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Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws. [ABSTRACT FROM AUTHOR]

Background Increased microbial translocation (MT) into the systemic circulation is associated with liver disease progression. Microbial translocation has yet to be completely defined in chronic hepatitis B virus (HBV) and chronic hepatitis delta virus (HDV). Methods Our aim was to characterize MT and associated immune response in chronic HBV and HDV at various stages of disease. Serum from 53 HBV, 43 HDV, and 36 healthy control (HC) subjects was obtained. Subjects were categorized by aspartate aminotransferase-to-platelet ratio index into mild (<0.5), moderate, and severe (>1.0) disease. Cytokines, microbial products, and microbial deoxyribonucleic acid (DNA) levels were assessed in a single treatment-naive time point for each patient. Next-generation sequencing identified bacterial species present within patient sera. Results The HBV and HDV subjects display higher serum concentrations of Gram-negative (G−) bacterial lipopolysaccharide and fungal beta-glucan compared with HC (all P <.01). Gram-positive (G+) bacterial peptidoglycan is higher in HBV compared to HDV and HC (both P <.0001). Within both disease cohorts, peptidoglycan correlates with interleukin (IL)-1b, IL-8, IL-12p70, and IL-13 (all Spearman's rho >0.45; P <.05). Next-generation sequencing from 7 subjects with detectable serum bacterial DNA revealed changes in abundance of bacterial taxa and a higher proportion of Gram-positive genera in severe disease. Greater G+/G− taxa ratio is associated with higher cytokine levels and disease markers. Conclusions The HBV and HDV patients display increased translocation of bacterial and fungal products into serum. An increased proportion of Gram-positive genera is associated with disease progression. Correlations of peptidoglycan with antimicrobial cytokines suggest that particular microbial classes may contribute to systemic inflammation and possibly disease progression. [ABSTRACT FROM AUTHOR]

Background: The damaging effect of rheumatoid arthritis (RA) on cartilage, bone, ligaments, and tendons has raised the importance of the disease activity and severity assessment to enable therapeutic decisions and to evaluate disease outcome. Aim: The aim is to compare the clinical examination of the Disease Activity Score (DAS)-28 with the musculoskeletal ultrasonography (US) examination in RA patients. Moreover, finding if we can use ultrasonographical results as a tool for predicting subsequent radiological damage. Patients and Methods: It is a longitudinal study included 60 adult RA patients. Patients were under assessment at baseline, 6 months, and 12 months from the recruitment time. Twenty-eight joints of DAS were assessed for tenderness and swelling. US gray scale (GS) and US power Doppler (PD) score also was done at each visit. Results: DAS-28, with its parameters, is positively and highly significantly correlated to synovitis severity both by US GS and US-PD score along the study follow-up visits. There was highly significant difference between the number of 28 swollen and tender joints by clinical examination with both US GS and US-PD. Linear regression analysis to predict the number of swollen and tender joints after 12 months showed significance between US PD with swollen and tender joints' numbers. The correlation was positive and significant between Larsen score at 12 months with GS US and PD US assessment, but linear regression analysis was only significant for Larsen score with only GS US. Conclusion: GS US and PD is a sensitive and reliable noninvasive method complementary to standard clinical assessment and could be a tool for predicting subsequent joints' damage. [ABSTRACT FROM AUTHOR]

Background and Purpose Cyclosporine ( CSA) and non-steroidal anti-inflammatory drugs ( NSAIDs) are co-prescribed for some arthritic conditions. We tested the hypothesis that this combined regimen elicits exaggerated nephrotoxicity in rats via the up-regulation of endothelin ( ET) receptor signalling. Experimental Approach The effects of a 10 day treatment with CSA (20 mg·kg−1·day−1), indomethacin (5 mg·kg−1·day−1) or their combination on renal biochemical, inflammatory, oxidative and structural profiles were assessed. The roles of ETA receptor and COX-2 pathways in the interaction were evaluated. Key Results Oral treatment with CSA or indomethacin elevated serum urea and creatinine, caused renal tubular atrophy and interstitial fibrosis, increased renal TGF-β1, and reduced immunohistochemical expressions of ETA receptors and COX-2. CSA, but not indomethacin, increased renal ET-1, the lipid peroxidation product malondialdehyde ( MDA) and GSH activity. Compared with individual treatments, simultaneous CSA/indomethacin exposure caused: (i) greater elevations in serum creatinine and renal MDA; (ii) loss of the compensatory increase in GSH; (iii) renal infiltration of inflammatory cells and worsening of fibrotic and necrotic profiles; and (iv) increased renal ET-1 and decreased ETA receptor and COX-2 expressions. Blockade of ETA receptors by atrasentan ameliorated the biochemical, structural, inflammatory and oxidative abnormalities caused by the CSA/indomethacin regimen. Furthermore, atrasentan partly reversed the CSA/indomethacin-evoked reductions in the expression of ETA receptor and COX-2 protein. Conclusions and Implications The exaggerated oxidative insult and associated dysregulation of the ETA receptor/ COX-2/ TGF-β1 signalling might account for the aggravated nephrotoxicity caused by the CSA/indomethacin regimen. The potential renoprotective effect of ETA receptor antagonism might be exploited therapeutically. [ABSTRACT FROM AUTHOR]

Background: Racial differences and broad spectrum response to anti-hepatitis C (anti-HCV) therapy suggest a possible role for host genetic diversity in treatment outcomes. We aim to determine the association and predictive value of certain human leukocyte antigen (HLA) class I alíeles with either susceptibility to viral clearance or persistence following pegylated interferon (Peg-IFN) plus ribavirin therapy in chronic hepatitis C (HCV) genotype 4 patients in Egypt. Methods: This study included 200 unrelated chronic HCV patients who received Peg-IFN plus ribavirin therapy [112 patients with sustained virological response (SVR) and 88 non-responders (NR)]. Serological testing of HLA class I antigens (HLA-A and HLA-B alíeles) were performed by standard complement-dependent microlymphocytotoxicity assay. Results: The frequency of HLA-A01 was significantly higher in SVR than in NR cases [OR: 0.51 ; 95% CI: 0.27-0.981 ; P = 0.042], while the frequency of alíeles B38 (P = 0.011), B40 (P < 0.001) and B41 (P < 0.001) was significantly higher in NR cases (OR/95% CI: 7.05/ (1.39-18.01), 10.31/3.14-36.1 . On logistic regression analysis, presence of the HLA-A01 alíele was associated with SVR (OR: 0.50; 95% CI: 0.28-0.89; P= 0.02) and HLA-B38 can predict non response to therapy (OR: 7.92; 95% CI: 1.67-37.54; P = 0.009) with an overall accuracy of 60%.Severe fibrosis (OR: 3.035; 95% CI: 1.521-6.091; P= 0.002), high viremia (OR: 2.69; 95% CI: 1.11-6.53; P= 0.005) and steatosis (OR: 2.1 ; 95% CI: 1.002-3.90; P = 0.041 ) predicted no response with an overall accuracy of 81.8%. Conclusion: HLA-A01 and HLA-B38 alíeles are associated with and may have a role in the outcome of response to Peg-IFN plus ribavirin therapy in Egyptian patients diagnosed with chronic HCV infection. The use of immunologie markers to predict the outcome of treatment may help pharmacogenetic personalization of treatment for HCV infection. [ABSTRACT FROM AUTHOR]

Background: Bile acids mediate gut-liver cross-talk through bile acid receptors. Serum, hepatic, and microbial bile acid metabolism was evaluated in HCV-compensated chronic liver disease., Methods: Patients underwent liver biopsy; portal and peripheral blood were obtained before (HCVi), and 6 months after sustained virologic response (SVR), splenic blood was obtained only after SVR. The fecal microbiome and liver transcriptome were evaluated using RNA-Seq. Twenty-four bile acids were measured in serum, summed as free, taurine-conjugated bile acids (Tau-BAs), and glycine-conjugated bile acids., Results: Compared to SVR, HCVi showed elevated conjugated bile acids, predominantly Tau-BA, compounded in HCVi cirrhosis. In the liver, transcription of bile acids uptake, synthesis, and conjugation was decreased with increased hepatic spillover into systemic circulation in HCVi. There was no difference in the transcription of microbial bile acid metabolizing genes in HCVi. Despite an overall decrease, Tau-BA remained elevated in SVR cirrhosis, mainly in splenic circulation. Only conjugated bile acids, predominantly Tau-BA, correlated with serum proinflammatory markers and hepatic proinflammatory pathways, including NLRP3 and NFKB. Among hepatic bile acid receptors, disease-associated conjugated bile acids showed the strongest association with hepatic spingosine-1-phosphate receptor 2 (S1PR2)., Conclusions: Enhanced expression of hepatic S1PR2 in HCVi and HCVi-cirrhosis and strong associations of S1PR2 with Tau-BAs suggest pathological relevance of Tau-BA-hepatic S1PR2 signaling in chronic liver disease. These findings have therapeutic implications in chronic liver diseases.