Biology subjects

Cox, A.

Publications and source records attributed to Cox, A..

26 records · Page 2Linked to original sources

bioRxiv2021

SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

Diarrhea occurs in 2-50% of cases of COVID-19 ([~]8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for [~]24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by [~]8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of [~]10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

physiology↗

bioRxiv2021

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism

BackgroundDogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. Methodology/Principal FindingsWe examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with moderate disease showed significantly higher transmission to the vector than dogs with very mild or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. Conclusions/SignificanceTogether, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies. AUTHOR SUMMARYSand flies transmit Leishmania parasites between infected dogs and humans leading to the life-threatening tropical disease Visceral Leishmaniasis (VL). Identifying which dogs transmit parasites well to sand flies is important to curb disease spread. The offspring of both dogs and humans can also be infected vertically while in utero. Despite this, the infectiousness of dogs that receive parasites in utero to sand flies has not been thoroughly investigated. Thus, we allowed sand flies to feed on a group of vertically infected dogs at varying stages of VL disease severity and measured sand fly parasite uptake. We found vertically infected dogs were readily able to transmit parasites to the sand flies. Dogs that were most infectious had moderate clinical disease and relatively high levels of parasite infection in their blood and skin. However, the level of skin infection was significantly higher than that observed in the blood, and the skin parasite load had the strongest correlation with sand fly parasite uptake. This implicates the skin may be an underappreciated driver of canine infectiousness to the sand fly vector. In addition, this work highlights that vertically infected dogs are very important parts of the transmission cycle and must be considered in all public health efforts addressing VL.

microbiology↗

bioRxiv2020

The D614G Mutation Enhances the Lysosomal Trafficking of SARS-CoV-2 Spike

The spike D614G mutation increases SARS-CoV-2 infectivity, viral load, and transmission but the molecular mechanism underlying these effects remains unclear. We report here that spike is trafficked to lysosomes and that the D614G mutation enhances the lysosomal sorting of spike and the lysosomal accumulation of spike-positive punctae in SARS-CoV-2-infected cells. Spike trafficking to lysosomes is an endocytosis-independent, V-ATPase-dependent process, and spike-containing lysosomes drive lysosome clustering but display poor lysotracker labeling and reduced uptake of endocytosed materials. These results are consistent with a lysosomal pathway of coronavirus biogenesis and raise the possibility that a common mechanism may underly the D614G mutations effects on spike protein trafficking in infected cells and the accelerated entry of SARS-CoV-2 into uninfected cells.

cell biology↗

bioRxiv2019

Herpes Simplex Virus type 1 Inflammasome Activation in Human Macrophages is Dependent on NLRP3, ASC, and Caspase-1

The pro-inflammatory cytokines interleukin (IL)-1{beta} and IL-18 are products of activation of the inflammasome, an innate sensing system, and important in the pathogenesis of herpes simplex type 1 (HSV-1). The release of IL-18 and IL-1{beta} from monocytes/macrophages is critical for protection from HSV-1 based on animal models of encephalitis and genital infection, yet if and how HSV-1 activates inflammasomes in human macrophages is unknown. To investigate this, we utilized both primary human monocyte derived macrophages and human monocytic cell lines (THP-1 cells) with various inflammasome components knocked-out. We found that HSV-1 activates inflammasome signaling in pro-inflammatory primary human macrophages. Additionally, HSV-1 inflammasome activation is dependent on nucleotide-binding domain and leucine-rich repeat-containing receptor 3 (NLRP3), apoptosis-associated speck-like molecule containing a caspase recruitment domain (ASC), and caspase-1, but not on absent in melanoma 2 (AIM2), or gamma interferon-inducible protein 16 (IFI16). Ultraviolet irradiation of HSV-1 enhanced inflammasome activation, demonstrating that viral replication suppresses inflammasome activation. These results confirm that HSV-1 is capable of activating the inflammasome in human macrophages through an NLRP3 dependent process and that the virus has an NLRP3 specific mechanism to inhibit inflammasome activation in monocytes and macrophages.\n\nAuthor SummaryThe inflammasome is a multi-protein complex that forms in response to pathogens and cellular damage. Active inflammasomes recruit pro-caspase-1 via ASC and cleave the cytokine precursors pro-IL-1{beta} and pro-IL-18 into mature IL-1{beta} and IL-18. These cytokines serve to activate other immune cells that either repair the damage or attempt to clear the invading pathogen. Upon activation, the inflammasome also promotes an inflammatory form of cell death called pyroptosis. Herpes simplex virus type 1 (HSV-1) is a common human pathogen that can cause cold sores, genital ulcers, encephalitis, and blindness. HSV-1 infection leads to induction of IL-1{beta} and IL-18, but whether it is capable of activating inflammasomes in macrophages, which play a role in severe forms of HSV-1 infection, was unclear. Here, we infected both primary human macrophages and a macrophage/monocytic cell line, THP-1 cells, with HSV-1. We found that HSV-1 does activate inflammasome signaling in macrophages in a process dependent on NLRP3, ASC, and caspase-1. This is important because it illustrates the mechanism by which HSV-1 infection leads to inflammasome activation in macrophages, known to be crucial for protection from severe disease in mouse models.

microbiology↗

bioRxiv2019

Common breast cancer risk loci predispose to distinct tumor subtypes

BackgroundGenome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER), but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear. MethodsAmong 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes. ResultsEighty-five of 173 variants were associated with at least one tumor feature (false discovery rate <5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at P<0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions. ConclusionThis report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction.

genetics↗

bioRxiv2019

Missense mutations in the MLKL brace region lead to lethal neonatal inflammation in mice and are present in high frequency in humans.

We have isolated a mouse strain with a single missense mutation in the gene encoding MLKL, the essential effector of necroptotic cell death. The resulting substitution lies within the two-helix brace and confers constitutive, RIPK3 independent, killing activity to MLKL. Mice homozygous for MlklD139V develop lethal inflammation within days of birth, implicating the salivary glands and pericardium as hotspots for necroptosis and inflammatory infiltration. The normal development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, CRMO.

cell biology↗

bioRxiv2018

Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap-/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis: transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap-/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improved Yap-deficiency phenotypes, indicating functional importance of glucose-fuelled nucleotide biosynthesis. Yap-regulated Glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signalling in metabolic homeostasis.

developmental biology↗

bioRxiv2017

Fine-mapping of genetic loci driving spontaneous clearance of hepatitis C virus infection

Approximately three quarters of acute HCV infections evolve to a chronic state, while one quarter are spontaneously cleared. Genetic predispositions strongly contribute to the development of chronicity. We have conducted a genome-wide association study to identify genomic variants underlying HCV spontaneous clearance using Immunochip in European and African ancestries. We confirmed two previously reported significant associations, in the IL28B/IFNL41,2 and MHC regions, with spontaneous clearance in the European population. We further fine-mapped the MHC association to a region of about 50 kilo base pairs, down from 1 mega base pairs in the previous study. Additional analyses suggested that the association in the MHC locus might be significantly stronger for virus subtype 1a than 1b, suggesting that viral subtype may have influenced the genetic mechanism underlying the clearance of HCV.

immunology↗