Mitochondrial dysfunction in inflammatory bowel disease alters intestinal epithelial metabolism of hepatic acylcarnitines.

As the interface between the gut microbiota and the mucosal immune system, there has been great interest in the maintenance of colonic epithelial integrity through mitochondrial oxidation of butyrate, a short-chain fatty acid produced by the gut microbiota. Herein, we showed that the intestinal epithelium could also oxidize long-chain fatty acids, and that luminally delivered acylcarnitines in bile could be consumed via apical absorption by the intestinal epithelium, resulting in mitochondrial oxidation. Finally, intestinal inflammation led to mitochondrial dysfunction in the apical domain of the surface epithelium that may reduce the consumption of fatty acids, contributing to higher concentrations of fecal acylcarnitines in murine Citrobacter rodentium-induced colitis and human inflammatory bowel disease.

Linking long-term dietary patterns with gut microbial enterotypes.

Diet strongly affects human health, partly by modulating gut microbiome composition. We used diet inventories and 16S rDNA sequencing to characterize fecal samples from 98 individuals. Fecal communities clustered into enterotypes distinguished primarily by levels of Bacteroides and Prevotella.

The human gut virome: inter-individual variation and dynamic response to diet.

Immense populations of viruses are present in the human gut and other body sites. Understanding the role of these populations (the human "virome") in health and disease requires a much deeper understanding of their composition and dynamics in the face of environmental perturbation. Here, we investigate viromes from human subjects on a controlled feeding regimen.

High-fat diet determines the composition of the murine gut microbiome independently of obesity.

Background & Aims: The composition of the gut microbiome is affected by host phenotype, genotype, immune function, and diet. Here, we used the phenotype of RELMbeta knockout (KO) mice to assess the influence of these factors.

Methods: Both wild-type and RELMbeta KO mice were lean on a standard chow diet, but, upon switching to a high-fat diet, wild-type mice became obese, whereas RELMbeta KO mice remained comparatively lean.

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways.

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype.

Absence of bacterially induced RELMbeta reduces injury in the dextran sodium sulfate model of colitis.

Although inflammatory bowel disease (IBD) is the result of a dysregulated immune response to commensal gut bacteria in genetically predisposed individuals, the mechanism(s) by which bacteria lead to the development of IBD are unknown. Interestingly, deletion of intestinal goblet cells protects against intestinal injury, suggesting that this epithelial cell lineage may produce molecules that exacerbate IBD. We previously reported that resistin-like molecule beta (RELMbeta; also known as FIZZ2) is an intestinal goblet cell-specific protein that is induced upon bacterial colonization whereupon it is expressed in the ileum and colon, regions of the gut most often involved in IBD.

Activation of RegIIIbeta/gamma and interferon gamma expression in the intestinal tract of SCID mice: an innate response to bacterial colonisation of the gut.

Background And Aims: The mechanisms by which commensal bacteria provoke intestinal inflammation in animal models of inflammatory bowel disease (IBD) remain incompletely defined, leading to increasing interest in the innate immune response of the colonic mucosa to bacterial colonisation.

Methods: Using gene expression profiling of colonic RNA from C.B17.

RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract.

Gastrointestinal (GI) nematode infections are an important public health and economic concern. Experimental studies have shown that resistance to infection requires CD4(+) T helper type 2 (Th2) cytokine responses characterized by the production of IL-4 and IL-13. However, despite >30 years of research, it is unclear how the immune system mediates the expulsion of worms from the GI tract.

An open-label trial of the PPAR-gamma ligand rosiglitazone for active ulcerative colitis.

Objectives: Previous research has demonstrated that ligands for the gamma subtype of peroxisome proliferator-activated receptors (PPARs) reduce inflammation in two different murine models of colitis. This study was designed to examine the potential efficacy of rosiglitazone, a ligand for the gamma subtype of PPARs, as a therapy for active ulcerative colitis.

Methods: Fifteen patients with mild to moderately active ulcerative colitis despite therapy with 5-aminosalicylic acid compounds were enrolled in an open-label study of rosiglitazone (4 mg b.

High-level expression of I kappa B-beta in the surface epithelium of the colon: in vitro evidence for an immunomodulatory role.

The intestinal epithelium is spatially segregated into two compartments, one containing undifferentiated cells in a proliferative state and one with non-proliferative differentiated cells. Although this epithelium can produce many immunemodulating substances, emerging evidence suggests that the differentiated cell compartment is less immune responsive. Indeed, it is the differentiated cellular compartment that represents the interface between the highly antigenic luminal environment and the mucosal immune system.

A novel therapy for colitis utilizing PPAR-gamma ligands to inhibit the epithelial inflammatory response.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-gamma protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-gamma ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-kappaB via an IkappaB-alpha-dependent mechanism.

Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues.

GAP-43 is an abundant protein in axonal growth cones of developing and regenerating neurons. We found that GAP-43 was enriched in detergent-resistant membranes (DRMs) isolated by Triton X-100 extraction from PC12 pheochromocytoma cells and could be detected in detergent-insoluble plasma membrane remnants after extraction of cells in situ. GAP-43 is palmitoylated at Cys-3 and Cys-4.

Evidence that the two C1q binding membrane proteins, gC1q-R and cC1q-R, associate to form a complex.

Two types of widely coexpressed, highly acidic, cell membrane binding proteins that display preferential domain specificity for C1q have been described: a 60-kDa calreticulin homologue, designated cC1q-R, that binds to the collagen-like "stalk" and a 33-kDa glycoprotein with affinity for the globular "heads" (gC1q-R). Although the two molecules are known to be coexpressed on all cell types examined to date and often coelute during purification, there is no direct evidence showing that they associate with each other either on the membrane or when examined in a purified system. In this report we present the first evidence that 1) biotinylated cC1q-R binds to recombinant as well as native gC1q-R, as assessed by solid phase ELISA; 2) binding sites for cC1q-R are located within N-terminal residues 76 through 93 of the mature form of gC1q-R and within residues 204 through 218; 3) this interaction is inhibited by two mAbs, 60.

The C1q-binding cell membrane proteins cC1q-R and gC1q-R are released from activated cells: subcellular distribution and immunochemical characterization.

Two types of widely coexpressed cell surface C1q-binding proteins (C1q-R): a 60-kDa calreticulin-homolog which binds to the collagen-like "stalk" of C1q and a 33-kDa protein with affinity for the globular "heads" of the molecule, have been described. In this report, we show that the two molecules are also secreted by Raji cells and peripheral blood lymphocytes and can be isolated in soluble form from serum-free culture supernatant by HPLC purification using a Mono-Q column. The two purified soluble proteins had immunochemical and physical characteristics similar to their membrane counterparts in that both bound to intact C1q and to their respective C1q ligands, cC1q and gC1q.

Effect of 2',3'-dideoxycytidine on oxidative phosphorylation in the PC12 cell, a neuronal model.

Peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) could result from the previously shown inhibition of mtDNA replication by the action of ddC on the mitochondrial enzyme DNA polymerase gamma. Such inhibition would be expected to impair oxidative phosphorylation, and this was demonstrated in the present study for the PC12 cell, a model of a peripheral neuron. The dramatic rise in lactate formation upon exposure of the cell to ddC indicated that increased glycolysis was needed to produce ATP.

Cellular targets of 3'-azido-3'-deoxythymidine: an early (non-delayed) effect on oxidative phosphorylation.

Previous results demonstrated that incubation of the Friend murine erythroleukemic cell with 5 microM AZT for several days leads to a decrease in the rate of cell growth, inhibition of mtDNA replication, reduction of mtDNA per cell and per mitochondrion, and an increase in mitochondria per cell. As shown here, such treatment also leads to changes in lactate and ATP synthesis and in O2 uptake, suggesting impairment of oxidative phosphorylation. Direct measurement of ATP synthesis in mitochondria isolated from AZT-treated cells confirmed this view.

Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain.

Recent cloning of auxiliary subunits associated with voltage-gated ion channels and their subsequent coexpression with the channel forming alpha-subunits has revealed that the expression level, gating and conductance properties of the expressed channels can be profoundly affected by the presence of an auxiliary subunit polypeptide. In the present study, we raised antibodies against the beta-subunit associated with the bovine dendrotoxin sensitive K(+)-channel complex and used these antibodies to characterize the related beta-subunit polypeptides in rat brain. The anti-beta-subunit antibodies displayed a specific reaction on immunoblots of rat brain membranes with a major 38 kDa polypeptide, and a minor 41 kDa polypeptide, which correspond closely to the predicted sizes of the Kv beta 2 and Kv beta 1 beta-subunit polypeptides, respectively, recently cloned from rat brain.

Mechanisms of toxicity of 3'-azido-3'-deoxythymidine. Its interaction with adenylate kinase.

Recent experiments from our laboratory have indicated that the inhibitory effect of 3'-azido-3'-deoxythymidine (AZT) on oxidative phosphorylation may occur directly, in addition to being brought about by its inhibition of mtDNA replication. We report here studies on the effect of AZT on adenylate kinase, an enzyme crucial to oxidative phosphorylation. AZT decreased the aromatic residues fluorescence of rabbit muscle adenylate kinase, indicating binding of AZT to the enzyme.

Anti-human immunodeficiency virus type 1 therapy and peripheral neuropathy: prevention of 2',3'-dideoxycytidine toxicity in PC12 cells, a neuronal model, by uridine and pyruvate.

A strategy for preventing or delaying the peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) therapy in patients with acquired immunodeficiency syndrome was suggested by findings, in two laboratories, that cultured avian and mammalian cells devoid of mitochondrial DNA continue to replicate at virtually normal rates, provided that the medium is supplemented with uridine and pyruvate. Inasmuch as it is likely that a depletion of mitochondrial DNA also takes place in neuronal cells exposed to ddC, we used PC12 cells, the neuronal model we have reported on previously, in an attempt to rescue these cells from the deleterious effects of ddC. We first show, using undifferentiated PC12 cells, that DNA replication is impaired in mitochondria isolated from cells grown in the presence of ddC.