Leucine Rich Repeat Containing 8A (LRRC8A) is a required component of volume-regulated anion channels (VRACs). In vascular smooth muscle cells, tumor necrosis factor-α (TNFα) activates VRAC via type 1 TNFα receptors (TNFR1), and this requires superoxide (O2•−) production by NADPH oxidase 1 (Nox1). VRAC inhibitors suppress the inflammatory response to TNFα by an unknown mechanism. We hypothesized that LRRC8A directly supports Nox1 activity, providing a link between VRAC current and inflammatory signaling. VRAC inhibition by 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) impaired NF-κB activation by TNFα. LRRC8A siRNA reduced the magnitude of VRAC and inhibited TNFα-induced NF-κB activation, iNOS and VCAM expression, and proliferation of VSMCs. Signaling steps disrupted by both siLRRC8A and DCPIB included; extracellular O2•− production by Nox1, c-Jun N-terminal kinase (JNK) phosphorylation and endocytosis of TNFR1. Extracellular superoxide dismutase, but not catalase, selectively inhibited TNFR1 endocytosis and JNK phosphorylation. Thus, O2•− is the critical extracellular oxidant for TNFR signal transduction. Reducing JNK expression (siJNK) increased extracellular O2•− suggesting that JNK provides important negative feedback regulation to Nox1 at the plasma membrane. LRRC8A co-localized by immunostaining, and co-immunoprecipitated with, both Nox1 and its p22phox subunit. LRRC8A is a component of the Nox1 signaling complex. It is required for extracellular O2•− production, which is in turn essential for TNFR1 endocytosis. These data are the first to provide a molecular mechanism for the potent anti-proliferative and anti-inflammatory effects of VRAC inhibition.

The protozoan Leishmania chagasi can cause disseminated, fatal visceral leishmaniasis (VL) or asymptomatic infection in humans. We hypothesized that host genetic factors contribute to this variable response to infection. A family study was performed in neighborhoods of endemicity for L. chagasi near Natal in northeastern Brazil. Study subjects were assessed for the presence of VL or asymptomatic infection, which was defined by a positive delayed-type hypersensitivity (DTH) skin test response to Leishmania antigen without disease symptoms. A genomewide panel of 385 autosomal microsatellite markers in 1254 subjects from 191 families was analyzed to identify regions of linkage. Regions with potential linkage to the DTH response on chromosomes 15 and 19, as well as a novel region on chromosome 9 with potential linkage to VL, were identified. Understanding the genetic factors that determine whether an individual will develop symptomatic or asymptomatic infection with L. chagasi may identify proteins essential for immune protection against this parasitic disease and reveal strategies for immunotherapy or prevention.

Visceral leishmaniasis (VL) caused by Leishmania chagasi is endemic to northeast Brazil. A positive delayed-type hypersensitivity skin test response (DTH þ ) is a marker for acquired resistance to disease, clusters in families and may be genetically controlled. Twenty-three single nucleotide polymorphisms (SNPs) were genotyped in the cytokine 5q23.3-q31.1 region IRF1-IL5-IL13-IL4-IL9-LECT2-TGFBI in 102 families (323 DTH þ ; 190 DTHÀ; 123 VL individuals) from a VL endemic region in northeast Brazil. Data from 20 SNPs were analyzed for association with DTH þ /À status and VL using family-based, stepwise conditional logistic regression analysis. Independent associations were observed between the DTH þ phenotype and markers in separate linkage disequilibrium blocks in LECT2 (OR 2.25; P ¼ 0.005; 95% CI ¼ 1.28-3.97) and TGFBI (OR 1.94; P ¼ 0.003; 95% CI ¼ 1.24-3.03). VL child/parent trios gave no evidence of association, but the DTHÀ phenotype was associated with SNP rs2070874 at IL4 (OR 3.14; P ¼ 0.006; 95% CI ¼ 1.38-7.14), and SNP rs30740 between LECT2 and TGFBI (OR 3.00; P ¼ 0.042; 95% CI ¼ 1.04-8.65). These results indicate several genes in the immune response gene cluster at 5q23.3-q31.1 influence outcomes of L. chagasi infection in this region of Brazil.

Visceral leishmaniasis is a potentially fatal infectious disease caused by the protozoan parasite Leishmania infantum/chagasi in the New World, or by L. donovani or L. infantum/chagasi in the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. We reasoned that events occurring during the initial few hours when the parasite encounters cells of the innate and adaptive immune systems are likely to influence the eventual immune response that develops. Therefore, we performed gene expression analysis using Affymetrix U133Plus2 microarray chips to investigate a model of early infection with human monocyte-derived macrophages (MDMs) challenged with wild-type L. chagasi parasites, with or without subsequent co-culture with Leishmania-naïve, autologous T-cells. Microarray data generated from total RNA were analyzed with software from the Bioconductor Project and functional clustering and pathway analysis were performed with DAVID and Gene Set Enrichment Analysis (GSEA), respectively. Many transcripts were down-regulated by infection in cultures containing macrophages alone, and the pattern indicated a lack of a classically activated phenotype. By contrast, the addition of autologous Leishmania-naïve T cells to infected macrophages resulted in a pattern of gene expression including many markers of type 1 immune cytokine activation (IFN-γ, IL-6, IL-1α, IL-1β). There was simultaneous up-regulation of a few markers of immune modulation (IL-10 cytokine accumulation; TGF-β Signaling Pathway). We suggest that the initial encounter between L. chagasi and cells of the innate and adaptive immune system stimulates primarily type 1 immune cytokine responses, despite a lack of classical macrophage activation. This local microenvironment at the site of parasite inoculation may determine the initial course of immune T-cell development.

BackgroundPrevious findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome.Methodology/Principal FindingsTo understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response.ConclusionsResults show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48–96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

SummaryVisceral leishmaniasis (VL) in northeast Brazil is a disease caused by infection with the protozoan Leishmania chagasi. Infection leads to variable clinical outcomes ranging from asymptomatic infection to potentially fatal disease. Prior studies suggest the genetic background of the host contributes to the development of different outcomes after infection, although it is not known if ancestral background itself influences outcomes. VL is endemic in peri-urban areas around the city of Natal in northeast Brazil. The population of northeast Brazil is a mixture of distinct racial and ethnic groups. We hypothesized that some sub-populations may be more susceptible than others to develop different clinical outcomes after L. chagasi infection. Using microsatellite markers, we examined whether admixture of the population as a whole, or markers likely inherited from a distinct ethnic background, differed between individuals with VL, individuals with an asymptomatic infection, or individuals with no infection. There was no apparent significant difference in overall population admixture proportions among the three clinical phenotype groups. However, one marker on Chr. 22 displayed evidence of excess ancestry from putative ancestral populations among different clinical phenotypes, suggesting this region may contain genes determining the course of L. chagasi infection.

In this report, the authors describe the case of a teenage boy who presented with hypertensive emergency, posterior reversible encephalopathy syndrome, and hydrocephalus due to fourth ventricle outlet obstruction. Posterior reversible encephalopathy syndrome is a well-characterized but uncommon syndrome in children that is generally triggered by severe hypertension. The unusual clinical picture of this patient, who had isolated cerebellar edema leading to obstructive hydrocephalus, has been rarely described in children.

Background/aim Low-cost commercial bCPAP devices have been deployed in resource-limited settings to treat neonatal respiratory failure. The use of these devices has increased access to pediatric respiratory support for infants. However, constrained resources may result in substitution of recommended consumables and/or use in older age groups. We hypothesized that commercially available bCPAP devices, the standard WHO-style device and various improvised adaptations would all generate effective, safe positive pressure at the patient interface. Methods Performance of 2 commercially available bCPAP devices was tested against the standard WHO-style bCPAP device, as well as several improvised modifications of these devices, by measuring positive pressure delivered at the patient interface. Variables tested included different flow rates, patient interfaces and respiratory circuit tubing. Results Both commercial devices utilized according to manufacturer recommendations generated the expected positive pressure at the patient interface. When testing the recommended WHO-style bCPAP device with recommended materials as well as other improvised modifications, we found variable and potentially unpredictable generation of positive pressure at the patient interface. Conclusions Modified or improvised bCPAP devices should be used with extreme caution as the support provided may be more or less than expected depending on respiratory tubing and flow rates employed. Our data support the effectiveness of bCPAP in newborns and young infants. But, to our knowledge, there are no bCPAP patient interfaces for older children effective with low liter flow devices. Therefore, based on these results, we recommend against using WHO-style bCPAP devices for non-infant patients with respiratory failure and instead recommend using standard oxygen therapy with nasal cannulae or face-masks, as well as early consideration of transfer to a higher level of care.