decreased and caspase3 activity was reduced in MEG3-shRNA-treated ICH rats, while we observed the opposite result in ICH+MEG3-shRNA+miR-181b inhibitor rats. Further analyses revealed that MEG3-shRNA inhibited inflammatory cytokines release and reduced oxidative stress.
All these results were reversed by miR-181b inhibitor. In addition, MEG3-shRNA activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, which was reversed by miR-181b inhibitor. CONCLUSIONS MEG3-shRNA restrained oxidative stress and inflammation following ICH in an miR-181b-dependent manner.

CircPRKCI alleviates lipopolysaccharide (LPS)-induced HK-2 cell injury by regulating miR-106b-5p/GAB1 axis

Circular RNAs (circRNAs) act as vital regulators in diverse diseases. However, the investigation of circRNAs in sepsis-engendered acute kidney injury (AKI) remains dismal. We aimed to explore the effects of circPRKCI in lipopolysaccharide (LPS)-mediated HK2 cell injury.
Sepsis in vitro model was established by LPS treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was conducted for determining the levels of circPRKCI, microRNA-106b-5p (miR-106b-5p) and growth factor receptor binding 2-associated binding protein 1 (GAB1). Cell viability and apoptosis were evaluated using Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis, respectively.
The concentrations of interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) were measured with Enzyme linked immunosorbent assay (ELISAkits. The levels of oxidative stress markers were determined using relevant commercial kits. Western blot assay was conducted for B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax) and GAB1 protein levels.
Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized to verify the association between miR-106b-5p and circPRKCI or GAB1. We found CircPRKCI level was decreased in sepsis patients and LPS-induced HK-2 cells. LPS exposure inhibited cell viability and facilitated apoptosis, inflammation and oxidative stress in HK-2 cells.
CircPRKCI overexpression abrogated the effects of LPS on cell apoptosis, inflammation and oxidative stress in HK-2 cells. Furthermore, circPRKCI was identified as the sponge for miR-106b-5p to positively regulate GAB1 expression. Overexpression of circPRKCI relieved LPS-mediated HK-2 cell damage by sponging miR-106b-5p.
MiR-106b-5p inhibition ameliorated the injury of HK-2 cells mediated by LPS, while GAB1 knockdown reversed the effect. Collectively, CircPRKCI overexpression attenuated LPS-induced HK-2 cell injury by regulating miR-106b-5p/GAB1 axis.

Analysis of daily variation in the release of faecal eggs and coproantigen of Fasciola hepatica in naturally infected dairy cattle and the impact on diagnostic test sensitivity

The liver fluke, Fasciola hepatica (F. hepatica) is a widespread parasite infection in dairy cattle in Victoria, South-eastern Australia. Robust diagnosis of fluke infection is needed in dairy cattle to identify sub-clinical infections which often go unnoticed, causing significant production losses. We tested the coproantigen ELISA (cELISA) and the FlukeFinder faecal egg count kit® on naturally infected cows in a fluke endemic region of Victoria.
The aim of the study was to investigate the variation in the release of coproantigens and eggs into faeces over a 5-day period, at the morning (AM) and afternoon (PM) milkings, and to assess the impact of the timing of faecal sample collection on diagnostic test sensitivity.
Ten cows were enrolled into the study based on positive F. hepatica faecal egg counts (LFEC) and faecal samples from the ten cows were collected twice daily, at the 7-9 AM and 4-6 PM milking, for five consecutive days. At the conclusion of the sampling period, the cows were euthanized and F. hepatica burden determined at necropsy. A moderate negative correlation between cow age and cELISA optical density (OD) was observed using data from all samples (R -0.63; 95 % CI -0.68 to -0.57).
Over the 5-day sampling period, we observed within-animal variation between days for both the cELISA OD (2.6-8.9 fold) and LFEC (5-16 fold), with more variation in values observed in the PM samples for both tests. The correlation with total fluke burden was higher in the AM sampling using both the cELISA and LFEC (R 0.64 and 0.78, respectively). The sensitivity was 100 % for the cELISA using various cut offs from the literature (0.014 OD, 0.030 OD, and 1.3 % or 1.6 % of the positive control).
The sensitivity of the FlukeFinder kit® (based on 588 faecal samples and not accounting for lack of independence in the data) was 88 % (95 % CI 85 %-90 %). Seventy one false negatives were recorded from the 588 LFEC tests all of which were observed in the cows with fluke burdens <14 flukes; 42 of the 71 false negative LFECs occurred in one individual cow which had the lowest burden of nine flukes.
In dairy cows, the cut-off for production losses due to fasciolosis is estimated at> 10 fluke. Both the cELISA and the LFEC identified all cows that had burdens equal to or greater than this cut-off. Five of the ten cows also exhibited relatively high paramphistome egg counts.

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