Nitric oxide as a mediator of gastrointestinal mucosal injury?—Say it ain't so

Nitric oxide has been suggested as a contributor to tissue injury in various experimental models of gastrointestinal inflammation. However, there is overwhelming evidence that nitric oxide is one of the most important mediators of mucosal defence, influencing such factors as mucus secretion, mucosal blood flow, ulcer repair and the activity of a variety of mucosal immunocytes. Nitric oxide has the capacity to down-regulate inflammatory responses in the gastrointestinal tract, to scavenge various free radical species and to protect the mucosa from injury induced by topical irritants. Moreover, questions can be raised regarding the evidence purported to support a role for nitric oxide in producing tissue injury. In this review, we provide an overview of the evidence supporting a role for nitric oxide in protecting the gastrointestinal tract from injury.     

Does the microbiota regulate immune responses outside the gut?

Perturbations in the gastrointestinal (GI) microbiota composition that occur as a result of antibiotics and diet in "westernized" countries are strongly associated with allergies and asthma ("hygiene hypothesis"). The microbiota ("microflora") plays a crucial role in the development of mucosal tolerance, including the airways. Significant attention has been focused on the role of the microbiota in GI development, immune adaptation and initiation of GI inflammatory diseases. This review covers the post-developmental functions that the microbiota plays in regulating immunological tolerance to allergen exposure outside the GI tract and proposes the question: is the microbiota a major regulator of the immune system?     

Bacterial translocation from the intestines

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract through the mucosal epithelium to other sites, such as the mesenteric lymph nodes, spleen, liver and blood. This paper reviews results from animal models utilized to obtain information concerning the defense mechanisms operating in the healthy host to confine bacteria to the GI tract. Gnotobiotic and antibiotic-decontaminated mice colonized with particular bacteria demonstrated that the indigenous GI flora maintains an ecologic equilibrium to prevent intestinal bacterial overgrowth and translocation from the GI tract. Studies with athymic (nu/nu) mice, thymus-grafted (nu/nu) mice, neonatally thymectomized mice, and mice injected with immunosuppressive agents demonstrated that the host immune system is another defense mechanism inhibiting bacterial translocation from the GI tract. Ricinoleic acid given orally to mice disrupted the intestinal epithelial barrier allowing indigenous bacteria to translocate from the GI tract. Thus, bacterial translocation from the GI tract of healthy adult mice is inhibited by: (a) an intact intestinal epithelial barrier, (b) the host immune defense system, and (c) an indigenous GI flora maintaining ecological equilibrium to prevent bacterial overgrowth. Deficiencies in host defense mechanisms act synergistically to promote bacterial translocation from the GI tract as demonstrated by animal models with multiple alterations in host defenses. Bacterial translocation occurred to a greater degree in mice with streptozotocin-induced diabetes, mice receiving nonlethal thermal injury, and mice receiving the combination of an immunosuppressive agent plus an oral antibiotic than in mice with only a primary alteration in host defenses. The study of bacterial translocation in these complex models suggests that opportunistic infections from the GI tract occur in discrete stages. In the healthy adult animal, bacterial translocation from the GI tract either does not occur or occurs at a very low level and the host immune defenses eliminate the translocating bacteria. Bacterial translocation does take place if one of the host defense mechanisms is compromised, such as a deficiency in the immune response, bacterial overgrowth in the intestines, or an increase in the permeability of the intestinal barrier. In this first stage, the bacteria usually translocate in low numbers to the mesenteric lymph node, and sometimes spleen or liver, but do not multiply and spread systemically.(ABSTRACT TRUNCATED AT 400 WORDS)     

MyD88 signalling plays a critical role in host defence by controlling pathogen burden and promoting epithelial cell homeostasis during Citrobacter rodentium-induced colitis

Myeloid differentiation factor (MyD)88, an adaptor protein shared by the Toll-interleukin 1 receptor superfamily, plays a critical role in host defence during many systemic bacterial infections by inducing protective inflammatory responses that limit bacterial growth. However, the role of innate responses during gastrointestinal (GI) infections is less clear, in part because the GI tract is tolerant to commensal antigens. The current study investigated the role of MyD88 following infection by the murine bacterial pathogen, Citrobacter rodentium . MyD88-deficient mice suffered a lethal colitis coincident with colonic mucosal ulcerations and bleeding. Their susceptibility was associated with an overwhelming bacterial burden and selectively impaired immune responses in colonic tissues, which included delayed inflammatory cell recruitment, reduced iNOS and abrogated production of TNF-α and IL-6 from MyD88-deficient macrophages and colons cultured ex vivo . Immunostaining for Ki67 and BrDU revealed that MyD88 signalling mediated epithelial hyper-proliferation in response to C. rodentium infection. Thus, MyD88-deficient mice could not promote epithelial cell turnover and repair, leading to deep bacterial invasion of colonic crypts, intestinal barrier dysfunction and, ultimately, widespread mucosal ulcerations. In conclusion, MyD88 signalling within the GI tract plays a critical role in mediating host defence against an enteric bacterial pathogen, by controlling bacterial numbers and promoting intestinal epithelial homeostasis.     

Testing Stem Cell Therapy in a Rat Model of Inflammatory Bowel Disease: Role of Bone Marrow Stem Cells and Stem Cell Factor in Mucosal Regeneration

Background

The gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD).

Methods

BMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls.

Results

PKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group.

Conclusion

BMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.     

肠上皮快速复原过程中的细胞信号传递: 多胺和K+通道的影响

胃肠道粘膜上皮细胞具有重要的屏障作用,可以保护次上皮组织抵御一系列的有害物质,包括过敏原、病毒以及微生物病原体。粘膜损伤后的修复有赖于上皮细胞对信号网络的调节,而这一网络系统控制着基因的表达、细胞的存活、迁移及增殖。近几年的研究结果显示,在胃肠道粘膜的修复中,多胺起到关键作用;且细胞多胺的调控是众多信号传递路径的焦点。本文简要综述了多胺在肠粘膜上皮快速复原中的功能和机制,特别是对K^ 通道活性的影响。     

Personal reminiscences about Morton Grossman and the founding of the Center for Ulcer Research and Education (CURE)

The Center for Ulcer Research and Education (CURE) from its onset was primarily the work of one man: Professor Morton Grossman, or "Mort" as he was known and called by all. Mort's legacy includes a large body of scientific publications, the first National Institutes of Health Digestive Diseases Center (CURE), and, most importantly, a group of scientists who have become academic leaders and who have made important contributions in the fields of upper gastrointestinal (GI) tract secretion, hormones and receptors, mucosal defense mechanisms, the design and conduct of randomized clinical trials, and ulcer epidemiology. Indeed, Mort is considered to be a founding father of modern academic GI research. I was fortunate to have known and worked with Mort and would like to memorialize his contributions so that his memory can inspire the next generation of academicians.     

Biomarkers of leukocyte traffic and activation in the vaginal mucosa

Development of novel vaginal spermicides and anti-human immunodeficiency virus (HIV) microbicides requires careful assessment of their potential to recruit and activate CD4+ HIV-1 host cells in the female genital tract mucosa, two events that facilitate HIV-1 infection. Leukocyte traffic and activation are mediated by proinflammatory cytokines and chemokines, e.g. interleukin (IL)-1, IL-6 and IL-8, which have been detected in vaginal secretions in association with epithelial damage and infections. These proinflammatory mediators, however, have bidirectional, destructive as well as beneficial, effects on the mucosal barrier, and may be counterbalanced by endogenous inhibitors. Here we propose additional biomarkers for the evaluation of compound-induced cervicovaginal mucosal inflammation. Displaying different temporal patterns of detection, the levels of soluble E-selectin, vascular adhesion molecule-1, CD14 and myeloperoxidase in vaginal secretions reflected the mucosal leukocyte reaction to proinflammatory compounds being evaluated for safety in an improved rabbit vaginal irritation model. These biomarkers, which were also detected in human vaginal secretions, may be used to enhance the characterization of mucosal safety of vaginally applied compounds, both in animal as well as clinical studies.     

Biomarkers of leukocyte traffic and activation in the vaginal mucosa.

Development of novel vaginal spermicides and anti-human immunodeficiency virus (HIV) microbicides requires careful assessment of their potential to recruit and activate CD4+ HIV-1 host cells in the female genital tract mucosa, two events that facilitate HIV-1 infection. Leukocyte traffic and activation are mediated by proinflammatory cytokines and chemokines, e.g. interleukin (IL)-1, IL-6 and IL-8, which have been detected in vaginal secretions in association with epithelial damage and infections. These proinflammatory mediators, however, have bidirectional, destructive as well as beneficial, effects on the mucosal barrier, and may be counterbalanced by endogenous inhibitors. Here we propose additional biomarkers for the evaluation of compound-induced cervicovaginal mucosal inflammation. Displaying different temporal patterns of detection, the levels of soluble E-selectin, vascular adhesion molecule-1, CD14 and myeloperoxidase in vaginal secretions reflected the mucosal leukocyte reaction to proinflammatory compounds being evaluated for safety in an improved rabbit vaginal irritation model. These biomarkers, which were also detected in human vaginal secretions, may be used to enhance the characterization of mucosal safety of vaginally applied compounds, both in animal as well as clinical studies.     

Role of leukotrienes and platelet activating factor in gastric mucosal damage and repair.

Gastric mucosal integrity depends upon the balance between "aggressive" factors and "defensive" mechanisms. The formation of mucosal lesions results from the disruption of defense lines, including the breaking of unstirred mucus layer, the reduction of surface hydrophobicity, extensive exfoliation of surface epithelium, penetration of offending agents deeply into the mucosa and damage to the microvessels. The release of proinflammatory and vasoactive mediators such as leukotrienes (LT), thromboxanes, platelet activating factor (PAF), endothelins and others has been thought to be involved in the pathomechanism of mucosal injury, especially damage to the microvascular endothelium, increased vascular permeability, reduction in mucosal blood flow, vascular stasis, tissue ischemia and glandular cell necrosis. This paper reviews the mechanisms and possible pathogenetic implication of two related compounds, LT and PAF in acute mucosal injury by topical irritants such as ethanol, aspirin, bile salts and by stress. LT and PAF arise from similar membrane phospholipids and may regulate the biosynthesis of one another in the damaged mucosa. Although pharmacological studies have clearly demonstrated the noxious effects of cysteinyl LT and PAF on the mucosa, especially when exposed to topical irritants, recent publications have challenged the primary role of these mediators in the pathogenesis of mucosal lesions and ulcerations because the treatment with agents that selectively antagonize their biosynthesis or the receptor sites at the target cells did not always interrupt the chain of events leading to mucosal injury. The role of these mediators in the mucosal repair processes has been little studied but both cysteinyl LT and PAF seem to delay the restitution and healing of the mucosa. Further studies are necessary to clarify to what extent the biosynthesis of LT and PAF and the pharmacological inhibition of their action on the target tissues is related to noxious, protective and reparative events in the mucosa exposed to mild irritants and ulcerogens.     

No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation

Diseases of mucosal inflammation represent important causes of morbidity and mortality, and have led to intense research efforts to understand the factors that lead to their development. It is well accepted that a breakdown of the normally impermeant epithelial barrier of the intestine, the lung, and the kidney is associated with the development of inflammatory disease in these organs, yet significant controversy exists as to how this breakdown actually occurs, and how such a breakdown may lead to inflammation. In this regard, much work has focused upon the role of the epithelium as an "innocent bystander," a target of a leukocyte-mediated inflammatory cascade that leads to its destruction in the mucosal inflammatory process. However, recent evidence from a variety of laboratories indicates that the epithelium is not merely a passive component in the steps that lead to mucosal inflammation, but is a central participant in the process. In addressing this controversy, we and others have determined that epithelial cells express Toll-like receptors (TLRs) of the innate immune system, and that activation of TLRs by endogenous and exogenous ligands may play a central role in determining the balance between a state of "mucosal homeostasis," as is required for optimal organ function, and "mucosal injury," leading to mucosal inflammation and barrier breakdown. In particular, activation of TLRs within intestinal epithelial cells leads to the development of cellular injury and impairment in mucosal repair in the pathogenesis of intestinal inflammation, while activation of TLRs in the lung and kidney may participate in the development of pneumonitis and nephritis respectively. Recent work in support of these concepts is extensively reviewed, while essential areas of further study that are required to determine the significance of epithelial TLR signaling during states of health and disease are outlined.     

Gastropathy and defense mechanisms in common bile duct ligated portal hypertensive rats

Portal hypertensive gastropathy is associated with a broad spectrum of gastric mucosal damage inspite of decreased gastric acid secretion, suggestive of compromised endogenous protective mechanisms. To determine the mechanisms of damage in portal hypertensive gastropathy we measured lipid peroxidation, glutathione, antioxidant and lysosomal enzymes in gastric mucosal homogenates from male Wistar rats with elevated intrasplenic pulp pressure, eighteen days after common bile duct ligation. Thiobarbituric acid-reactive substances and lysosomal enzymes (-glucuronidase and acid phosphatase) were increased in the common bile duct ligated group as compared to the sham-operated group. The levels of antioxidant defense enzymes, superoxide dismutase, glutathione peroxidase, catalase and glutathione were decreased as compared to the sham-operated controls. Pre-operative vitamin E administration decreased mucosal lipid peroxidation increased the levels of antioxidant defense enzymes and lowered the lysosomal enzymes. The plasma vitamin E levels in this group were lower when compared to animals receiving it post-operatively. In conclusion, free radical and lysosomal enzyme mediated damage may play a role in portal hypertensive gastropathy.     

Mucosal damage and neutropenia are required for Candida albicans dissemination

Candida albicans fungemia in cancer patients is thought to develop from initial gastrointestinal (GI) colonization with subsequent translocation into the bloodstream after administration of chemotherapy. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but we have hypothesized that both neutropenia and GI mucosal damage are critical for allowing widespread invasive C. albicans disease. We investigated these parameters in a mouse model of C. albicans GI colonization that led to systemic spread after administration of immunosuppression and mucosal damage. After depleting resident GI intestinal flora with antibiotic treatment and achieving stable GI colonization levels of C. albicans, it was determined that systemic chemotherapy with cyclophosphamide led to 100% mortality, whereas selective neutrophil depletion, macrophage depletion, lymphopenia or GI mucosal disruption alone resulted in no mortality. Selective neutrophil depletion combined with GI mucosal disruption led to disseminated fungal infection and 100% mortality ensued. GI translocation and dissemination by C. albicans was also dependent on the organism's ability to transform from the yeast to the hyphal form. This mouse model of GI colonization and fungemia is useful for studying factors of innate host immunity needed to prevent invasive C. albicans disease as well as identifying virulence factors that are necessary for fungal GI colonization and dissemination. The model may also prove valuable for evaluating therapies to control C. albicans infections.     

The modulation of inflammatory gene expression by lipids: mediation through Toll-like receptors

Toll-like receptors (TLRs) were evolved to detect invading pathogens and to induce innate immune responses in order to mount host defense mechanisms. It becomes apparent that the activation of certain TLRs is also modulated by endogenous molecules including lipid components, fatty acids. Results from epidemiological and animal studies demonstrated that saturated and polyunsaturated dietary fatty acids can differentially modify the risk of development of many chronic diseases. Inflammation is now recognized as an important underlying etiologic condition for the pathogenesis of many chronic diseases. Therefore, if the activation of TLRs and consequent inflammatory and immune responses are differentially modulated by types of lipids in vivo, this would suggest that the risk of the development of chronic inflammatory diseases and the host defense against microbial infection may be modified by the types of dietary fat consumed.     

Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+ T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+ T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+ T cells during AEI. HIV-1 RNA was detected in both "activated" and "nonactivated" mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+ T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.     

Distribution of CD4pos -, CD8pos – and Regulatory T Cells in the Upper and Lower Gastrointestinal Tract in Healthy Young Subjects

The gastrointestinal immune system is involved in the development of several autoimmune-mediated diseases, including inflammatory bowel disease, multiple sclerosis, and type 1 diabetes mellitus. Alterations in T-cell populations, especially regulatory T cells (Tregs), are often evident in patients suffering from these diseases. To be able to detect changes in T-cell populations in diseased tissue, it is crucial to investigate T-cell populations in healthy individuals, and to characterize their variation among different regions of the gastrointestinal (GI) tract. While limited data exist, quantitative data on biopsies systematically drawn from various regions of the GI tract are lacking, particularly in healthy young humans. In this report, we present the first systematic assessment of how T cells—including Tregs—are distributed in the gastrointestinal mucosa throughout the GI tract of healthy young humans by means of multi-parameter FACS analysis. Gastroduodenoscopy and colonoscopy were performed on 16 healthy volunteers aged between 18 and 32. Biopsies were drawn from seven GI regions, and were used to determine the frequencies of CD8⁺-, CD4⁺- and Tregs in the gastrointestinal mucosa by means of multi-parameter FACS analysis. Our data show that there is significant variation in the baseline T-cell landscape along the healthy human gastrointestinal tract, and that mucosal T-cell analyses from a single region should not be taken as representative of the entire gastrointestinal tract. We show that certain T-cell subsets in the gastrointestinal mucosa vary significantly among regions; most notably, that Tregs are enriched in the appendiceal orifice region and the ascending colon, and that CD8^pos T cells are enriched in the gastric mucosa.     

Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV

Human Reg and Reg-related genes constitute a multi-gene family belonging to the calcium (C-type) dependent lectin superfamily. Regenerating gene family members are expressed in the proximal gastrointestinal (GI) tract and ectopically at other sites in the setting of tissue injury. By high-throughput sequence analysis of a large inflammatory bowel disease library, two cDNAs have been isolated which encode a novel member of this multigene family. Based on primary sequence homology, tissue expression profiles, and shared exon-intron junction genomic organization, we assign this gene to the regenerating gene family. Specific protein structural differences suggest that the current three regenerating gene subtypes should be expanded to four. We demonstrate that Reg IV has a highly restricted tissue expression pattern, with prominent expression in the gastrointestinal tract. Reg IV mRNA expression is significantly up-regulated by mucosal injury from active Crohn's disease or ulcerative colitis.     

The blood-brain barrier: connecting the gut and the brain

The BBB prevents the unrestricted exchange of substances between the central nervous system (CNS) and the blood. The blood-brain barrier (BBB) also conveys information between the CNS and the gastrointestinal (GI) tract through several mechanisms. Here, we review three of those mechanisms. First, the BBB selectively transports some peptides and regulatory proteins in the blood-to-brain or the brain-to-blood direction. The ability of GI hormones to affect functions of the BBB, as illustrated by the ability of insulin to alter the BBB transport of amino acids and drugs, represents a second mechanism. A third mechanism is the ability of GI hormones to affect the secretion by the BBB of substances that themselves affect feeding and appetite, such as nitric oxide and cytokines. By these and other mechanisms, the BBB regulates communications between the CNS and GI tract.     

Differential effects of bacterial toxins on mitogenic actions of sodium fluoride and those of aluminum fluoride in human TE85 osteosarcoma cells

Free radical mediated effects on the gastrointestinal (GI) tract were studied by supplementing 8 mg of iron orally for 15 days to groups of both control (C⁺) and iron deficient (D⁺) rats. They were compared with their respective unsupplemented groups C and D. Incorporation of ³H-thymidine into the isolated mucosal cells, as a measure of cell turn over, was lowered significantly in both the D⁺ and C⁺ groups compared to their respective controls D and C. It was observed that a single dose of 8 mg of iron given orally to control rats could cause apoptosis of GI tract mucosal cells as shown by the ladder pattern of DNA on electrophoresis. Continuous administration of the same dose of iron for a period of 15 days resulted in necrosis of the GI tract absorptive surface in D⁺ and C⁺ rats. In addition to this, a reduction of microvillus height in C⁺ and complete erosion of the same in D⁺ were observed by the transmission electron microscopy. EPR spectroscopy identified production of hydroxyl and methoxyl radicals in both the luminal and mucosal contents in the GI tract of rats. These results suggest that when iron is orally administered, free radicals are formed at the site of absorption causing damage to the GI tract mucosa.