Month: February 2019

Co-infection with helminths is also known to attenuate murine models of autoimmunity and inflammatory bowel disease. Despite being classed as pathogens, helminth parasites are being proposed as treatments for allergic and autoimmune diseases. Preliminary observations suggest that intentional infection with the zoophilic Trichuris suis is safe, and reduces the activity of inflammatory bowel disease. However, dosing with T. suis every three weeks is required to maintain ongoing infection because the human host does not sustain development of T. suis to maturity. Necator americanus is a long-lived hematophagous, humanspecific gastrointestinal nematode that infects over 500 million people in developing countries where heavy infection causes iron deficiency anemia, and is associated with reduced physical and intellectual development. Experimental infection of healthy volunteers with NA infective third-stage larvae may cause an acute, painful enteropathy. Recent published data indicate that low-dose inocula of NA are better tolerated, and because they do not proliferate in humans, a defined dose can be administered and later fully eliminated with anthelmintic therapy. A further advantage is infected individuals pose no risk to others because hookworms are soil-transmitted and cannot be propagated in modern sanitary environments. We chose NA and celiac disease to explore the relationship between helminth infection and intestinal inflammation due to a well 2-O-galloylhyperin characterised dietary antigen, gluten. Our previous studies with this hookworm have provided us with a pure source of infective larvae. Individuals carrying chronic infection with hookworms in endemic settings demonstrate parasite-specific TH2 responses, but TH1 and TH2 immune responses to other antigens are diminished. Celiac disease is uniquely suited to explore the effects of helminth infection; it is common and remission is achieved with elimination of dietary gluten allowing host-parasite interaction to be studied free of potential artefacts caused by medications. Clinical studies in volunteers with CD also have the advantage that the effects of deliberate gluten exposure can be measured by symptom response,LOUREIRIN-B in blood and intestinal tissue. Over 90% of people with CD possess genes encoding the major histocompatability Class II molecule, HLA-DQ2, and HLA DQ2-restricted CD4 + T cells specific for deamidated gluten peptides can be isolated from intestinal tissue. HLA DQ2restricted CD4 + TH1 cells specific for deamidated gluten including the immunodominant a-gliadin 17-mer p57-73 peptide are also present in blood after oral wheat challenge. In this study, we undertook a clinical trial to test whether NA infection reduces the immunotoxic effects of gluten in CD. All vials and pipettes were examined following inoculation to ensure that there were no residual hookworm larvae. We propose that chronic helminthiasis, such as hookworm infection, may be immunomodulatory and alter pathogenic immune responses in vivo. In this Phase 1b/2a trial, we have established an experimental model allowing us to explore how hookworm infection alters the effects of gluten in CD. Chronic hookworm infection can be reliably and safely established in subjects with well controlled disease. Lacking precedent, the size of this demanding study was small. We observed at best weak trends towards reduced numbers of gluten peptide-specific T cells in blood and histological damage following wheat challenge in CD. As has been reported in another trial, standard fecal microscopy is relatively insensitive in light infection; a negative test does not exclude colonization. All patients inoculated acquired adult hookworms in the intestine.

The pathophysiological significance of this role in intestinal barrier protection is underscored by the impact of GUCY2C signaling on colitis and systemic genotoxicity and tumorigenesis. In turn, these considerations highlight the translational opportunities for GUCY2C ligands in the prevention and treatment of inflammatory bowel disease and extra-intestinal malignancies. This therapeutic potential is underscored by the recent advancement of the oral GUCY2C ligand linaclotide for FDA approval for the treatment of constipation-type irritable bowel syndrome. In mammals, there are four Par-1 homologs that comprise the MARK family. This family consists of four closely related proteins that have been shown to play a role in cell polarity, microtubule stability, protein stability, and cell cycle control. Although similar in structure, the MARK proteins have different subcellular localizations. Phosphorylation of many MARK targets generates a 14-3-3 binding site. 14-3-3 regulates the subcellular localization of many proteins. Two mark2-/- mouse lines have been independently generated that implicate MARK2 in the regulation of immune homeostasis, fertility, learning, memory, growth and metabolism. C-TAK1 has been implicated in pancreatic, liver, and colorectal cancers, hippocampal function, and metabolism. In C. elegans Par-1 plays Corosolic-acid a negative role in vulva induction and may function by negatively regulating the scaffolding protein KSR1. In mammalian cells, C-TAK1 has been shown to negatively regulate KSR1 by phosphorylation of Ser392. Phosphorylation of this site sequesters KSR1 in the cytoplasm. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase pathway. KSR1 enhances Raf-1 activity in a kinase-independent manner. Proteomic analys reveal that MARK2 interacts with KSR1 and we have shown that MARK2, similar to C-TAK1, is able to phosphorylate KSR1 in vitro on S392. This phosphorylation site has been shown previously to be a negative regulatory site of KSR1. This result predicts that MARK2 negatively regulates KSR1 as an ERK scaffold. However, it is also possible that KSR1 serves as a scaffold for MARK2 similar to the interaction of KSR1 with ERK. As the MARK family contains multiple members, it is possible that other members of the MARK family are able to compensate for the loss of MARK2. However,Acetylcorynoline though the family has a high degree of homology, the subcellular localization varies. MARK1, MARK2, and C-TAK1 are all basolateral, but C-TAK1 is also found on the apical surface. MARK4 does not display asymmetric localization, but interacts with filamentous structures. The family members are also differentially regulated. MARK2 localizes to the cytoplasm upon overexpression of PKCf. However, MARK1 and C-TAK1 do not alter their localization when PKCf is overexpressed. This observation suggests that other members of the MARK family may not fully compensate for the loss of MARK2. These data raise the possibility that different stimuli could selectively recruit related members of a kinase family to impair KSR1 function through phosphorylation of a common site. In this model, KSR1 would integrate different signals to the same effect. These mechanisms may allow KSR1 to respond to signals in different cell types or in multiple subcellular compartments. The potential of KSR1 to receive input at the same site from multiple kinases may also affect the intensity and/or duration of KSR1mediated signaling by increasing the stoichiometry of KSR1 phosphorylation on Ser392. Observational studies supporting this hypothesis include a report that infection with the nematode Strongyloides stercoralis is associated with protection against autoimmune liver disease, and another showing that unspecified helminth infections with peripheral eosinophilia are associated with reduced autoantigen-specific responses and disease progression in multiple sclerosis.

Indeed, genetic or chemical disruption of the intestinal barrier associated with inflammation produces systemic DNA damage and genotoxic stress, including in circulating leukocytes. Here, barrier disruption and hyperpermeability produced by silencing GUCY2C was associated with elevated levels of oxidative DNA damage in circulating leukocytes. Conversely, defending barrier integrity and restricting permeability by activating GUCY2C with oral ST decreased systemic genotoxicity in hepatocytes produced by DSS. Further, systemic genotoxicity produced by barrier disruption reflecting GUCY2C silencing was associated with increased spontaneous and carcinogen-induced extra-intestinal tumorigenesis, specifically in lung, liver and lymph nodes. In the context of the role of GUCY2C as a tissue-specific tumor suppressing receptor, the present observations expand that function beyond transformation in the colorectum, to neoplasia in lymph nodes, liver, and lung through maintenance of epithelial barrier integrity. Moreover, it is tempting to speculate that loss of paracrine hormone expression and silencing of GUCY2C in inflammatory bowel disease produces systemic genotoxicity which contributes to the pattern of extra-intestinal cancer in these patients specifically in lymph nodes, liver,Herbacetin and lung. The present study further clarifies the evolving role of the GUCY2C paracrine hormone system in modulating the intestinal barrier. A role for GUCY2C in regulating epithelial barrier function was recently demonstrated, and the present work confirms that phenomenon. Those studies revealed that silencing GUCY2C disrupted the barrier and induced inflammation, associated with production of interferon gamma and IL-12. In turn, those events were associated with increased expression of epithelial cell myosin light chain kinase, an established regulator of tight junction structure and function, and suppression of JAM-A and claudin 2 expression. These phenomena were presumed to be the molecular mechanisms mediating GUCY2C-dependent barrier disruption. However, canonical regulation of myosin light chain phosphorylation by cGMP is mediated by myosin light chain phosphatase, rather than myosin light chain kinase. Moreover, these observations are complicated by the contribution of inflammatory cytokines,Diosgenin-glucoside including interferon gamma, to regulation of expression of myosin light chain kinase in intestinal epithelial cells in vivo and in vitro. Thus, the contribution of these components may not reflect the primary mechanism underlying GUCY2C regulation of barrier integrity but, rather, an epiphenomenon related to the associated inflammation. In contrast, the present study demonstrates a mechanistic role for GUCY2C as a primary modulator of the intestinal epithelial barrier, mediated by AKT1, occludin and claudin 4, but not JAM-A and claudin 2, in mouse models in vivo, and in human intestinal cell monolayers in vitro in the absence of confounding by inflammation. Paradoxically, subsequent work from the same group suggested that eliminating GUCY2C signaling protects the intestinal mucosa from inflammatory injury. Beyond proliferation, metabolism, and chromosomal integrity, the present studies reveal an additional dimension of AKT1dependent epithelial homeostatic regulation by GUCY2C. Indeed, it is tempting to speculate that one essential function of GUCY2C signaling is maintenance of intestinal barrier integrity. In that context, regulation of epithelial tight junctions contributing to macromolecular permeability described here can be added to other established GUCY2C functions, including fluid and electrolyte secretion accelerating lumenal clearance; differentiation of Paneth cells producing antimicrobial peptides; and differentiation of goblet cells producing intestinal mucus that contribute to the separation of systemic and environmental compartments across the mucosal interface.

The present studies extend this homeostatic role of GUCY2C beyond proliferation, differentiation, migration, DNA damage sensing and repair, and metabolism to maintenance of macromolecular permeability and the intestinal barrier. Elimination of GUCY2C signaling produced basal macromolecular hyperpermeability and potentiated barrier disruption resulting in colitis induced by DSS. Conversely, genetic or pharmacological induction of GUCY2C signaling reduced basal permeability and DSS-induced hyperpermeability and inflammation. Results in mouse models were recapitulated in monolayers of human intestinal epithelial cells in vitro, in the absence of other confounding physiological mechanisms like inflammation,Epigoitrin demonstrating a primary mechanistic role for GUCY2C in regulating the epithelial barrier and macromolecular permeability. Moreover, protection of the epithelial barrier by GUCY2C in vivo and in vitro was associated with alterations in steady state concentrations of tight junction proteins, including occludin and claudin 4. Regulation of steady-state concentrations of these components are one established mechanism modulating the dynamic assembly and deployment of tight junction complexes, barrier integrity and macromolecular permeability. Integration of crypt-villus homeostatic programs mediated by GUCY2C is coordinated through AKT1. Ligand activation of GUCY2C and accumulation of cGMP produces dephosphorylation of AKT1, modulating downstream signaling circuits regulating proliferation and metabolism. In the present study,Isoforskolin homeostatic regulation of epithelial barrier function and permeability by GUCY2C was mediated by AKT1. In that context, specifically reducing AKT1, but not AKT2, blocked alterations in occludin and claudin 4, but not JAM-A and claudin 2, and macromolecular hyperpermeability induced by eliminating GUCY2C signaling in mice. Further, in monolayers of human intestinal epithelial cells in vitro, eliminating AKT1 expression induced steady state levels of occludin and claudin 4, but not JAMA and claudin 2, and barrier function to levels achieved by ST activation of GUCY2C. Moreover, a constitutively activated form of AKT1 produced resistance to, while eliminating AKT1 expression from these cells mimicked, the effects of GUCY2C activation on epithelial cell permeability. Together, these results demonstrate a key mechanistic role for suppression of AKT1 signaling in the regulation of epithelial cell barrier function by GUCY2C. These results reinforce the established role of AKT in regulating the expression of junctional proteins, disrupting epithelial cell membrane complexes, and inducing barrier dysfunction. Further, they identify occludin and claudin 4, but not JAM-A and claudin 2, as specific downstream targets of AKT1 contributing to the regulation of epithelial barrier permeability in vitro and in vivo. They underscore the integrated homeostatic role of GUCY2C signaling, centrally coordinated through AKT, in regulating intestinal epithelial structure and function. There is an established mechanistic relationship between disruption of the intestinal epithelial barrier, inflammation resulting from systemic immune exposure to normally compartmentalized lumenal antigens, and the generation of reactive oxygen species producing DNA damage and genotoxic stress. In turn, reactive oxygen species producing DNA damage comprise one mechanism contributing to tumorigenesis generally, and to the evolution of colon cancer in the context of inflammatory bowel disease, specifically. Importantly, there is emerging recognition that this oxidative stress extends beyond the primary site of barrier disruption and inflammation, and is disseminated systemically.

These observations suggest that bacterial cells might sense the molecular ratio between UMP and intermediates in the de novo UMP biosynthesis such as carbamoyl-L-aspartate, which accumulates in the pyrC mutant strain, as a signal of the relative balance between the two UMP biosynthetic pathways. An unbalance towards UMP biosynthesis via the SKI II pyrimidine salvage pathway triggers cellulose production, and this effect relies on the activity of the diguanylate cyclase YedQ. The interplay between nucleotide salvage pathway and cellulose production might be connected to the role of cellulose and other EPS in the response to environmental stresses such as desiccation and resistance to bacteriophages. In bacterial biofilms, events leading to extensive cell lysis, such as exposure to antibiotics or attack by bacteriophages, would release cell components into the local environment: thus, a sudden increase in concentrations of exogenous nucleotides due to bacterial lysis might function as an ����alarm signal���� to neighboring cells, which would react by producing EPS as a defense mechanism against environmental stresses. For intracellular pathogenic enterobacteria, sensing an increase of exogenous nucleotide concentration might instead signal Qingyangshengenin-B stress events in the host cell, such as leakage of nucleotides from the nuclear compartment. Consistent with our observations, it has been reported that allosteric inhibition of the CarB protein by exogenous uracil strongly influences production of extracellular structures and negatively affects expression of type III secretion systems in the intracellular pathogen Shigella flexneri. In Pseudomonas fluorescens, a spontaneous mutation in the carB gene affects the proportion of capsulated and non-capsulated subpopulations via yet unknown molecular mechanisms. Our results complement and expand these observations, and underline the importance of the interplay linking biofilm formation, bacterial virulence, production of extracellular structures, and nucleotide biosynthetic pathways: better understanding of these connections at the molecular level will allow us to improve our strategies in preventing bacterial biofilms. In this perspective, our results provide strong evidence to confirm previous findings suggesting that drugs targeting nucleotide biosynthetic pathways have a strong potential as antibiofilm agents. The mammalian placenta is the first organ to be developed during gestation and carries out multiple functions required for normal embryonic development in the uterine environment. Impaired placental development is associated with many complications to both moms and babies during pregnancy, including preeclampsia, intrauterine growth retardation and fetal loss.