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DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of β-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

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.

Mice are the primary animal of choice for modeling aGVHD. One aspect of these mouse models that may limit translation of promising therapeutic strategies to patient treatment is the use inbred mice as surrogates for genetically diverse human populations. Objective Quantify and compare tissue inflammation and injury in inbred vs. genetically diverse outbred mice using a well-characterized model of aGVHD-mediated bone marrow (BM) failure and spleen hypoplasia. Methods C57Bl6 (Bl6) CD4+ T cells (20,000 T cells/g b.w.) were injected (i.p.) into sub-lethally irradiated Bl6-H2-Ab1bm12(BM12) recipients (Bl6→BM12) or into Collaborative Cross (CC) or CD1 outbred recipients. Results Adoptive transfer of allogeneic Bl6 T cells into BM12 recipients induced marked weight loss (25%) at 20 days post T cell transfer that was associated with dramatic reductions in hematocrit as well as large and significant decreases in circulating granulocytes, platelets and erythrocytes when compared to their syngeneic controls (BM12→BM12). Total cell numbers in BM and spleen were also found to be dramatically reduced in these mice. Histopathological inspection confirmed severe hypocellularity in BM and spleen whereas no aGVHD was present in the lungs, liver, skin and colon. In contrast, adoptive transfer of Bl6 T cells into sub-lethally irradiated CC or CD1 or CC mice induced little or no weight loss at 25–30 days post T cell transfer with no significant reductions in hematocrit, circulating leukocytes, erythrocytes or platelets. BM- and spleen-associated cell numbers were not reduced when compared their CC controls. Conclusions Genetically diverse outbred mice are resistant to aGVHD-mediated BM failure and spleen hypoplasia.

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.

Anti-adhesion therapies that target α(4) integrins (e.g., natalizumab) are thought to work by blocking T-cell recruitment to the intestinal tissues in patients with Crohn's disease (CD); however, little direct evidence is available to confirm this contention. We wished to evaluate the importance of T cell-associated α(4) integrins in a chronic colitis model in mice and to determine the effect of natalizumab treatment on intestinal tissue T-cell accumulation in human CD. Adoptive transfer of T cells lacking α(4) (α(4)(-/-)) but not β(1) integrin into immunodeficient mice produced significantly attenuated disease. This was correlated with reduced numbers of colon CD4 T cells compared with the control mice; however, tissue distribution of T helper type 1 (Th1) and T helper type 17 (Th17) cells and regulatory T cells (Tregs) was not affected by the lack of α(4). Furthermore, α(4)(-/-) T cells demonstrated defective homing to the chronically inflamed small intestines and colons. Finally, patients treated with natalizumab showed significant reduction in mucosal CD4 T cells and no skewing in the foxp3(+) Treg or T-bet(+)Th1 fractions thereof. These results demonstrate a direct role for T cell-associated α(4)β(7) but not α(4)β(1) integrins during initiation and perpetuation of chronic colitis. Moreover, our data demonstrated that natalizumab treatment reduced mucosal CD4 T-cell accumulation in CD patients.

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.

Intracellular generation of reactive oxygen species (ROS) is an inescapable consequence of aerobic metabolism. Although some of these oxygen-derived metabolites are well-documented mediators of cell and tissue damage, others have been shown to be crucial for cell survival and homeostasis. One ROS that has been identified as a major second messenger in redox signaling is hydrogen peroxide (H2O2). This small, membrane-permeable oxidant is produced transiently in physiological (nontoxic) amounts by a variety of different enzymes residing within different subcellular compartments and organelles. There is an accumulating literature demonstrating that the reversible, H2O2-mediated oxidation of different signaling proteins is an important posttranslational mechanism that regulates a number of different biological processes including cell proliferation, differentiation, motility and apoptosis. Although several, well-characterized methods have been developed to quantify the generation of extracellular H2O2, the ability to unequivocally detect and quantify this important signaling molecule within living cells has been relatively limited. Fortunately, a great deal of progress has been made over the past few years in developing H2O2-selective probes that are capable of detecting physiological levels of this signaling molecule. This overview presents a critical evaluation of the established as well as the more recently developed methods to detect and quantify extracellular and intracellular H2O2 produced by living cells.

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.

T cells critically regulate inflammatory bowel disease (IBD), with T-cell-dependent experimental colitis models gaining favor in identifying potential pathogenic mechanisms; yet limited understanding of specific pathogenic molecules or pathways still exists.In this study we sought to identify changes in whole genome expression profiles using the CD4CD45Rbhi T-cell transfer colitis model compared to genome expression differences from Crohn's disease (CD) tissue specimens. Colon tissue was used for histopathological and genome expression profiling analysis at 0, 2, 4, or 6 weeks after adoptive T-cell transfer.We identified 1775 genes that were significantly altered during disease progression, with 361 being progressively downregulated and 341 progressively upregulated. Gene expression changes were validated by quantitative real-time polymerase chain reaction (qRT-PCR), confirming genome expression analysis data. Differentially expressed genes were clearly related to inflammation/immune responses but also strongly associated with metabolic, chemokine signaling, Jak-STAT signaling, and angiogenesis pathways. Ingenuity network analysis revealed 25 unique network associations that were associated with functions such as antigen presentation, cell morphology, cell-to-cell signaling and interaction, as well as nervous system development and function. Moreover, many of these genes and pathways were similarly identified in CD specimens.These findings reveal novel, complex, and dynamic changes in gene expression that may provide useful targets for future therapeutic approaches.

Active episodes of the inflammatory bowel diseases are associated with the infiltration of large numbers of myeloid cells including neutrophils, monocytes, and macrophages. The objective of this study was to systematically characterize and define the different populations of myeloid cells generated in a mouse model of chronic gut inflammation. Using the T cell transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic cytokines (IL-17 and G-CSF), increased production of neutrophils and monocytes, and infiltration of large numbers of myeloid cells into the mesenteric lymph nodes (MLNs) and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1+Ly6ChighGr-1low/neg cells (monocytes), Mac-1+Ly6CintGr-1+ cells (neutrophils), and Mac-1+Ly6Clow/negGr-1low/neg leukocytes (macrophages, dendritic cells, and eosinophils). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared with neutrophils obtained from the blood, the MLNs, and the spleen of colitic mice. Furthermore, we found that colonic neutrophils had acquired APC function that enabled these granulocytes to induce proliferation of OVA-specific CD4+ T cells in an Ag- and MHC class II-dependent manner. Finally, we observed a synergistic increase in proinflammatory cytokine and chemokine production following coculture of T cells with neutrophils in vitro. Taken together, our data suggest that extravasated neutrophils acquire APC function within the inflamed bowel where they may perpetuate chronic gut inflammation by inducing T cell activation and proliferation as well as by enhancing production of proinflammatory mediators.

The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results.

Most therapeutic agents used in clinical practice today were originally developed and tested in animal models so that drug toxicity and safety, dose-responses and efficacy could be determined. Retrospective analyses of preclinical intervention studies using animal models of different diseases demonstrate that only a small percentage of the interventions reporting promising effects translate to clinical efficacy. The failure to translate therapeutic efficacy from bench to bedside may be due, in part, to shortcomings in the design of the clinical studies; however, it is becoming clear that much of the problem resides within the preclinical studies. One potential strategy for improving our ability to identify new therapeutics that may have a reasonable chance of success in well-controlled clinical trials is to identify the most relevant mouse models IBD and pharmacologic strategies that most closely mimic the clinical situation. To begin this process, we present a critical evaluation of the different mouse models and pharmacological approaches that may be used in intervention studies as well as discuss emerging issues related to study design and data interpretation of preclinical studies.

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.

Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. NO is involved in many physiological processes including regulation of blood pressure, immune response, and neural communication. Therefore its accurate detection and quantification are critical to understanding health and disease. Due to the extremely short physiological half-life of this gaseous free radical, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of NO metabolites in biological samples provides valuable information with regard to in vivo NO production, bioavailability, and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The methods described in this review is not an exhaustive or comprehensive discussion of all methods available for the detection of NO but rather a description of the most commonly used and practical methods which allow accurate and sensitive quantification of NO products/metabolites in multiple biological matrices under normal physiological conditions.

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.

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.