Immunomodulatory role of phagocyte-derived chloramines involving lymphocyte glutathione

This study shows that human lymphocytes markedly decrease chloramines (long-lived oxidants) generated by polymorphonuclear neutrophils (PMN) after stimulation by phorbol-myristate-acetate or opsonized zymosan. In a cell-free model, reduced glutathione (GSH) scavenged chloramines, giving rise to oxidized glutathione (GSSG). In the cell system, treatment of lymphocytes with autologous PMN-derived chloramines induced a profound decrease in their total and reduced glutathione (GSH) content and markedly inhibited their proliferate responses to concanavalin-A and, to a lesser extent, phytohaemagglutinin. It is concluded that (i) lymphocytes may play a defensive role against phagocyte-derived oxidative stress by scavenging chloramines, and (ii) as this effect which is mediated by GSH affects lymphocyte proliferative responses, it may help to elucidate the still obscure mechanisms of oxidative stress associated immunodeficiency

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target for N-acetylcysteine treatment in dialysis patients

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target forN-acetylcysteine treatment in dialysis patients.BackgroundOxidative stress largely contributes to hemodialysis-associated lethal complications, thus explaining the urgent need of antioxidant-based therapeutic strategies in hemodialysis patients. We previously identified advanced oxidation protein products (AOPP) in the uremic plasma as exquisite markers of oxidative stress and potent mediators of monocyte activation. The present study was aimed at searching whether (1) AOPP can also trigger activation of polymorphonuclear neutrophils (PMN), and (2) whether AOPP-induced activation could be inhibited by N-acetylcysteine (NAC), a widely used compound which has been shown to prevent oxidative injury to kidney.MethodsBoth human serum albumin (HAS) AOPP (i.e., HOCl-modified HSA in vitro preparations and AOPP extracted from plasma of hemodialysis patients) were tested for their capacity to trigger phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent activities as measured by lucigenin- and luminol-amplified chemiluminescence (CL), respectively, as compared to receptor-dependent [opsonized zymosan or receptor-independent phorbol myristate acetate (PMA)]. The effect of PMN priming by platelet-activating factor (PAF), and the effect of NAC on normal monocyte and on normal or hemodialysis patient's (N = 16) PMN oxidative responses were compared.ResultsHSA-AOPP triggered in a HOCl dose-dependent manner both NADPH-oxidase- and MPO-dependent CL of PMN. This latter was further enhanced by PAF priming. Plasma-derived AOPP obtained from hemodialysis patients also triggered PMN respiratory burst. NAC significantly reduced HSA-AOPP–mediated responses of normal monocyte and of normal and uremic PMN but had no significant effect on opsonized zymosan- or PMA-induced CL responses.ConclusionThis dual potential of NAC to inhibit phagocyte oxidative responses induced by HSA-AOPP without affecting those mediated by compounds mimicking pathogens supports the proposal of a therapeutic trial with NAC aimed at reducing oxidative stress–related inflammation in hemodialysis patients

Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

International audienceHepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis

Myeloperoxidase Promoter Polymorphism −463G Is Associated With More Severe Clinical Expression of Cystic Fibrosis Pulmonary Disease

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the −463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation

Comparison of two RT-qPCR methods targeting BK polyomavirus microRNAs in kidney transplant recipients

BackgroundBK polyomavirus replication leads to progressive tubulointerstitial nephritis and ureteral stenosis, with a considerable risk of subsequent graft failure in kidney transplant recipients. Since specific antiviral therapies are lacking, new tools are required to enhance the biological monitoring of the infection. Viral microRNAs are promising new biomarkers, but the performance of RT-qPCR methods limits the clinical application and the validation of a standard method for quantification.MethodsWe compared TaqMan microRNA Assays and TaqMan Advanced miRNA Assays for bkv-miR-B1-3p and bkv-miR-B1-5p quantification in synthetic microRNA templates and in 44 urine samples belonging to 14 consecutive kidney transplant recipients with BK polyomavirus replication from Amiens University Medical Center in a 1-year span.ResultsCycle threshold values were constantly higher with TaqMan Advanced MicroRNA Assays. TaqMan microRNA Assays showed better performance in predicting the good prognosis of BK polyomavirus nephropathy.ConclusionOverall, TaqMan MicroRNA Assays appeared to be a more sensitive and accurate RT-qPCR method than TaqMan Advanced MicroRNA Assays to quantify bkv-miR-B1-3p and bkv-miR-B1-5p BKPyV miRNAs in patients’ urine samples

A prospective, observational study of fidaxomicin use for Clostridioides difficile infection in France.

To describe the characteristics, management and outcomes of hospitalised patients with Clostridioides difficile infection (CDI) treated with and without fidaxomicin. This prospective, multicentre, observational study (DAFNE) enrolled hospitalised patients with CDI, including 294 patients treated with fidaxomicin (outcomes recorded over a 3-month period) and 150 patients treated with other CDI therapies during three 1-month periods. The primary endpoint was baseline and CDI characteristics of fidaxomicin-treated patients. At baseline, the fidaxomicin-treated population included immunocompromised patients (39.1%) and patients with severe (59.2%) and recurrent (36.4%) CDI. Fidaxomicin was associated with a high rate of clinical cure (92.2%) and low CDI recurrence (16.3% within 3 months). Clinical cure rates were ≥90% in patients aged ≥65 years, those receiving concomitant antibiotics and those with prior or severe CDI. There were 121/296 (40.9%) patients with adverse events (AEs), 5.4% with fidaxomicin-related AEs and 1.0% with serious fidaxomicin-related AEs. No fidaxomicin-related deaths were reported. Fidaxomicin is an effective and well-tolerated CDI treatment in a real-world setting in France, which included patients at high risk of adverse outcomes.Trial registration: Description of the use of fidaxomicin in hospitalised patients with documented Clostridium difficile infection and the management of these patients (DAFNE), NCT02214771, www.ClinicalTrials.gov

Development of a rat model for glioma-related epilepsy

Seizures are common in patients with high-grade gliomas (30–60%) and approximately 15–30% of glioblastoma (GB) patients develop drug-resistant epilepsy. Reliable animal models are needed to develop adequate treatments for glioma-related epilepsy. Therefore, fifteen rats were inoculated with F98 GB cells (GB group) and four rats with vehicle only (control group) in the right entorhinal cortex. MRI was performed to visualize tumor presence. A subset of seven GB and two control rats were implanted with recording electrodes to determine the occurrence of epileptic seizures with video-EEG recording over multiple days. In a subset of rats, tumor size and expression of tumor markers were investigated with histology or mRNA in situ hybridization. Tumors were visible on MRI six days post-inoculation. Time-dependent changes in tumor morphology and size were visible on MRI. Epileptic seizures were detected in all GB rats monitored with video-EEG. Twenty-one days after inoculation, rats were euthanized based on signs of discomfort and pain. This study describes, for the first time, reproducible tumor growth and spontaneous seizures upon inoculation of F98 cells in the rat entorhinal cortex. The development of this new model of GB-related epilepsy may be valuable to design new therapies against tumor growth and associated epileptic seizures

HIV-1-infected patients from the French National Observatory experiencing virological failure while receiving enfuvirtide

Objectives We studied gp41 mutations associated with failing enfuvirtide salvage therapy. Methods This multicentre study involved patients with HIV-1 plasma viral load (pVL) > 5000 copies/mL after at least 3 months of uninterrupted enfuvirtide therapy and with plasma samples available at inclusion (T0), at initial enfuvirtide failure (T1) and at last follow-up visit during continued failing enfuvirtide therapy (T2). The HR-1 and HR-2 domains of the gp41 gene were sequenced at T0, T1 and T2. Results Ninety-nine patients were enrolled. At baseline, the median pVL and CD4 cell count were 5.1 log copies/mL and 72 cells/mm3, respectively. Based on the ANRS Resistance Group algorithm, the proportion of patients harbouring viruses with enfuvirtide resistance mutations increased significantly between T0 and T1. In the HR-1 domain, the V38A/M, Q40H, N42T, N43D and L45M mutations wereselected (P < 0.02). In the HR-2 domain, no mutations were significantly selected during the follow-up. None of the mutations was associated with a CD4 cell count increment. Conclusions Mutations selected during failing enfuvirtide salvage therapy are mainly located in the HR-1 domain of the gp41 gene, between codons 38 and 45. No mutations were associated with an increase in the CD4 cell coun

The treatment response of chronically hepatitis C virus-infected patients depends on interferon concentration but not on interferon gene expression in peripheral blood mononuclear cells.

International audienceThe current treatment of chronic hepatitis C is based on pegylated alpha interferon (PEG-IFN-α) and ribavirin. The aim of this study was to identify biological and clinical variables related to IFN therapy that could predict patient outcome. The study enrolled 47 patients treated with PEG-IFN and ribavirin combined therapy. The interferon concentration was measured in serum by a bioassay. The expression of 93 interferon-regulated genes in peripheral blood mononuclear cells was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) before and after 1 month of treatment. The interferon concentration in the serum was significantly lower in nonresponders than in sustained virological responders. Moreover, a significant correlation was identified between interferon concentration and interferon exposition as well as body weight. The analysis of interferon-inducible genes in peripheral blood mononuclear cells among the genes tested did not permit the prediction of treatment outcome. In conclusion, the better option seems to be to treat patients with weight-adjusted PEG-IFN doses, particularly for patients with high weight who are treated with PEG-IFN-α2a. Although the peripheral blood mononuclear cell samples are the easiest to obtain, the measurement of interferon-inducible genes seems not be the best strategy to predict treatment outcome