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Background The use of inbred mice housed under standardized environmental conditions has been critical in identifying immuno-pathological mechanisms in different infectious and inflammatory diseases as well as revealing new therapeutic targets for clinical trials. Unfortunately, only a small percentage of preclinical intervention studies using well-defined mouse models of disease have progressed to clinically-effective treatments in patients. The reasons for this lack of bench-to-bedside transition are not completely understood; however, emerging data suggest that genetic diversity and housing environment may greatly influence muring immunity and inflammation. Results Accumulating evidence suggests that certain immune responses and/or disease phenotypes observed in inbred mice may be quite different than those observed in their outbred counterparts. These differences have been thought to contribute to differing immune responses to foreign and/or auto-antigens in mice vs. humans. There is also a growing literature demonstrating that mice housed under specific pathogen free conditions possess an immature immune system that remarkably affects their ability to respond to pathogens and/or inflammation when compared with mice exposed to a more diverse spectrum of microorganisms. Furthermore, recent studies demonstrate that mice develop chronic cold stress when housed at standard animal care facility temperatures (i.e. 22–24 °C). These temperatures have been shown alter immune responses to foreign and auto-antigens when compared with mice housed at their thermo-neutral body temperature of 30–32 °C. Conclusions Exposure of genetically diverse mice to a spectrum of environmentally-relevant microorganisms at housing temperatures that approximate their thermo-neutral zone may improve the chances of identifying new and more potent therapeutics to treat infectious and inflammatory diseases.

BackgroundHematopoietic stem cell transplantation is a potential cure for certain life-threatening malignant and nonmalignant diseases. However, experimental and clinical studies have demonstrated that pre-transplant myeloablative conditioning damages the gut leading to translocation of intestinal bacteria and the development of acute graft vs. host disease (aGVHD). The overall objective of this study was to determine whether administration of broad spectrum antibiotics (Abx) affects the onset and/or severity of aGVHD in lymphopenic mice that werenotsubjected to toxic, pre-transplant conditioning.ResultsWe found that treatment of NK cell-depleted recombination activating gene-1-deficient (-NK/RAG) recipients with an Abx cocktail containing vancomycin and neomycin for 7 days prior to and 4 weeks following adoptive transfer of allogeneic CD4+T cells, exacerbated the development of aGVHD-induced BM failure and spleen damage when compared to untreated–NK/RAG recipients engrafted with syngeneic or allogeneic T cells. Abx-treated mice exhibited severe anemia and monocytopenia as well as marked reductions in BM- and spleen-residing immune cells. Blinded histopathological analysis confirmed that Abx-treated mice engrafted with allogeneic T cells suffered significantly more damage to the BM and spleen than did untreated mice engrafted with allogeneic T cells. Abx-induced exacerbation of BM and spleen damage correlated with a dramatic reduction in fecal bacterial diversity, marked loss of anaerobic bacteria and remarkable expansion of potentially pathogenic bacteria.ConclusionsWe conclude that continuous Abx treatment may aggravate aGVHD-induced tissue damage by reducing short chain fatty acid-producing anaerobes (e.g.Clostridium,Blautia) and/or by promoting the expansion of pathobionts (e.g.Akkermansia) and opportunistic pathogens (Cronobacter).

It is well known that alterations in splanchnic organ perfusion and/or immune regulation may produce inflammatory tissue injury similar to that observed in several human disorders such as ischaemia and reperfusion injury, food allergies, diabetes, inflammatory bowel disease and graft-versus-host disease. Mouse models have been tremendously important in defining the roles of the circulation, leukocyte trafficking, inflammatory mediator generation, immune regulation and the intestinal microbiota in the pathogenesis of acute and chronic inflammation. However, few of the promising interventions or therapeutics reported in mouse models of inflammatory diseases have been translated to clinically effective treatments in patients. There is growing concern that because of the significant differences that exist between the murine and human immune systems, mouse models may not adequately recapitulate the immuno-pathogenesis of inflammatory diseases. This inconvenient reality has prompted a number of investigators to undertake a series of studies to humanize the murine immune system via adoptive transfer of human lymphoid or progenitor cells into a new generation of immuno-deficient recipients. In this review, we summarize the recent advances that have been made in the development of humanized mice and describe how these mouse models are being used to study the pathophysiology of splanchnic organ inflammation. In addition, we discuss the limitations of the different approaches and present potential solutions for the continued improvement of these important animal models.

The intestinal mucosal surface in all vertebrates is exposed to enormous numbers of microorganisms that include bacteria, archaea, fungi and viruses. Coexistence of the host with the gut microbiota represents an active and mutually beneficial relationship that helps to shape the mucosal and systemic immune systems of both mammals and teleosts (ray-finned fish). Due to the potential for enteric microorganisms to invade intestinal tissue and induce local and/or systemic inflammation, the mucosal immune system has developed a number of protective mechanisms that allow the host to mount an appropriate immune response to invading bacteria, while limiting bystander tissue injury associated with these immune responses. Failure to properly regulate mucosal immunity is thought to be responsible for the development of chronic intestinal inflammation. The objective of this review is to present our current understanding of the role that intestinal bacteria play in vertebrate health and disease. While our primary focus will be humans and mice, we also present the new and exciting comparative studies being performed in zebrafish to model host-microbe interactions.

The inflammatory diseases ulcerative colitis and Crohn's disease constitute the two main forms of inflammatory bowel disease (IBD). They are characterized by chronic, relapsing inflammation of the gastrointestinal tract, significantly impacting on patient quality of life and often requiring prolonged treatment. Existing therapies for IBD are not effective for all patients, and an unmet need exists for additional therapies to induce and maintain remission. Here we describe the mechanism of action of the Janus kinase (JAK) inhibitor, tofacitinib, for the treatment of IBD and the effect of JAK inhibition on the chronic cycle of inflammation that is characteristic of the disease. The pathogenesis of IBD involves a dysfunctional response from the innate and adaptive immune system, resulting in overexpression of multiple inflammatory cytokines, many of which signal through JAKs. Thus JAK inhibition allows multiple cytokine signaling pathways to be targeted and is expected to modulate the innate and adaptive immune response in IBD, thereby interrupting the cycle of inflammation. Tofacitinib is an oral, small molecule JAK inhibitor that is being investigated as a targeted immunomodulator for IBD. Clinical development of tofacitinib and other JAK inhibitors is ongoing, with the aspiration of providing new treatment options for IBD that have the potential to deliver prolonged efficacy and clinically meaningful patient benefits.

One of the best characterized mouse models of the inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) is the CD4(+)CD45RB(high) T cell transfer model of chronic colitis. Following our relocation to Texas Tech University Health Sciences Center (TTUHSC), we observed a dramatic reduction in the incidence of moderate-to-severe colitis from a 16-year historical average of 90% at Louisiana State University Health Sciences Center (LSUHSC) to

The recruitment of leukocytes from peripheral blood into the intestine is required for the maintenance of intestinal health and protection from microbial insult. However, during immune-mediated gastrointestinal conditions (e.g., inflammatory bowel disease (IBD), celiac disease), the recruitment process becomes dysregulated. Several families of molecules regulate the influx of these cells into immune sites both during homeostasis and inflammation. Interference with some of these molecules has already shown efficacy in the clinics and antibodies that target the molecules involved have been approved by the FDA for use in IBD. In this chapter, we discuss basic aspects of the diverse families of relevant molecules that mediate the leukocyte adhesion cascade under physiological and inflammatory conditions to relevant immune compartments. Finally, we discuss the translational applications of targeting-specific steps of the leukocyte adhesion cascade for therapeutic purposes in human IBD.

Animal models of disease have been used extensively by the research community for the past several decades to better understand the pathogenesis of different diseases as well as assess the efficacy and toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to translate promising interventions or therapeutics into clinically-effective treatments in patients. Although several possible reasons have been suggested to account for this generalized failure to translate therapeutic efficacy from the laboratory bench to the patient’s bedside, it is becoming increasingly apparent that the mouse immune system may not adequately recapitulate the immuno-pathological mechanisms observed in human diseases. Indeed, it is well-known that >80 major differences exist between mouse and human immunology; all of which contribute to significant differences in immune system development, activation and responses to challenges in innate and adaptive immunity. This inconvenient reality has prompted investigators to attempt to humanize the mouse immune system in order to address important, human-specific questions that are impossible to study in patients. The successful long-term engraftment of human hemato-lymphoid cells in mice would provide investigators with a relatively inexpensive, small animal model to study clinically-relevant mechanisms as well as facilitate the evaluation of human-specific therapies in vivo. The discovery that targeted mutation of the IL-2 receptor common gamma chain in lymphopenic mice allows for the long-term engraftment of functional human immune cells has advanced greatly our ability to humanize the mouse immune system. The objective of this review is to present a brief overview of the recent advances that have been made in the development and use of humanized mice with special emphasis on autoimmune and chronic inflammatory diseases. In addition, we discuss current challenges and possible solutions for utilizing these unique mouse models to define the human-specific immuno-pathological mechanisms responsible for the induction and perpetuation of chronic gut inflammation.

Mouse models of disease have been used extensively by the research community for the past three decades to better understand the pathogenesis of different diseases as well as to assess the efficacy and toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to translate promising interventions or therapeutics into clinically effective treatments in patients. This is especially true for preclinical investigations using mouse models of the inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis). One potential strategy for improving our ability to discover new therapeutics that may have a reasonable chance of success in clinical trials is to identify the most immunologically-relevant mouse models of human IBD. However, it is becoming increasingly appreciated that the mouse immune system differs substantially from that of humans, and thus, may not express the same pathogenetic mechanisms that are thought to occur in human IBD. This chapter presents a critical evaluation of selected mouse models of IBD and discusses their utility in preclinical studies. In addition, we describe recent advances in generating and utilizing “humanized” mice to study the pathogenesis and treatment of chronic gut inflammation.

Multiple sclerosis (MS) has been proposed to be an immune-mediated disease in the central nervous system (CNS) that can be triggered by virus infections. In Theiler's murine encephalomyelitis virus (TMEV) infection, during the first week (acute stage), mice develop polioencephalomyelitis. After 3 weeks (chronic stage), mice develop immune-mediated demyelination with virus persistence, which has been used as a viral model for MS. Regulatory T cells (Tregs) can suppress inflammation, and have been suggested to be protective in immune-mediated diseases, including MS. However, in virus-induced inflammatory demyelination, although Tregs can suppress inflammation, preventing immune-mediated pathology, Tregs may also suppress antiviral immune responses, leading to more active viral replication and/or persistence. To determine the role and potential translational usage of Tregs in MS, we treated TMEV-infected mice with ex vivo generated induced Tregs (iTregs) on day 0 (early) or during the chronic stage (therapeutic). Early treatment worsened clinical signs during acute disease. The exacerbation of acute disease was associated with increased virus titers and decreased immune cell recruitment in the CNS. Therapeutic iTreg treatment reduced inflammatory demyelination during chronic disease. Immunologically, iTreg treatment increased interleukin-10 production from B cells, CD4(+) T cells and dendritic cells, which may contribute to the decreased CNS inflammation.

BACKGROUND Approximately one-half of patients with inflammatory bowel disease (IBD) suffer from anemia, with the most prevalent cause being iron deficiency. Accompanying the anemia are increases in erythropoietin, a plasma protein that can initiate the feedback production of new red blood cells. Anemia also occurs in animal models that are used to investigate the mechanisms of IBD; however, the extent to which iron deficiency produces the anemia in these animal models is unknown. Also unknown in the different animal models of IBD is whether the anemia upregulates the production of erythropoietin or, alternatively, whether a decrease in erythropoietin contributes to the induction of anemia. METHODS Two mouse models of colitis were used in this study: (1) acute 6-day ingestion of dextran sodium sulfate and (2) T-cell transfer into lymphopenic recipient mice. Measurements included indices of colitis severity, hematocrit, blood hemoglobin, plasma erythropoietin, serum iron concentration, plasma iron-binding capacities, transferrin saturation, and tissue iron concentrations. RESULTS Both models of colitis induced significant decreases in hematocrit, blood hemoglobin, and transferrin saturation, with the spleen and liver showing a decrease in iron content in the T-cell transfer model. Additionally, both models of colitis demonstrated significant increases in plasma erythropoietin and plasma iron-binding capacities. CONCLUSIONS The measurements of iron, whether in acute (dextran sodium sulfate) or chronic (T-cell transfer) models of colitis, were generally consistent with iron-deficient anemia, with large increases in erythropoietin indicative of tissue hypoxia. These changes in animal models of colitis are similar to those found in human IBD.

Liver ischemia and reperfusion-induced injury is a major clinical complication associated with hemorrhagic or endotoxin shock and thermal injury as well as liver transplantation and resectional surgery. Data obtained from several different studies suggest that an important initiating event in the pathophysiology of ischemia and reperfusion-induced tissue injury is enhanced production of superoxide concomitant with a decrease in the bioavailability of endothelial cell-derived nitric oxide. This review will summarize the evidence supporting the hypothesis that the redox imbalance induced by alterations in superoxide and nitric oxide generation creates a more oxidative environment within the different cells of the liver that enhances the nuclear transcription factor-κB-dependent expression of a variety of different cytokines and mediators that may promote as well as limit ischemia and reperfusion-induced hepatocellular injury. In addition, the evidence implicating endothelial cell nitric oxide synthase-dependent and -independent generation of nitric oxide as important regulatory pathways that act to limit ischemia and reperfusion-induced liver injury and inflammation is also presented.

Intraepithelial lymphocytes (IELs) represent the first line of lymphocyte defense against the intestinal bacteria. Although previous studies have demonstrated a protective role of IELs in the development of colitis, the data supporting a regulatory role for IELs are limited. The objective of this study was to examine the suppressive activity of IELs in vitro and in vivo using a mouse model of chronic small and large bowel inflammation. Adoptive transfer of CD8α(+) IELs isolated from small intestines of wild-type (WT) mice into TCR βxδ-deficient (TCR βxδ(-/-)) recipients did not prevent or delay the onset of the disease induced by WT CD4(+)CD45RB(high) T cells. On the contrary, we observed a more rapid onset of wasting and clinical signs of intestinal inflammation when compared with animals injected with CD4(+)CD45RB(high) T cells alone. Histopathological scores of small and large bowel did not differ significantly between the two groups. Transfer of IELs alone did not produce any pathological changes. Real-time PCR analysis of intestinal tissues showed up-regulation of message for T(h)1- and macrophage-derived cytokines in colon and small bowel. Using Foxp3-GFP reporter mice, we were unable to detect any Foxp3(+) cells within the CD8α(+) IELs but did find a small population of Foxp3(+)CD4(+) IELs in the small and large bowel. Using in vitro suppression assay, we found that neither TCRαβ(+)CD8αα(+), TCRαβ(+)CD8αβ(+) nor TCRγδ(+)CD8αα(+) IELs were capable of suppressing CD4(+) T-cell proliferation. Taken together, our data do not support an immunoregulatory role for CD8α(+) IELs in a mouse model of small and large bowel inflammation.

The mobilization and recruitment of blood and lymphatic vasculatures are widely described in inflammatory bowel diseases (IBDs). Although angiogenesis contributes to intense gut inflammation, it remains unclear whether and when lymphangiogenesis amplifies or protects in IBD. The prolonged maintenance of lymphatic (over blood vessels) in inflammation indicates that lymphatic-blood vessel interactions may regulate IBD pathogenesis and restitution. Although lymphatic expansion helps to restore fluid balance and clear cytokines and immune cells, lymphatic failure results in accumulation of these factors and exacerbates IBD. Lymphatic obstruction and remodeling may impair lymphatic pumping, leading to repeated rounds of lymphangiogenesis. Early descriptions of Crohn's disease and ulcerative colitis describe colon lymphatic congestion, remodeling, expansion, and many other features that are recapitulated in experimental IBD and also by intestinal lymphatic obstruction, supporting lymphangitis as a cause and consequence of IBD. Growth factors, cytokines, gut flora, Toll receptors, and leukocytes all regulate inflammation and gut lymphatic remodeling in IBD. This review summarizes the importance of lymphatics and lymphangiogenesis in IBD etiology that may be useful in diagnosis and therapy of gut inflammation.

The etiologies of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) have not been fully elucidated. However, there is very good evidence implicating T cell and T cell trafficking to the gut and its associated lymphoid tissue as important components in disease pathogenesis. The objective of this review is to provide an overview of the mechanisms involved in naive and effector T cell trafficking to the gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes and intestine in response to commensal enteric antigens under physiological conditions as well as during the induction of chronic gut inflammation. In addition, recent data suggests that the GALT may not be required for enteric antigen-driven intestinal inflammation in certain mouse models of IBD. These new data suggest a possible paradigm shift in our understanding of how and where naive T cells become activated to yield disease-producing effector cells.

Background: Adoptive transfer of naive T-lymphocyte subsets into lymphopenic mice initiates chronic gut inflammation that mimics several aspects of inflammatory bowel disease (IBD). Patients with IBD can have profound alterations in intestinal blood flow, but whether the same is true in the T-cell transfer model has yet to be determined. Methods: In the current study, chronic intestinal inflammation was induced in recombinase-activating gene-1-deficient (RAG−/−) mice by adoptive transfer of CD4+ T-lymphocytes obtained from interleukin-10 deficient (IL-10−/−) mice. Results: Four weeks later, widespread colonic inflammation was observed in the reconstituted recipients, in contrast to 2 control sets of mice injected with a different subset of lymphocytes or with vehicle alone. We observed that the resulting pathology induced in the reconstituted RAG−/− mice was divided distinctly into 2 subsets: 1 with blood flow near normal with very high inflammation scores, and the other with severely attenuated blood flow but with much lower signs of inflammation. Colonic and ileal blood flow rates in the latter subset of CD4+ mice averaged only ≈30% compared to the mice with higher inflammation scores. The lower blood flow rates were associated with greatly reduced red blood cell concentrations in the tissue, suggesting a possible loss of vascular density. Conclusions: In this model of chronic intestinal inflammation, mild inflammation was associated with significant decreases in blood flow. Inflamm Bowel Dis 2009

The inflammatory bowel diseases (Crohn's disease; ulcerative colitis) are idiopathic chronic inflammatory disorders of the intestine and/or colon. A major advancement in our understanding of the pathogenesis of these diseases has been the development of mouse models of chronic gut inflammation. One model that has been instrumental in delineating the immunological mechanisms responsible for the induction as well as regulation of intestinal inflammation is the T cell transfer model of chronic colitis. This paper presents a detailed protocol describing the methods used to induce chronic colitis in mice. Special attention is given to the immunological concepts that explain disease pathogenesis in this model, considerations and potential pitfalls in using this model, and finally different “tricks” that we have learned over the past 12 years that have allowed us to develop a more simplified version of this model of experimental IBD.

SUMMARY 1 Hepatic resection with concomitant periods of ischaemia and reperfusion (I/R) is required to perform reduced-size liver (RSL) transplantation procedures, such as living donor or split liver transplantation. Although a great deal of progress has been made using these types of surgical procedures, a significant number of patients develop tissue injury from these procedures, ultimately resulting in graft failure. 2 Because of this, there is a real need to understand the different mechanisms responsible for the tissue injury induced by I/R of RSL transplantation (RSL + I/R), with the ultimate goal to develop new and improved therapeutic agents that may limit the tissue damage incurred during RSL transplantation. 3 The present paper reviews the recent studies that have been performed examining the role of reactive metabolites of oxygen and nitrogen in a mouse model of RSL + I/R. In addition, we present data demonstrating how the pathophysiological mechanisms identified in this model compare with those observed in a model of RSL transplantation in rats.

The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate-buffered saline (PBS) developed clinical evidence of colitis at 6-8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Th1 and macrophage-derived cytokines including interferon gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-12, and IL-18 lymphotoxin-beta. In addition, message levels and vascular surface expression of ICAM-1, VCAM-1, and MAdCAM-1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

The adaptive immune system plays an important role in host defense against invading micro-organisms. Yet, mice deficient in T- and B-cells are surprisingly healthy and develop few spontaneous infections when raised under specific pathogen-free conditions (SPF). The objective of this study was to ascertain what role phagocyte-associated NADPH oxidase or myeloperoxidase (MPO) plays in host defense in mice lacking both T- and B-cells. To do this, we generated lymphopenic mice deficient in either NADPH oxidase or MPO by crossing gp91 phox -deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko). We found that neither gp91 ko, MPO ko mice nor lymphocyte-deficient RAG ko mice developed spontaneous infections when raised under SPF conditions and all mice had life spans similar to wild-type (WT) animals. In contrast, gp91xRAG double-deficient (DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Infections in the gp91xRAG DKO mice were characterized by granulomatous inflammation of the skin, liver, heart, brain, kidney, and lung. Addition of antibiotics to the drinking water attenuated the spontaneous infections and increased survival of the mice. Oyster glycogen-elicited polymorphonuclear neutrophils (PMNs) and macrophages obtained from gp91 ko and gp91xRAG DKO mice had no detectable NADPH oxidase activity whereas WT, RAG ko, and MPOxRAG DKO PMNs and macrophages produced large and similar amounts of superoxide in response to phorbol myristate acetate. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase activity (iNOS) as total numbers of elicited PMNs and macrophages as well as PMN- and macrophage-derived production of nitric oxide-derived metabolites in these mice were similar and not reduced when compared to that of WT mice. Taken together, our data suggest that that NADPH oxidase but not MPO (nor iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each other’s deficiency in providing resistance to spontaneous bacterial infections.

Experimental models of colitis in mice have been used extensively for analyzing the molecular events that occur during inflammatory bowel disease (IBD) development. However, it is uncertain to what extent the experimental models reproduce features of human IBD. This is largely due to the lack of precise methods for direct and comprehensive comparison of mouse and human inflamed colon tissue at the molecular level. Here we use global gene expression patterns of two sets of pediatric IBD and two mouse models of colitis to obtain a direct comparison of the genome signatures of mouse and human IBD. By comparing the two sets of pediatric IBD microarray data, we found 83 genes were differentially expressed in a similar manner between pediatric Crohn’s disease (CD) and ulcerative colitis (UC). Up regulation of the chemokine (C-C motif) ligand 2 (CCL2) gene that maps to 17q12, a confirmed IBD susceptibility loci, indicates that our comparison study can reveal known genetic associations with IBD. In comparing pediatric IBD and experimental colitis microarray data, we found common signatures amongst them including: 1) up regulation of CXCL9 and S100A8; 2) cytokine-cytokine receptor pathway dysregulation; and 3) over-represented IRF1 and IRF2 transcription binding sites in the promoter region of up regulated genes, and HNF1A and Lhx3 binding sites were over-represented in the promoter region of the down regulated genes. In summary, this study provides a comprehensive view of transcriptome changes between different pediatric IBD populations in comparison with different colitis models. These findings reveal several new molecular targets for further study in the regulation of colitis.

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract with unknown multifactorial etiology that, among other things, result in alteration and dysfunction of the intestinal microvasculature. Clinical observations of increased colon microvascular density during IBD have been made. However, there have been no reports investigating the physiological or pathological importance of angiogenic stimulation during the development of intestinal inflammation. Here we report that the dextran sodium sulfate and CD4+CD45RBhigh T-cell transfer models of colitis stimulate angiogenesis that results in increased blood vessel density concomitant with increased histopathology, suggesting that the neovasculature contributes to tissue damage during colitis. We also show that leukocyte infiltration is an obligatory requirement for the stimulation of angiogenesis. The angiogenic response during experimental colitis was differentially regulated in that the production of various angiogenic mediators was diverse between the two models with only a small group of molecules being similarly controlled. Importantly, treatment with the anti-angiogenic agent thalidomide or ATN-161 significantly reduced angiogenic activity and associated tissue histopathology during experimental colitis. Our findings identify a direct pathological link between angiogenesis and the development of experimental colitis, representing a novel therapeutic target for IBD.

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) are required to perform partial liver transplantation procedures such as split liver or living donor transplantation. Although great progress has been made using these types of surgeries, there remains substantial risk to both donors and recipients, with a significant number of patients developing liver injury and failure during the course these operations. Therefore, there is need to investigate the different mechanisms responsible for the tissue injury induced by ischemia and reperfusion of a reduced-size liver (RSL+I/R) with the ultimate objective of developing new therapeutic agents that may limit hepatocellular damage induced during partial liver transplantation. This review summarizes recent studies that have been performed in a mouse model of RSL+I/R. In addition, we present data demonstrating how the pathophysiological mechanisms identified in this model compare to those observed in a rat model of RSL transplantation.

The b2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) is important for lymphocyte trafficking and activation as well as recruitment to sites of tissue inflammation. The objective of this study was to assess the role of ‘T-cell-associated’ LFA-1 in the pathogenesis of chronic colitis in vivo. Transfer of CD4 1 CD25 � T cells isolated from wild-type (wt) mice into immunodeficient recipients [recombinase-activating gene-1-deficient (RAG-1 � /� )] produced moderate to severe colitis, whereas RAG-1 � /� mice injected with CD11a-deficient (CD11a � /� ; LFA-1 � /� ) donor T cells displayed minimal macroscopic and histological evidence of colitis. Surface expression of L-selectin, a4, a4b7 and chemokine receptor-7 were similar for wt and CD11a � /� donor T cells. Attenuated disease in the CD11a � /� ! RAG-1 � /� animals was associated with decreased numbers of CD4 1 T cells in the mesenteric lymph nodes (MLNs), spleen and intestinal lamina propria (LP). In addition, significant reductions in Th1 cytokines were observed following ex vivo stimulation of mononuclear cells obtained from the MLNs and colonic LP. Interestingly, mononuclear cells obtained from the spleens of CD11a � /� ! RAG-1 � /� exhibited enhanced pro-inflammatory cytokine production compared with splenocytes obtained from wt ! RAG-1 � /� colitic mice. Taken together, our data suggest that T-cell-associated CD11a (LFA-1) expression plays a dual role in the initiation of chronic gut inflammation by facilitating naive T-cell priming/activation and expansion within MLNs and by augmenting pro-inflammatory cytokine production following secondary stimulation by antigenpresenting cells in the colonic interstitium.

The major risk factors for intracerebral hemorrhage (ICH) are hypertension and aging. A fundamental mechanism for hypertension- and aging-induced vascular injury is oxidative stress. We hypothesize that oxidative stress has a crucial role in ICH. To test our hypothesis, we used bacterial collagenase to produce ICH in wild-type C57BL/6 and gp91phox knockout (gp91phox KO) mice (deficient in gp91phox subunit of the superoxide-producing enzyme NADPH oxidase). All animals were studied at 20-35 weeks of age, resembling an older patient population. We found that collagenase produced less bleeding in gp91phox KO mice than wild-type mice. Total oxidative product was lower in gp91phox KO mice than in wild-type mice, both under basal conditions and after ICH. Consistent with the ICH volume, brain edema formation, neurological deficit and a high mortality rate was noted in wild-type but not in gp91phox KO mice. This ICH-induced brain injury in wild-type mice is associated with enhanced expression of the gp91phox subunit of NADPH oxidase. In conclusion, the oxidative stress resulting from activation of NADPH oxidase contributes to ICH induced by collagenase and promotes brain injury.

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) is required to perform reduced size liver transplantation such as split liver or liver donor transplantation. Although great progress has been made using these types of surgeries, there remains substantial risk to both donors and recipients, with a significant number of patients developing liver injury and failure. The objective of this study was to assess the roles of superoxide (O(2)(-)) and tumor necrosis factor-alpha (TNF-alpha) in the pathophysiology of a mouse model of reduced size liver combined with ischemia and reperfusion (RSL+I/R). We found that all male mice subjected to RSL+I/R died within 3-5 days following surgery. Mortality was always preceded by dramatic increases in liver injury and TNF-alpha expression in the absence of neutrophil infiltration. Using a long-lived, polycationic form of human manganese superoxide dismutase (pcMnSOD), NADPH oxidase-deficient mice (gp91(-/-)) or a monoclonal antibody directed against mouse TNF-alpha, we demonstrated that hepatocellular injury (and mortality) were significantly attenuated. In addition, we found that pcMnSOD administration or NADPH deficiency reduced expression of TNF-alpha. Taken together, our data suggest that NADPH oxidase-derived O(2)(-) plays an important role in the pathophysiology of RSL+I/R-induced liver injury via its ability to enhance expression of TNF-alpha. We propose that therapies directed toward scavenging of O(2)(-), inhibiting NADPH oxidase, and/or immuno-neutralizing TNF-alpha may prove useful in limiting the liver injury induced by surgical procedures that require resection and I/R such as split liver or living donor liver transplantation.

Cessation of blood flow to the liver is required during liver transplantation and resectional surgery. A growing body of experimental evidence suggests that restoration of blood flow to the ischemic liver initiates hepatocellular injury which may lead, in some cases, to severe liver injury and graft failure. A large number of studies have implicated reactive oxygen species as potential mediators of post-ischemic tissue injury. Recent developments in genetic engineering as well as chemical modeling, have allowed for the production of novel free radical scavengers including mutated forms of superoxide dismutase (SOD) and low molecular weight SOD mimics with extended circulating half-lives and/or significant membrane permeability's. Application of these newly developed free radical scavengers show promising results in animal models of liver I/R and may become powerful tools in the treatment of post-ischemic liver injury that occurs in liver transplantation.

The dextran sulfate (DSS) model of colitis causes intestinal injury sharing many characteristics with inflammatory bowel disease, e.g., leukocyte infiltration, loss of gut epithelial barrier, and cachexia. These symptoms are partly mediated by entrapped leukocytes binding to multiple endothelial adhesion molecules (MAdCAM-1, VCAM-1, ICAM-1, and E-selectin). Pravastatin, an 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, has anti-inflammatory potency in certain inflammation models; therefore, in this study, we measured the effects of pravastatin in DSS-induced colitis. The administration of pravastatin (1 mg/kg) relieved DSS-induced cachexia, hematochezia, and intestinal epithelial permeability, with no effect on serum cholesterol. Histopathologically, pravastatin prevented leukocyte infiltration and gut injury. Pravastatin also blocked the mucosal expression of MAdCAM-1. DSS treatment promoted mucosal endothelial nitric-oxide synthase (eNOS) mRNA degradation, an effect that was blocked by pravastatin. Importantly, the protective effects of pravastatin in DSS-induced colitis were not found in eNOS-deficient mice. Our results demonstrate that HMG-CoA reductase inhibitors preserve intestinal integrity in colitis, most likely via increased eNOS expression and activity, independent of cholesterol metabolism.

The inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.

Pulmonary injury with leukocyte infiltration is a frequent occurrence in obstructive cholangitis patients. We wished to evaluate the roles of reactive oxygen species and vascular cell adhesion molecule-1 (VCAM-1) in this distant organ failure. Wild type (WT) and transgenic (SODtg) mice overexpressing superoxide dismutase underwent bile duct ligation and transection (BDL). VCAM-1 expression was quantified, and histopathology was assessed for the liver and lung. BDL resulted in increased leukocyte infiltration to the lung at five days in WT mice. VCAM-1 expression significantly increased in WT mouse liver at three days and WT mouse lung at five days. When these same experiments were performed in SODtg mice, these increases in leukocyte infiltration and VCAM-1 expression in lung were significantly attenuated. These data suggest that reactive oxygen species produced in response to BDL may up-regulate VCAM-1 expression in the lung and play an important role in the pathophysiology of this pulmonary injury.

Free radicals play an important role in the initiation and progression of inflammatory bowel disease (IBD). Therefore, the reduction or elimination of adverse oxidant effects can provide novel therapy for IBD. Here, the antioxidant capacity and protective effects of a new class of chemically modified hetastarch (polynitroxyl starch, or PNS) plus 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol or TPL) (PNS/TPL) were assessed in a model of colitis. The superoxide scavenging capacity of PNS/TPL—that is, the inhibition of the reduction of cytochrome c in the presence of xanthine/xanthine oxidase (X/XO)—was evaluated in vitro. The effects of PNS/TPL on X/XO–induced neutrophil endothelial adhesion in vitro were investigated. Also, this study tested the protection produced by PNS/TPL in a mouse model of trinitrobenzene sulfonic acid (TNBS)–induced colitis. PNS/TPL was given intravenously immediately before (

The overall objective of this proposal is to evaluate the therapeutic efficacy of human, bone marrow-derived mesenchymal stem cells (MSCs) in a well-characterized mouse model of inflammatory bowel disease (IBD). Shortly after my relocation from LSU Health Sciences Center (LSUHSC) to my current position at Texas Tech Health Sciences Center (TTUHSC), we encountered an unexpected problem with our mouse model of IBD. We found that the incidence of intestinal inflammation in these mice was only 30-40% compared to our 15 year historical incidence of 85% we observed at LSUHSC. Following a year of investigations, we identified the problem and were successful in reestablishing the model with an incidence of 95%. Although these investigations significantly delayed the start of the studies outlined in Task 1, we were able to begin experiments to ascertain the immuno-suppressive activity of human MSCs in our new and improved mouse model of IBD. These studies are currently ongoing. In addition, we made substantial progress in developing a more immunologically-relevant in vitro system to assess suppressive activity and mechanisms of MSCs that more closely models the cellular and immunological interactions that are thought to occur in our mouse model of IBD in vivo. Furthermore, we present exciting new data demonstrating that activation of MSCs with pro-inflammatory cytokines that are known to be overproduced in mouse and human IBD, induces the dramatic up-regulation of several different immuno-suppressive mediators. Finally, we present new data that utilizes a novel, highly sensitive and quantitative method for simultaneously measuring the homing of human MSCs to several different mouse tissues during the development of chronic gut inflammation.

Inflammatory bowel diseases (IBD) are chronic, relapsing conditions of multifactorial etiology. The two primary diseases of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Both entities are hypothesized to occur in genetically susceptible individuals due to microbial alterations and environmental contributions. The exact etiopathogenesis, however, is not known for either disease. A variety of mouse models of CD and UC have been developed to investigate the pathogenesis of these diseases and evaluate treatment modalities. Broadly speaking, the mouse models can be divided into 4 categories: genetically engineered, immune manipulated, spontaneous and erosive/chemically induced. No one mouse model completely recapitulates the immunopathology of CD or UC, however each model possesses particular similarities to human IBD and offers advantageous for specific details of IBD pathogenesis. Here we discuss the more commonly used models in each category and critically evaluate how the immunopathology induced compares to CD or UC, as well as the advantages and disadvantages associated with each model.

There is growing clinical evidence suggesting that certain secondary lymphoid tissues (e.g., appendix and spleen) contribute to the initiation and/or perpetuation of ulcerative colitis. In this study, the importance of secondary lymphoid tissues in inducing colitis was assessed experimentally by removing the spleen and/or appendix (or sham operation) prior to inducing colitis in mice. Feeding 2.5% dextran sulphate sodium (DSS) in drinking water over 7 days induced colitis. Clinical disease activity was assessed based on weight loss, stool consistency, and presence of blood in stools. Additional measurements included white blood cell count and hematocrit, and myeloperoxidase activity (MPO) in colon samples. Colonic injury was assessed by histology and computerized image analysis. DSS treatment in sham-operated mice produced colitis associated with weight loss, bloody diarrhea, and mucosal ulceration. Clinical assessment of DSS-treated mice subjected to appendectomy or combined appendectomy/splenectomy exhibited a delayed onset and course of disease activity. Histomorphologic examination revealed significantly lower damage scores and a reduction in ulcerated mucosal surface area. Colonic MPO activity, which correlated with tissue injury and disease activity, was lowest in appendectomized mice. No beneficial effects of splenectomy were observed after 7 days of colitis. These findings support the hypothesis that appendicular lymphoid tissue, but not the spleen, contributes to the development of colitis.

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) is a common occurrence in resectional surgery as well as reduced-size liver transplantation (e.g., split liver or living donor transplantation). However, the I/R induced by these types of surgical manipulations may impair liver regeneration, ultimately leading to liver failure. The objectives of the study were to develop a murine model of reduced-size liver I/R and assess the role of gender in this model of hepatocellular injury. We found that 100% of female mice survived the surgery indefinitely, whereas all male mice had greater initial liver injury and died within 5 days after surgery. The protective effect observed in females appeared to be due to ovarian 17β-estradiol, as ovariectomy of females or administration of a selective estrogen antagonist to female mice resulted in enhanced liver injury and greater mortality following reduced-size liver I/R. Conversely, 17β-estradiol-treated male mice exhibited less hepatocellular damage and survived indefinitely. Taken together, these data demonstrate an estrogen-mediated protective pathway(s) that limits or attenuates hepatocellular injury induced by reduced-size liver I/R.

The mucosal interstitia of the intestine and colon are continuously exposed to large amounts of dietary and microbial antigens. Fortunately, the mucosal immune system has evolved efficient mechanisms to distinguish potentially pathogenic from nonpathological antigens. There are, however, situations in which this immune regulation fails, resulting in chronic gut inflammation.

Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient (−/−) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox−/− mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NOX and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS−/− and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox−/− mice, enhanced protection was noted in 1400W-treated p47phox−/− mice. Interestingly, SODTg mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestine and/or colon of unknown etiology in which patients suffer from severe diarrhea, rectal bleeding, abdominal pain, fever, and weight loss. Active episodes of IBD are characterized by vasodilation, venocongestion, edema, infiltration of large numbers of inflammatory cells, and erosions and ulcerations of the bowel. It is becoming increasingly apparent that chronic gut inflammation may result from a dysregulated immune response toward components of the normal intestinal flora, resulting in a sustained overproduction of proinflammatory cytokines and mediators. Many of these Th1 and macrophage-derived cytokines and lipid metabolites are known to activate microvascular endothelial cells, thereby promoting leukocyte recruitment into the intestinal interstitium. This review discusses the basic immune mechanisms involved in the regulation of inflammatory responses in the gut and describes how a breakdown in this protective response initiates chronic gut inflammation.

A number of laboratories have sought to elucidate the role of nitric oxide (NO) in both acute and chronic inflammatory diseases. It is now well appreciated that NO can influence many aspects of the inflammatory cascade ranging from its own expression to recruitment of leucocytes to the effected tissue. With the advent of mice selectively deficient in the various isoforms of nitric oxide synthase (NOS), the role that NO may play in various disease states can now be examined in vivo. One such syndrome that has gained much attention in recent years is ischaemia and reperfusion-induced tissue injury. Ischaemia-reperfusion (I/R) injury is an important clinical consideration in situations such as transplantation, trauma, liver or bowel resection and haemorrhagic shock. A hallmark of I/R is the production of reactive oxygen species (ROS) during the reperfusion phase and it is thought that the production of ROS mediate much of the post-ischaemic tissue injury. This review will examine the current state of knowledge regarding the regulatory mechanisms by which NO can influence various aspects of the inflammatory cascade as well as its role in a model of I/R injury in vivo.

Background : Enhanced MAdCAM-1 (mucosal addressin cell adhesion molecule-1) expression is associated with the aetiology of inflammatory bowel disease, but little is known about MAdCAM-1: regulation, or how inflammatory bowel disease therapies modulate MAdCAM-1. Aim : To examine how agents currently used to treat inflammatory bowel disease affect MAdCAM-1: induced by tnf-α in an in vitro model of inflammatory bowel disease. Methods : Endothelial monolayers were pretreated with dexamethasone (DEX): 5-aminosalicylic acid (5-ASA), 6-mercaptopurine (6-MP), sulfasalazine or interleukin-10: (IL-10: prior to TNF-α (20 ng/mL), and MAdCAM-1: measured by Western blotting, RT-PCR, EMSA and lymphocyte adhesion assays. Results : MAdCAM-1: was induced dose- and time-dependently by TNF-α on endothelial cells. Either dexamethasone or IL-10: reduced TNF-α-induced MAdCAM-1: protein, mRNA and lymphocyte adhesion. However, neither 5-ASA, sulfasalazine nor 6-MP blocked MAdCAM-1 induction. Conclusions : Our data indicate that dexamethasone or IL-10 can exert therapeutic activity in inflammatory bowel disease through MAdCAM-1 inhibition. 5-ASA, sulfasalazine and 6-MP, while beneficial in inflammatory bowel disease, do not directly control MAdCAM-1, and are beneficial through inhibition of other inflammatory processes.

The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1-6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using L-N(6)(iminoethyl)-lysine (L-NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury.

Objectives: 1) To determine if endothelial expression of adhesion molecules involved in leukocyte recruitment is increased in the brain and other organs in four different models of experimental colitis, and 2) to investigate whether leukocyte infiltration occurs in the brain of colitic animals. Methods: Endothelial vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression was quantified, using the dual radiolabeled antibody technique in rats with trinitrobenzenesulfonic acid (TNBS)-induced colitis, in mice with dextran sulfate sodium (DSS)-induced colitis, in SCID mice reconstituted with CD45RB high T-cells, and in IL-10 ˛/˛ mice. Leukocyte infiltration in the brain of TNBS-induced colitic rats was assessed by myeloperoxidase activity and immunohistochemical staining with antiCD45 monoclonal antibody. Results: Marked upregulation of brain endothelial VCAM-1 (2- to 5.5-fold) was consistently found in colitic animals in the four models studied. Brain VCAM-1 strongly correlated with colon VCAM-1 and colon weight. By contrast, upregulation of brain ICAM-1 in colitic animals was only observed in the CD45RB high transfer (3-fold) and the TNBS-induced (1.5-fold models). Heart and muscle VCAM-1 and ICAM-1 were not upregulated in colitic animals in the majority of models studied. There was no leukocyte infiltration into the brain of TNBSinduced colitic rats. Conclusions: Our study demonstrates a marked and specific upregulation of endothelial VCAM-1 in the brain of colitic animals. This activation of cerebral endothelial cells was not associated with an infiltration of leukocytes into brain tissue. Microcirculation (2001) 8, 105‐114.

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.

Malaria is a life-threatening re-emerging disease, yet it is still not clear how bloodstage malarial parasites are killed. Nitric oxide (NO), which has potent anti-microbial activity, may represent an important killing mechanism. The production of NO during descending Plasmodium chabaudi parasitemia, a period when parasites are killed by the immune response, supports this concept. However, NOS20/0 and NOS30/0 mice as well as mice treated with NO synthase 2 (NOS2) inhibitors do not develop exacerbated malaria, indicating that NO production is not necessary for the suppression of P. chabaudi parasitemia. It is possible due to the plasticity in the immune response during malaria that Ab-mediated immunity is enhanced in the absence of NO, thereby explaining the lack of exacerbated malaria in NOS-deficient mice even though NO may function in protection. However, NOS2- and B cell-deficient mice, which cannot use Ab-mediated immunity, suppress their parasitemia with a similar time course as B cell-deficient controls. C57BL/6 mice treated with Propionibacterium acnes to elicit high levels of macrophage-derived NO have a similar time course of P. chabaudi parasitemia as P. acnes-treated NOS20/0 mice, which do not produce NO; this indicates that NO is not sufficient for parasite killing. Collectively, these results indicate that NO is not necessary or sufficient to resolve P. chabaudi malaria.

The objective of this study was to quantify E-selectin surface expression in the colon as well as other tissues in a CD4(+) T-cell model of chronic colitis in mice using the newly developed dual radiolabel monoclonal antibody technique. Male SCID mice were reconstituted with either 5 x 10(5) CD4(+) CD45RB(low) or CD45RB(high) T-cells isolated from normal CB-17 donor mouse spleens and subsequently monitored for clinical signs of colitis. We found that animals injected with CD45RB(high) but not CD45RB(low) T-cells nor PBS developed colitis at 6-8 weeks following reconstitution as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of E-selectin compared to SCID mice injected with PBS or reconstituted with CD45RB(low) T-cells, both of which did not develop colitis. We also observed significant increases in E-selectin expression in cecum, small intestine, mesentery, and liver of colitic mice. Our data confirm that reconstitution of SCID mice with CD45RB(high) but not CD45RB(low) T-cells induces chronic colitis and demonstrate that this chronic colitis is associated with enhanced expression of an endothelial cell-specific adhesion molecule. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RB(high) T-cells enhances E-selectin expression in a variety of tissues distant from the site of active inflammation.

Inhaled nitric oxide (NO) reduces pulmonary hypertension and dampens various aspects of lung inflammation; however, its effects are thought to be restricted to the lung because of its short half-life in biological systems. More recently, however, NO was shown to nitrosylate hemoglobin, albumin, and other plasma molecules to form stable nitrosothiol derivatives and could have an impact on the periphery. We examined whether inhaled NO could have an impact on the two compartments of distal organs, namely, the intravascular and extravascular spaces. The feline intestine was exposed to 1 h of ischemia and 1 h of reperfusion, and intestinal blood flow and mucosal dysfunction were measured in animals ventilated with room air and inhaling 0 or 80 ppm NO. A decrease in intestinal blood flow and an increase in mucosal barrier leakiness were noted in animals not exposed to inhaled NO. The intestinal blood flow impairment was entirely reversed in animals breathing 80 ppm NO, but the mucosal dysfunction was not affected. We further examined whether inhaled NO could reach the extravascular space by simply inhibiting NO in the intestine with the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME) that causes an increase in mucosal permeability that is rapidly reversed with NO donors. However, inhaled NO had no effect on the rise in mucosal permeability. l-NAME reduced lymph nitrosothiol concentrations, but inhaled NO could not replenish these levels. To further explore the intravascular impact of inhaled NO, we used intravital microscopy to visualize the microvasculature and demonstrated that inhaled NO could be initiated after reperfusion and still reduced microvascular disturbances, including reversing the impairment in blood flow and increasing leukocyte adhesion. The effects of inhaled NO persisted for an additional hour after termination of NO inhalation, consistent with a dramatic increase in nitrate within 1 h of NO inhalation, which persisted for 1 h after the termination of NO inhalation. These data suggest that inhaled NO can reach distal organs to dramatically improve reperfusion-induced microvascular but not extravascular dysfunction.

Myocardial ischemia and reperfusion (MI/R) initiates a cascade of polymorphonuclear neutrophil (PMN)-mediated injury, the magnitude of which may be influenced by the bioavailability of nitric oxide (NO). We investigated the role of endothelial cell nitric oxide synthase (ecNOS) in MI/R injury by subjecting wild-type and ecNOS-deficient (−/−) mice to 20 min of coronary artery occlusion and 120 min of reperfusion. Myocardial infarct size represented 20.9 ± 2.9% of the ischemic zone in wild-type mice, whereas the ecNOS −/− mice had significantly ( P < 0.01) larger infarcts measuring 46.0 ± 3.8% of the ischemic zone. Because P-selectin is thought to be involved with the pathogenesis of neutrophil-mediated I/R injury, we assessed the effects of MI/R on P-selectin expression in the myocardium of wild-type and ecNOS −/− mice. P-selectin expression measured with a radiolabeled monoclonal antibody (MAb) technique after MI/R in wild-type mice was 0.037 ± 0.009 μg MAb/g tissue, whereas ecNOS −/− coronary vasculature was characterized by significantly ( P < 0.05) higher P-selectin expression (0.080 ± 0.013 μg MAb/g tissue). Histological examination of the postischemic myocardium revealed significantly ( P < 0.01) more neutrophils in the ecNOS −/− (29.5 ± 2.5 PMN/field) compared with wild-type (5.0 ± 0.9 PMN/field) mice. A similar trend in infarct size and neutrophil accumulation was observed when wild-type and ecNOS −/− mice were subjected to 30 min of ischemia and 120 min of reperfusion. These novel in vivo findings demonstrate a cardioprotective role for ecNOS-derived NO in the ischemic-reperfused mouse heart.

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Ischemia and reperfusion (I/R) are thought to play an important role in the pathophysiology of ischemic diseases of the heart. It is now well appreciated that leukocyte-endothelial cell interactions are important determinants for I/R-induced microvascular injury and dysfunction. There is a growing body of experimental data to suggest that reactive metabolites of oxygen and nitrogen are important physiological modulators of leukocyte-endothelial cell interactions. A number of investigators have demonstrated that I/R enhances oxidant production within the microcirculation resulting in increases in leukocyte adhesion and transendothelial cell migration. Several other studies have shown that exogenous nitric oxide (NO) donors may attenuate leukocyte and platelet adhesion and/or aggregation in a number of different inflammatory conditions including I/R. The objective of this review is to discuss the physiological chemistry of reactive metabolites of oxygen and nitrogen with special attention given to those interactions that may modulate leukocyte-endothelial cell interactions, provide an overview of the evidence implicating reactive metabolites of oxygen and nitrogen as modulators of leukocyte-endothelial cell interactions in vivo, and discuss how these mechanisms may be involved in the pathophysiology of ischemic heart disease.