Phylogenetic analysis of the NS5 gene of dengue viruses isolated in Ecuador

Dengue virus (DENV) is a member of the genus Flavivirus of the family Flaviviridae. DENV causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). There is not knowledge of the genetic relations among DENV circulating in Ecuador. Given the emerging behaviour of DENV, a single tube RT-PCR assay using a pair of consensus primers to target the NS5 coding region has been recently validated for rapid detection of flaviviruses. In order to gain insight into the degree of genetic variation of DENV strains isolated in Ecuador, DENV NS5 sequences from 23 patients were obtained by direct sequencing of PCR fragments using the mentioned one step RT-PCR assay. Phylogenetic analysis carried out using the 23 Ecuadorian DENV NS5 sequences, as well as 56 comparable sequences from DENV strains isolated elsewhere, revealed a close genetic relation among Ecuadorian strains and DENV isolates of Caribbean origin. The use of partial NS5 gene sequences may represent a useful alternative for a rapid phylogenetic analysis of DENV outbreaks.     

Mycobacterium bovis BCG-infected neutrophils and dendritic cells cooperate to induce specific T cell responses in humans and mice

Neutrophils are increasingly thought to modulate dendritic cell (DC) functions. We investigated the role of the neutrophil-DC partnership in the response to Mycobacterium bovis BCG-the vaccine used against tuberculosis. We compared neutrophil-DC crosstalk in humans and mice, searching for functional differences. In both species, neutrophils captured fluorescent BCG-enhanced green fluorescent protein (EGFP) and were more phagocytic than DC. Non-apoptotic BCG-infected neutrophils clustered with immature DC, establishing intimate contacts with dendrites, at which fluorescent intact bacilli were observed. Physical interactions between neutrophils and DC were required for DC activation. Human BCG-infected DC produced interleukin (IL)-10, an inhibitory cytokine, whereas DC exposed to BCG-infected neutrophils produced low to undetectable amounts of the cytokine. Mouse BCG-infected neutrophils induced sustained IL-2 production by DC. Human DC exposed to BCG-infected neutrophils stimulated recall T cell reactivity from vaccinated donors. Mouse DC infected with recombinant ovalbumin (OVA)-producing BCG (rBCG(ova)) elicited proliferation of TCR-OVA-transgenic CD4 and CD8 T cells. Moreover, exposing DC to rBCG(ova)-infected neutrophils enhanced OVA presentation. Thus, in mice and humans, neutrophils help DC to cross-present BCG antigens to T cells. Our results suggest that this "ménage à trois" involving neutrophils, DC and T cells plays a major role in the immune response to BCG.     

Microcystin accumulation and antioxidant responses in the freshwater clam Diplodon chilensis patagonicus upon subchronic exposure to toxic Microcystis aeruginosa

We investigated the accumulation and toxicity of microcystin-LR (MCLR) in the digestive gland of the freshwater clam Diplodon chilensis patagonicus. Treated clams were fed with a toxic strain of Microcystis aeruginosa (NPJB1) during 6 weeks and control clams received the non-toxic strain NPDC1. Filtration rate was estimated for both groups. Toxic effects were evaluated through the hepatosomatic index (HSI) and different oxidative stress biomarkers, lipid peroxidation (content of thiobarbituric reactive substances-TBARS), protein oxidation (carbonyl groups) and reduced glutathione (GSH) levels, and enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST). The extractable MCLR measured by ELISA in digestive gland extracts showed little or no change during the first 3 weeks and increased significantly at weeks 5 and 6. HSI was reduced by 30% in treated clams at weeks 5 and 6. No significant oxidative damage to lipids or proteins was. All the antioxidant defense parameters analyzed were significantly increased at week 5 or 6. GSH increased in treated clams at week 5, reaching 62% increase at week 6. SOD, CAT and GST activities were significantly increased in treated clams by 50%, 66% and 60%, respectively, at the end of the experiment. D. chilensis patagonicus can be exposed to prolonged cyanobacterial blooms accumulating significant quantities of MCLR, which could be a risk for mammals and birds, which feed on this species and, in a lesser extent, to humans.     

Modulation of the CD4+ T-cell response by Helicobacter pylori depends on known virulence factors and bacterial cholesterol and cholesterol α-glucoside content

Helicobacter pylori blocks the proliferation of human CD4(+) T cells, facilitated by vacuolating exotoxin (VacA) and γ-glutamyl transpeptidase (GGT). H. pylori-triggered T-cell reactions in mice correlate with bacterial cholesterol and cholesterol α-glucoside content but their role in human cells is unclear. We characterized the effect of VacA, GGT, and cholesterol on T-helper 1, T-helper 2, T-regulatory and T-helper 17 associated cytokines and T-cell proliferation. VacA, GGT, and bacterial cholesterol content exhibited differential and synergistic inhibitory effects on the expression of activation markers CD25 and CD69 and on interleukin 2, interleukin 4, interleukin 10, and interferon γ production. These factors did not affect the H. pylori-mediated abrogation of transforming growth factor β secretion or increased interleukin 6 production. Cholesterol α-glucosyltransferase-deficient bacteria exerted strongly reduced antiproliferative effects on primary human CD4(+) T cells. In conclusion, H. pylori shapes rather than suppresses human CD4(+) T-cell responses, and glucosylated cholesterol is a relevant bacterial component involved in this modulation.     

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.Noncoding DNA in animal genomes harbors regulatory information that controls the levels and the spatiotemporal activation of gene expression (1, 2). Information is precisely organized in the 3D chromatin favoring contacts between regulatory elements and target genes (3). Subdivision into topologically associating domains (TADs), megabase-scale domains in which sequences preferentially contact one another, represents a hierarchical level in chromatin 3D organization (4–9). The limited number of studies conducted to date show that a large fraction of TADs are invariant among cell types and largely conserved in mammals (4–6, 10), but the extent of their evolutionary conservation and functional organizer properties are largely unexplored.     

High prevalence of hepatic steatosis and vascular thrombosis in COVID-19: A systematic review and meta-analysis of autopsy data

BACKGROUNDCoronavirus disease 2019 (COVID-19) disease can frequently affect the liver. Data on hepatic histopathological findings in COVID-19 is scarce.AIMTo characterize hepatic pathological findings in patients with COVID-19.METHODSWe conducted a systematic review with meta-analysis registered on PROSPERO (CRD42020192813), following PRISMA guidelines. Eligible trials were those including patients of any age and COVID-19 diagnosis based on a molecular test. Histopathological reports from deceased COVID-19 patients undergoing autopsy or liver biopsy were reviewed. Articles including less than ten patients were excluded. Proportions were pooled using random-effects models. Q statistic and I² were used to assess heterogeneity and levels of evidence, respectively.RESULTSWe identified 18 studies from 7 countries; all were case reports and case series from autopsies. All the patients were over 15 years old, and 67.2% were male. We performed a meta-analysis of 5 studies, including 116 patients. Pooled prevalence estimates of liver histopathological findings were hepatic steatosis 55.1% [95% confidence interval (CI): 46.2-63.8], congestion of hepatic sinuses 34.7% (95%CI: 7.9-68.4), vascular thrombosis 29.4% (95%CI: 0.4-87.2), fibrosis 20.5% (95%CI: 0.6-57.9), Kupffer cell hyperplasia 13.5% (95%CI: 0.6-54.3), portal inflammation 13.2% (95%CI: 0.1-48.8), and lobular inflammation 11.6% (95%CI: 0.3-35.7). We also identified the presence of venous outflow obstruction, phlebosclerosis of the portal vein, herniated portal vein, periportal abnormal vessels, hemophagocytosis, and necrosis.CONCLUSIONWe found a high prevalence of hepatic steatosis and vascular thrombosis as major histological liver features. Other frequent findings included portal and lobular inflammation and Kupffer cell hyperplasia or proliferation. Further studies are needed to establish the mechanisms and implications of these findings.     

Spontaneous portosystemic shunt embolization in liver transplant recipients with recurrent hepatic encephalopathy

Introduction and objectivesSpontaneous portosystemic shunts (SPSS) are a common cause of recurrent hepatic encephalopathy (HE). Shunt occlusion is an effective and safe procedure when performed in patients with cirrhosis and preserved liver function. We aimed to describe our experience with SPSS embolization after liver transplantation (LT).PatientsWe identified five patients who underwent SPSS embolization after LT. Clinical, biochemical and technical procedure data were collected.ResultsAt presentation, all patients had developed graft cirrhosis and HE after LT. Median Model for End-stage Liver Disease (MELD) at embolization was 9 (range 7-12), median Child-Pugh was 8 (range 7-9). Splenorenal and mesocaval shunt were the most frequent types of SPSS found. Three patients have been completely free of HE. Of the two patients who had HE recurrence after embolization, one patient had two episodes of HE which was controlled well with medications. The other patient required three embolizations because of recurrent HE. Median follow-up was 4.4 years (range 1.0-5.0) and MELD score at last follow up was 13 (range 10-18) and median Child-Pugh score B, 7 points (range 5-12).ConclusionsSPSS can be considered as a cause of HE after LT. SPSS embolization is feasible and safe in LT recipients.     

Rational polytherapy with lacosamide in clinical practice: results of a Spanish cohort analysis RELACOVA

There has been little long-term success with polytherapy for patients with refractory partial-onset epilepsy. The rational combination of antiepileptic drugs based on their mechanism of action may help improve treatment efficacy and tolerability. Lacosamide, a novel sodium channel blocker (SCB), was investigated in 158 patients with partial-onset epilepsy in the prospective, multicenter, observational, RELACOVA cohort study conducted in Spain. After 12 months' treatment with lacosamide, 47% of patients were responders (≥50% reduction in seizure frequency) and 24% were seizure free. Lacosamide was well tolerated; dizziness was the most frequent adverse event. Efficacy was better (responder rate, 65% vs 38%; seizure free rate, 35% vs 17%) and there was a lower adverse event rate (33% vs 58%) in patients receiving non-SCBs (n=49) versus those receiving SCBs (n=104) as concomitant therapy at baseline. Further investigation of lacosamide combination therapy is warranted.