Discovery of Novel Oleamide Analogues as Brain-Penetrant Positive Allosteric Serotonin 5-HT2C Receptor and Dual 5-HT2C/5-HT2A Receptor Modulators.

The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR localize to the brain and share overlapping signal transduction facets that contribute to their roles in cognition, mood, learning, and memory. Achieving selective targeting of these receptors is challenged by the similarity in their 5-HT orthosteric binding pockets. A fragment-based discovery approach was employed to design and synthesize novel oleamide analogues as selective 5-HT2CR or dual 5-HT2CR/5-HT2AR positive allosteric modulators (PAMs). Compound 13 (JPC0323) exhibited on-target properties, acceptable plasma exposure and brain penetration, as well as negligible displacement to orthosteric sites of ∼50 GPCRs and transporters. Furthermore, compound 13 suppressed novelty-induced locomotor activity in a 5-HT2CR-dependent manner, suggesting 5-HT2CR PAM, but not 5-HT2AR, activity at the level of the whole organism at the employed doses of 13. We discovered new selective 5-HT2CR PAMs and first-in-class 5-HT2CR/5-HT2AR dual PAMs that broaden the pharmacological toolbox to explore the biology of these vital receptors.

Disruption of retinol-mediated IL-6 expression in colon cancer-associated fibroblasts: new perspectives on the role of vitamin A metabolism.

Stromal myo-/fibroblasts (MFs) account for up to 30% of lamina propria cells in the normal human colon and their number is dramatically increased in colon cancer (CRC). Fibroblasts from cancers, also known as cancer-associated fibroblasts (CAFs), differ from normal colonic MF (N-MFs) and support tumor-promoting inflammation, in part due to increased IL-6 secretion. In this editorial, we highlight recent data obtained regarding IL-6 regulation in colorectal cancer CAFs through vitamin A (retinol) metabolism, discuss current limitations in our understanding of the mechanisms leading to the CAF pro-inflammatory phenotype, and discuss potential approaches to target CAF retinoid metabolism during CRC treatment.

Loss of alcohol dehydrogenase 1B in cancer-associated fibroblasts: contribution to the increase of tumor-promoting IL-6 in colon cancer.

BACKGROUND: Increases in IL-6 by cancer-associated fibroblasts (CAFs) contribute to colon cancer progression, but the mechanisms involved in the increase of this tumor-promoting cytokine are unknown. The aim of this study was to identify novel targets involved in the dysregulation of IL-6 expression by CAFs in colon cancer. METHODS: Colonic normal (N), hyperplastic, tubular adenoma, adenocarcinoma tissues, and tissue-derived myo-/fibroblasts (MFs) were used in these studies. RESULTS: Transcriptomic analysis demonstrated a striking decrease in alcohol dehydrogenase 1B (ADH1B) expression, a gene potentially involved in IL-6 dysregulation in CAFs. ADH1B expression was downregulated in approximately 50% of studied tubular adenomas and all T1-4 colon tumors, but not in hyperplastic polyps. ADH1B metabolizes alcohols, including retinol (RO), and is involved in the generation of all-trans retinoic acid (atRA). LPS-induced IL-6 production was inhibited by either RO or its byproduct atRA in N-MFs, but only atRA was effective in CAFs. Silencing ADH1B in N-MFs significantly upregulated LPS-induced IL-6 similar to those observed in CAFs and lead to the loss of RO inhibitory effect on inducible IL-6 expression. CONCLUSION: Our data identify ADH1B as a novel potential mesenchymal tumor suppressor, which plays a critical role in ADH1B/retinoid-mediated regulation of tumor-promoting IL-6.

Characterization of Adipose-Derived Stem Cells Following Burn Injury.

Severe burns induce a prolonged inflammatory response in subcutaneous adipose tissue that modulates signaling in adipose-derived stem cells (ASCs), which hold potential for healing burn wounds or generating skin substitutes. Using a 60% rat scald burn model, we conducted a series of experiments to determine which cells isolated from the adipose tissue produced inflammatory mediators and how these changes affect ASC fate and function. The stromal vascular fraction (SVF), adipocytes, and ASCs were isolated from adipose tissue at varying times up to 4 weeks postburn and from non-injured controls. Endpoints included inflammatory marker expression, expression of ASC-specific cell-surface markers, DNA damage, differentiation potential, and proliferation. Inflammatory marker expression was induced in adipocytes and the SVF at 24 and 48 h postburn; expression of inflammatory marker mRNA transcripts and protein returned to normal in the SVF isolated 1 week postburn. In enriched ASCs, burns did not alter cell-surface expression of stem cell markers, markers of inflammation, differentiation potential, or proliferative ability. These results suggest that adipocytes and the SVF produce large quantities of inflammatory mediators, but that ASCs do not, after burns and that ASCs are unaffected by burn injury or culturing procedures.. They also suggest that cells isolated over 48 h after injury are best for cell culture or tissue engineering purposes.

Effects of the nephrilin peptide on post-burn glycemic control, renal function, fat and lean body mass, and wound healing.

The mechanisms underlying the effects of severe burn trauma are not well understood. We previously demonstrated the ability of nephrilin peptide (an iron-binding peptide believed to enter cells through iron-uptake pathways) to suppress aspects of the neuroinflammatory response in a rat scald model, as well as sepsis mortality in a mouse model. This study explores the effect of nephrilin on other clinically relevant outcomes in the rat scald model. In a rat scald model, animals were treated with nephrilin either in week 1 or week 2 post-burn. Measurements were made of serum glucose and creatinine as well as wound area by planimetry and body composition by DEXA. Given the potential role of iron, results were analyzed both for the entire cohort of animals and for the normoferremic (>100 ug/dL serum iron) subset of animals. Nephrilin improved body composition, wound healing, kidney function, and glycemic control. The first two effects were significant in normoferremic but not in hypoferremic animals suggesting an effect of iron status on burn injury outcomes. Nephrilin treatment modulates a number of relevant variables in the rat scald model.

Topically applied metal chelator reduces thermal injury progression in a rat model of brass comb burn.

UNLABELLED: Oxidative stress may be involved in the cellular damage and tissue destruction as burn wounds continues to progress after abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of Livionex formulation (LF) lotion, that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent or reduce burns. METHODS: We used an established brass comb burn model with some modifications. Topical application of LF lotion was started 5 min post-burn, and repeated every 8 h for 3 consecutive days. Rats were euthanized and skin harvested for histochemistry and immunohistochemistry. Formation of protein adducts of 4-hydroxynonenal (HNE), malonadialdehyde (MDA) and acrolein (ACR) and expression of aldehyde dehydrogenase (ALDH) isozymes, ALDH1 and ALDH2 were assessed. RESULTS: LF lotion-treated burn sites and interspaces showed mild morphological improvement compared to untreated burn sites. Furthermore, the lotion significantly decreased the immunostaining of lipid aldehyde-protein adducts including protein -HNE, -MDA and -ACR adducts, and restored the expression of aldehyde dehydrogenase isozymes in the unburned interspaces. CONCLUSION: This data, for the first time, demonstrates that a topically applied EDTA-containing lotion protects burns progression with a concomitant decrease in the accumulation of reactive lipid aldehydes and protection of aldehyde dehydrogenase isozymes. Present studies are suggestive of therapeutic intervention of burns by this novel lotion.

Role of COX-2 in the bioactivation of methylenedianiline and in its proliferative effects in vascular smooth muscle cells.

4,4'-Methylenedianiline (DAPM) is an aromatic diamine used directly in the production of polyurethane foams and epoxy resins, or as a precursor to MDI in the manufacture of some polyurethanes. In our prior experiments, we showed that chronic, intermittent treatment of female rats with DAPM resulted in vascular medial hyperplasia of pulmonary arteries. In addition, treatment of vascular smooth muscle cells (VSMC) in culture with DAPM increased the rates of proliferation in a manner that was inhibited by co-treatment with N-acetylcysteine but was not associated with oxidative stress. We thus hypothesized that NAC treatment inhibited DAPM toxicity by competing for binding reactive intermediates formed through DAPM metabolism. Because the peroxidase enzyme cyclooxygenase is constitutively expressed in VSMC, and because cyclooxygenase is known to metabolize similar aromatic amines to electrophilic intermediates, we further hypothesized that DAPM-induced VSMC proliferation was dependent upon COX-1/2-mediated bioactivation. To test this hypothesis, we treated VSMC with DAPM and measured cell proliferation, COX-2 expression, COX-1/2 activity, and levels of covalent binding. DAPM treatment resulted in a dose-dependent increase in proliferation that was abolished by co-treatment with the COX-2-selective inhibitor celecoxib. In addition, DAPM exposure increased the rates of proliferation in VSMC isolated from wild-type but not COX-2 (-/-) mice. Paradoxically, treatment with DAPM reduced the cellular production of PGE(2) and PGF(2α), but dose-dependently increased the COX-2 protein levels. Covalent binding of [(14)C]-DAPM to VSMC biomolecules was greater in wild-type than in COX-2 (-/-) cells. However, covalent binding of [(14)C]-DAPM was not altered by co-treatment with a nonselective inhibitor of cytochromes P450. These studies thus suggest that DAPM-induced VSMC proliferation may be due to bioactivation of DAPM, perhaps through the action of cyclooxygenase. The data furthermore suggest that DAPM's mechanism of action may possibly involve inhibition or suicide inactivation of COX-2. In addition, because we observed an increase in DAPM-induced VSMC proliferation in cells isolated from female compared to male rats, further studies into the potential interplay between DAPM, the estrogen receptor, and COX-2 seem warranted.

Indian hedgehog regulates intestinal stem cell fate through epithelial-mesenchymal interactions during development.

BACKGROUND & AIMS: Intestinal stem cells (ISCs) are regulated by the mesenchymal environment via physical interaction and diffusible factors. We examined the role of Indian hedgehog (Ihh) in mesenchymal organization and the mechanisms by which perturbations in epithelial-mesenchymal interactions affect ISC fate. METHODS: We generated mice with intestinal epithelial-specific disruption of Ihh. Gross and microscopic anatomical changes were determined using histologic, immunohistochemical, and in situ hybridization analyses. Molecular mechanisms were elucidated by expression profiling and in vitro analyses. RESULTS: Deletion of intestinal epithelial Ihh disrupted the intestinal mesenchymal architecture, demonstrated by loss of the muscularis mucosae, deterioration of the extracellular matrix, and reductions in numbers of crypt myofibroblasts. Concurrently, the epithelial compartment had increased Wnt signaling, disturbed crypt polarity and architecture, defective enterocyte differentiation, and increased and ectopic proliferation that was accompanied by increased numbers of ISCs. Mechanistic studies revealed that Hh inhibition deregulates bone morphogenetic protein signaling, increases matrix metalloproteinase levels, and disrupts extracellular matrix proteins, fostering a proliferative environment for ISCs and progenitor cells. CONCLUSIONS: Ihh regulates ISC self-renewal and differentiation. Intestinal epithelial Ihh signals to the mesenchymal compartment to regulate formation and proliferation of mesenchymal cells, which in turn affect epithelial proliferation and differentiation. These findings provide a basis for analyses of the role of the muscularis mucosae in ISC regulation.

Cancer stem cells: the other face of Janus.

Advances in the field of stem cell biology have provided renewed hopes that stem cells can be used to treat a wide range of genetic diseases and traumatic injuries. However, advances in the field of cancer cell biology have led to formulation of the cancer stem cell hypothesis, which posits that cancers arise from mutant stem cells. Further, this hypothesis proposes that these stem cells account for cancer recurrence, metastasis, and resistance to conventional treatments. Thus, although normal stem cells represent potential effective solutions to numerous clinical problems, when mutated, they may also represent the cause of many human malignancies.

Tumor necrosis factor (TNF)-alpha-induced IL-8 expression in gastric epithelial cells: role of reactive oxygen species and AP endonuclease-1/redox factor (Ref)-1.

TNF-alpha contributes to oxidative stress via induction of reactive oxygen species (ROS) and pro-inflammatory cytokines. The molecular basis of this is not well understood but it is partly mediated through the inducible expression of IL-8. As redox factor-1 (Ref-1), is an important mediator of redox-regulated gene expression we investigated whether ROS and Ref-1 modulate TNF-alpha-induced IL-8 expression in human gastric epithelial cells. We found that TNF-alpha treatment of AGS cells enhanced nuclear expression of Ref-1 and potently induced IL-8 expression. Overexpression of Ref-1 enhanced IL-8 gene transcription at baseline and after TNF-alpha treatment whereas Ref-1 suppression and antioxidant treatment inhibited TNF-alpha-stimulated IL-8 expression. TNF-alpha-mediated enhancement of other pro-inflammatory chemokines like MIP-3 alpha and Gro-alpha was also regulated by Ref-1. Although TNF-alpha increased DNA binding activity of Ref-1-regulated transcription factors, AP-1 and NF-kappaB, to the IL-8 promoter, promoter activity was mainly mediated by NF-kappaB binding. Silencing of Ref-1 in AGS cells inhibited basal and TNF-alpha-induced AP-1 and NF-kappaB DNA binding activity, but not their nuclear accumulation. Collectively, we provide the first mechanistic evidence of Ref-1 involvement in TNF-alpha-mediated, redox-sensitive induction of IL-8 and other chemokines in human gastric mucosa. This has implications for understanding the pathogenesis of gastrointestinal inflammatory disorders.

PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity.

BACKGROUND & AIMS: A prominent role for inhibitory molecules PD-L1 and PD-L2 in peripheral tolerance has been proposed. However, the phenotype and function of PD-L-expressing cells in human gut remains unclear. Recent studies suggest that colonic myofibroblasts (CMFs) and fibroblasts are important in the switch from acute inflammation to adaptive immunity. In the normal human colon, CMFs represent a distinct population of major histocompatibility complex class II(+) cells involved in the regulation of mucosal CD4(+) T-cell responses. METHODS: PD-L1 and PD-L2 expression on human CMFs was determined using Western blot, fluorescence-activated cell sorter analysis and confocal microscopy. Lymphoproliferation assays and cytokine enzyme-linked immunosorbent assays were used to evaluate the role of B7 costimulators expressed by CMFs with regard to the regulation of preactivated T-helper cell responses. RESULTS: We demonstrate here the expression of PD-L1/2 molecules by normal human CMF and fibroblasts in situ and in culture. Both molecules support suppressive functions of CMFs in the regulation of activated CD4(+) T-helper cell proliferative responses; blocking this interaction reverses the suppressive effect of CMFs on T-cell proliferation and leads to increased production of the major T-cell growth factor, interleukin (IL)-2. PD-L1/2-mediated CMF suppressive functions are mainly due to the inhibition of IL-2 production, because supplementation of the coculture media with exogenous IL-2 led to partial recovery of activated T-cell proliferation. CONCLUSIONS: Our data suggest that stromal myofibroblasts and fibroblasts may limit T-helper cell proliferative activity in the gut and, thus, might play a prominent role in mucosal intestinal tolerance.

Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors.

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease.

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.

Interleukin-8 induction by Helicobacter pylori in gastric epithelial cells is dependent on apurinic/apyrimidinic endonuclease-1/redox factor-1.

Helicobacter pylori infection causes inflammation and increases the expression of IL-8 in human gastric epithelial cells. H. pylori activates NF-kappaB and AP-1, essential transcriptional factors in H. pylori-induced IL-8 gene transcription. Although colonization creates a local oxidative stress, the molecular basis for the transition from infection to the expression of redox-sensitive cytokine genes is unknown. We recently reported that the expression of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE-1/Ref-1), which repairs oxidative DNA damage and reductively activates transcription factors including AP-1 and NF-kappaB, is increased in human gastric epithelia during H. pylori infection. In this study, we examine whether APE-1/Ref-1 functions in the modulation of IL-8 gene expression in H. pylori-infected human gastric epithelial cells. Small interfering RNA-mediated silencing of APE-1/Ref-1 inhibited basal and H. pylori-induced AP-1 and NF-kappaB DNA-binding activity without affecting the nuclear translocation of these transcription factors and also reduced H. pylori-induced IL-8 mRNA and protein. In contrast, overexpression of APE-1/Ref-1 enhanced basal and H. pylori-induced IL-8 gene transcription, and the relative involvement of AP-1 in inducible IL-8 promoter activity was greater in APE-1/Ref-1 overexpressing cells than in cells with basal levels of APE-1/Ref-1. APE-1/Ref-1 inhibition also reduced other H. pylori-induced chemokine expression. By implicating APE-1/Ref-1 as an important regulator of gastric epithelial responses to H. pylori infection, these data elucidate a novel mechanism controlling transcription and gene expression in bacterial pathogenesis.

Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.

The human gastrointestinal mucosa is exposed to a diverse normal microflora and dietary Ags and is a common site of entry for pathogens. The mucosal immune system must respond to these diverse signals with either the initiation of immunity or tolerance. APCs are important accessory cells that modulate T cell responses which initiate and maintain adaptive immunity. The ability of APCs to communicate with CD4+ T cells is largely dependent on the expression of class II MHC molecules by the APCs. Using immunohistochemistry, confocal microscopy, and flow cytometry, we demonstrate that alpha-smooth muscle actin(+), CD90+ subepithelial myofibroblasts (stromal cells) constitutively express class II MHC molecules in normal colonic mucosa and that they are distinct from professional APCs such as macrophages and dendritic cells. Primary isolates of human colonic myofibroblasts (CMFs) cultured in vitro were able to stimulate allogeneic CD4+ T cell proliferation. This process was dependent on class II MHC and CD80/86 costimulatory molecule expression by the myofibroblasts. We also demonstrate that CMFs, engineered to express a specific DR4 allele, can process and present human serum albumin to a human serum albumin-specific and DR4 allele-restricted T cell hybridoma. These studies characterize a novel cell phenotype which, due to its strategic location and class II MHC expression, may be involved in capture of Ags that cross the epithelial barrier and present them to lamina propria CD4+ T cells. Thus, human CMFs may be important in regulating local immunity in the colon.

Interleukin-1-induced NF-kappaB recruitment to the oxytocin receptor gene inhibits RNA polymerase II-promoter interactions in cultured human myometrial cells.

The myometrial oxytocin receptor (OTR) is highly regulated during pregnancy, reaching maximal concentrations near term. These levels are then abruptly reduced in advanced labour and the post-partum period. Our goal was to examine the molecular basis for this reduction, using chromatin immunoprecipitation (ChIP). Interleukin-1alpha (IL1A) treatment of cultured human myometrial cells has previously been shown to reduce steady-state levels of OTR mRNA. We show further that IL1A reduced RNA polymerase II cross-linking to the otr promoter, as reflective of transcriptional inhibition. IL1A also increased the recruitment of nuclear factor kappaB (NF-kappaB) to a site 955 bp upstream from the transcriptional start site. Inhibition of NF-kappaB activation negated the effects of IL1A on polymerase II dissociation, indicating a causal relationship, at least in part, between recruitment of NF-kappaB and detachment of polymerase from the otherwise constitutively active otr promoter. IL1A treatment also resulted in increased histone H4 acetylation in the otr promoter region. Whereas NF-kappaB recruitment and histone acetylation are generally associated with activation of gene expression, our findings show that both processes can be involved in dissociation of RNA polymerase II from an active promoter. The results of these studies suggest that the elevation of IL1 in the myometrium occurring at the end of pregnancy initiates the process of down-regulation of OTRs in advanced labour, resulting in the desensitization of the myometrium to elevated levels of OT in the blood during lactation.

Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis.

Prostaglandins (PG) are produced throughout the gastrointestinal tract and are critical mediators for a complex array of physiologic and pathophysiologic processes in the intestine. Intestinal myofibroblasts, which express cyclooxygenase (COX) and generate PGE(2), play important roles in intestinal epithelial proliferation, differentiation, inflammation, and neoplasia through secreting growth factors and cytokines. Here, we show that PGE(2) activated human intestinal subepithelial myofibroblasts (18Co) through Gs protein-coupled E-prostanoid receptors and the cyclic AMP/protein kinase A pathway. 18Co cells and primary colonic myofibroblast isolates expressed a number of growth factors; several of them were dramatically regulated by PGE(2). An epidermal growth factor-like growth factor, amphiregulin (AR), which was not expressed by untreated cells, was strongly induced by PGE(2). Expression of vascular endothelial growth factor A (VEGFA) was rapidly increased by PGE(2) exposure. Hepatocyte growth factor (HGF) was elevated in PGE(2)-treated myofibroblasts at both mRNA and protein levels. Thus, PGE(2)-activated myofibroblasts promoted the proliferation and migration of intestinal epithelial cells, which were attenuated by neutralizing antibodies to AR and HGF, respectively. Moreover, in the presence of PGE(2), myofibroblasts strongly stimulated the migration and tubular formation of vascular endothelial cells. Neutralizing antibody to VEGFA inhibited the observed stimulation of migration. These results suggest that myofibroblast-generated growth factors are important mediators for PGE(2)-induced intestinal epithelial proliferation and angiogenesis, which play critical roles in intestinal homeostasis, inflammation, and neoplasia.

Polarized expression of CD74 by gastric epithelial cells.

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.

Helicobacter pylori and H2O2 increase AP endonuclease-1/redox factor-1 expression in human gastric epithelial cells.

BACKGROUND & AIMS: Helicobacter pylori infection causes inflammation, accumulation of reactive oxygen species, and oxidative DNA damage in the gastric mucosa. Apurinic/apyrimidinic endonuclease-1 (APE-1)/redox factor-1 (Ref-1) repairs damaged DNA and reductively activates transcription factors, including activator protein-1. Considering that H. pylori generate reactive oxygen species and that reactive oxygen species modulate APE-1/Ref-1 in other cell types, we examined the effect of H. pylori, oxidative stress, and antioxidants on APE-1/Ref-1 expression in human gastric epithelial cells. METHODS: Human gastric epithelial cell lines or cells isolated from mucosal biopsy samples were stimulated with H. pylori, Campylobacter jejuni, and/or H 2 O 2 in the presence or absence of antioxidants. APE-1/Ref-1 expression was assayed by Western blot or reverse-transcription polymerase chain reaction, and its cellular distribution was determined by using indirect conventional and confocal immunofluorescence. New protein synthesis was detected by [S 35 ]methionine labeling. APE-1/Ref-1 function was assessed by using a luciferase-linked reporter construct containing 3 activator protein 1 binding sites. RESULTS: APE-1/Ref-1 protein and messenger RNA were detected in resting gastric epithelial cells. APE-1/Ref-1 protein expression was increased after stimulation with H 2 O 2 or live cag pathogenicity island-bearing H. pylori, but not cag pathogenicity island-negative H. pylori or C. jejuni. H. pylori - or reactive oxygen species-mediated increases in APE-1/Ref-1 expression involved de novo protein synthesis that was inhibited by antioxidants. H. pylori or H 2 O 2 also induced nuclear accumulation of APE-1/Ref-1, and overexpression of APE-1/Ref-1 increased activator protein 1 binding activity. CONCLUSIONS: The data show that H. pylori or reactive oxygen species enhance APE-1/Ref-1 protein synthesis and nuclear accumulation in human gastric epithelial cells and implicate APE-1/Ref-1 in the modulation of the pathogenesis of H. pylori infection.