Genomic, microbial and environmental standardization in animal experimentation limiting immunological discovery.

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.

Induction of acute graft vs. host disease in lymphopenic mice.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially life-saving treatment for refractory/relapsing hematological malignancies, blood disorders or autoimmune diseases. However, approximately 40-50% of patients undergoing allogeneic HSCT will develop a multi-organ, inflammatory disorder called acute graft vs. host disease (aGVHD). Experimental and clinical studies suggest that intestinal injury due to toxic, pre-transplant conditioning protocols (e.g. lethal irradiation and/or chemotherapy) may play a major role in the development of aGVHD. However, recent studies from our laboratory suggest that this may not be the case. The objective of this study was to quantify and compare the onset and severity of aGVHD induced by the adoptive transfer of allogeneic T cells into untreated lymphopenic mice. Four million allogeneic or syngeneic CD4+CD62L+CD25- T cells were transferred (i.p.) into NK cell-depleted RAG1-/- mice or RAG2-/-IL2rγ-/-double knock-out (DKO) mice and assessed daily for signs of aGVHD. We found that adoptive transfer of allogeneic but not syngeneic T cells into NK cell-depleted RAG1-/- or DKO mice induced many of the clinical and histological features of aGVHD including weight loss, inflammatory cytokine production and tissue inflammation. In addition, adoptive transfer of allogeneic T cells into each recipient induced severe anemia as well as dramatic reductions in bone marrow and spleen cellularity. Taken together, we conclude that allogeneic CD4+ T cells are both necessary and sufficient to induce aGVHD in lymphopenic recipients in the absence of toxic, pre-transplant conditioning.

Humanizing the mouse immune system to study splanchnic organ inflammation.

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.

Protective and pro-inflammatory roles of intestinal bacteria.

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.

Acquisition of antigen-presenting functions by neutrophils isolated from mice with chronic colitis.

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(+)Ly6C(high)Gr-1(low/neg) cells (monocytes), Mac-1(+)Ly6C(int)Gr-1(+) cells (neutrophils), and Mac-1(+)Ly6C(low/neg)Gr-1(low/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.

Immunopathological characterization of selected mouse models of inflammatory bowel disease: Comparison to human disease.

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.

Methods to detect hydrogen peroxide in living cells: Possibilities and pitfalls.

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.

Influence of Housing Temperature and Genetic Diversity on Allogeneic T Cell-Induced Tissue Damage in Mice.

The objective of this study was to determine how housing temperature and genetic diversity affect the onset and severity of allogeneic T cell-induced tissue damage in mice subjected to reduced intensity conditioning (RIC). We found that adoptive transfer of allogeneic CD4+ T cells from inbred donors into sub-lethally irradiated inbred recipients (I→I) housed at standard housing temperatures (ST; 22-24 °C) induced extensive BM and spleen damage in the absence of injury to any other tissue. Although engraftment of T cells in RIC-treated mice housed at their thermo-neutral temperature (TNT; 30-32 °C) also developed similar BM and spleen damage, their survival was markedly and significantly increased when compared to their ST counterparts. In contrast, the adoptive transfer of allogeneic T cells into RIC-treated outbred CD1 recipients failed to induce disease in any tissue at ST or TNT. The lack of tissue damage was not due to defects in donor T cell trafficking to BM or spleen but was associated with the presence of large numbers of B cells and myeloid cells within these tissues that are known to contain immunosuppressive regulatory B cells and myeloid-derived suppressor cells. These data demonstrate, for the first time, that housing temperature affects the survival of RIC-treated I→I mice and that RIC-conditioned outbred mice are resistant to allogeneic T cell-induced BM and spleen damage.

SARS-CoV-2 and HIV: Impact on Pulmonary Epithelial Cells.

The SARS-CoV-2 pandemic provides a natural opportunity for the collision of coronavirus disease-2019 (COVID-19) with chronic infections, which place numerous individuals at high risk of severe COVID-19. Infection with Human Immunodeficiency Virus (HIV), a global epidemic, remains a major public health concern. Whether prior HIV+ status exacerbates COVID-19 warrants investigation. Herein, we characterized the impact of SARS-CoV-2 in human bronchial epithelial cells (HBECs) previously exposed to HIV. We optimized the air-liquid interface (ALI) cell culture technique to allow for challenges with HIV at the basolateral cell surface and SARS-CoV-2 spike protein on the apical surface, followed by genetic analyses for cellular stress/toxicity and innate/adaptive immune responses. Our results suggest that the IL-10 pathway was consistently activated in HBECs treated with spike, HIV, or a combination. Recombinant spike protein elicited COVID-19 cytokine storms while HIV activated different signaling pathways. HIV-treated HBECs could no longer activate NF-kB, pro-inflammatory TRAF-6 ubiquitination nor RIP1 signaling. Combinations of HIV and SARS-CoV-2 spike increased gene expression for activation of endoplasmic reticulum-phagosome pathway and downregulated non-canonical NF-kB pathways that are key in functional regulatory T cells and RNA Polymerase II transcription. Our in vitro studies suggest that prior HIV infection may not exacerbate COVID-19. Further in vivo studies are warranted to advance this field.

Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension.

People living with HIV and who receive antiretroviral therapy have a significantly improved lifespan, compared to the early days without therapy. Unfortunately, persisting viral replication in the lungs sustains chronic inflammation, which may cause pulmonary vascular dysfunction and ultimate life-threatening Pulmonary Hypertension (PH). The mechanisms involved in the progression of HIV and PH remain unclear. The study of HIV-PH is limited due to the lack of tractable animal models that recapitulate infection and pathobiological aspects of PH. On one hand, mice with humanized immune systems (hu-mice) are highly relevant to HIV research but their suitability for HIV-PH research deserves investigation. On another hand, the Hypoxia-Sugen is a well-established model for experimental PH that combines hypoxia with the VEGF antagonist SU5416. To test the suitability of hu-mice, we combined HIV with either SU5416 or hypoxia. Using right heart catheterization, we found that combining HIV+SU5416 exacerbated PH. HIV infection increases human pro-inflammatory cytokines in the lungs, compared to uninfected mice. Histopathological examinations showed pulmonary vascular inflammation with arterial muscularization in HIV-PH. We also found an increase in endothelial-monocyte activating polypeptide II (EMAP II) when combining HIV+SU5416. Therefore, combinations of HIV with SU5416 or hypoxia recapitulate PH in hu-mice, creating well-suited models for infectious mechanistic pulmonary vascular research in small animals.

Antibiotic administration exacerbates acute graft vs. host disease-induced bone marrow and spleen damage in lymphopenic mice.

BACKGROUND: Hematopoietic 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 were not subjected to toxic, pre-transplant conditioning. RESULTS: We 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. CONCLUSIONS: We 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).

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

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 ß-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.

The Versatility of Sirtuin-1 in Endocrinology and Immunology.

Sirtuins belong to the class III family of NAD-dependent histone deacetylases (HDAC) and are involved in diverse physiological processes that range from regulation of metabolism and endocrine function to coordination of immunity and cellular responses to stress. Sirtuin-1 (SIRT1) is the most well-studied family member and has been shown to be critically involved in epigenetics, immunology, and endocrinology. The versatile roles of SIRT1 include regulation of energy sensing metabolic homeostasis, deacetylation of histone and non-histone proteins in numerous tissues, neuro-endocrine regulation via stimulation of hypothalamus-pituitary axes, synthesis and maintenance of reproductive hormones via steroidogenesis, maintenance of innate and adaptive immune system via regulation of T- and B-cell maturation, chronic inflammation and autoimmune diseases. Moreover, SIRT1 is an appealing target in various disease contexts due to the promise of pharmacological and/or natural modulators of SIRT1 activity within the context of endocrine and immune-related disease models. In this review we aim to provide a broad overview on the role of SIRT1 particularly within the context of endocrinology and immunology.

Hepatocellular protection by nitric oxide or nitrite in ischemia and reperfusion injury.

Ischemia and reperfusion (I/R)-induced liver injury occurs in several pathophysiological disorders including hemorrhagic shock and burn as well as resectional and transplantation surgery. One of the earliest events associated with reperfusion of ischemic liver is endothelial dysfunction characterized by the decreased production of endothelial cell-derived nitric oxide (NO). This rapid post-ischemic decrease in NO bioavailability appears to be due to decreased synthesis of NO, enhanced inactivation of NO by the overproduction of superoxide or both. This review presents the most current evidence supporting the concept that decreased bioavailability of NO concomitant with enhanced production of reactive oxygen species initiates hepatocellular injury and that endogenous NO or exogenous NO produced from nitrite play important roles in limiting post-ischemic tissue injury.

Differential Susceptibility to T Cell-Induced Colitis in Mice: Role of the Intestinal Microbiota.

One of the best characterized mouse models of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) is the CD4+CD45RBhigh 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 <30% at TTUHSC. We hypothesized that differences in the commensal microbiota at the 2 institutions may account for the differences in susceptibility to T cell-induced colitis. Using bioinformatic analyses of 16S rRNA amplicon sequence data, we quantified and compared the major microbial populations in feces from healthy and colitic mice housed at the 2 institutions. We found that the bacterial composition differed greatly between mice housed at LSUHSC vs TTUHSC. We identified several genera strongly associated with, and signficantly overrepresented in high responding RAG-/- mice housed at LSUHSC. In addition, we found that colonization of healthy TTUHSC RAG-/- mice with feces obtained from healthy or colitic RAG-/- mice housed at LSUHSC transferred susceptibility to T cell-induced colitis such that the recipients developed chronic colitis with incidence and severity similar to mice generated at LSUHSC. Finally, we found that the treatment of mice with preexisting colitis with antibiotics remarkably attenuated disease. Taken together, our data demonstrate that specific microbial communities determine disease susceptibility and that manipulation of the intestinal microbiota alters the induction and/or perpetuation of chronic colitis.

Methods to detect nitric oxide and its metabolites in biological samples.

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.

Collagen-binding integrin alpha1beta1 regulates intestinal inflammation in experimental colitis.

Central to inflammatory responses are the integrin-mediated adhesive interactions of cells with their ECM-rich environment. We investigated the role of the collagen-binding integrin alpha(1)beta(1) in intestinal inflammation using the mouse model of colitis induced by dextran sodium sulfate (DSS). mAb's directed against murine alpha(1) were found to significantly attenuate inflammation and injury in DSS-treated wild-type mice; similar protection was seen in mice deficient for alpha(1)beta(1) integrin. Blockade or loss of alpha(1)beta(1) was also associated with decreased mucosal inflammatory cell infiltrate and cytokine production. Importantly, we demonstrated that development and alpha(1)-mediated inhibition of DSS-induced colitis occurred independently of lymphocytes (Rag-2(-/-) mice), and identified the monocyte as a key alpha(1)beta(1)-expressing cell type involved in the development of colitis in this model. In response to DSS, both alpha(1) deficiency and anti-alpha(1) mAb treatment significantly reduced monocyte accumulation and activation within the lamina propria. In summary, the data demonstrate that engagement of leukocyte-associated alpha(1)beta(1) receptors with ECM plays a pivotal role in mediating intestinal inflammation via promotion of monocyte movement and/or activation within the inflamed interstitium. Therapeutic strategies designed to disrupt such interactions may prove beneficial in treating intestinal inflammation.

Divergent roles of superoxide and nitric oxide in reduced-size liver ischemia and reperfusion injury: Implications for partial liver transplantation.

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.

Detection of nitrosothiols and other nitroso species in vitro and in cells.

The nitric oxide (NO)-mediated nitrosation of peptides and proteins may play important roles in normobiology and pathobiology. With the realization that S-nitrosothiols (RSNOs) participate in the transport, storage, and delivery of NO, as well as posttranslational modifications in cell signaling and inflammatory processes, there is an increasing need for the detection of nitrosothiols (RSNOs) and other nitroso species in cells and tissues. In this chapter, we describe the utilization of a gas phase chemiluminescence-based assay and "biotin switch" method for the detection of nitroso species in cells. These methods are sensitive enough to quantify and contrast the different pools of nitroso species that may coexist under physiologically relevant conditions. They also provide the means to characterize and identify proteins that may represent specific targets for nitrosation reactions.

Endothelial nitric oxide synthase protects the post-ischemic liver: potential interactions with superoxide.

Hepatic ischemia and reperfusion (I/R) continues to represent a significant cause of post-transplant liver failure. The roles that certain free radicals including nitric oxide (NO) and superoxide (O(2)(-)) play in this process are not well understood. The present study was designed to assess the role of endothelial cell nitric oxide synthase (eNOS) in I/R-induced liver injury in a murine model of hepatic I/R. Forty five minutes of partial (70%) hepatic ischemia followed by 3 and 6 h of reperfusion resulted in a significant increase in liver injury which occurred in the absence of neutrophil infiltration. eNOS-deficient mice displayed enhanced liver injury when compared to their wild type controls again in the absence of neutrophil infiltration. Interestingly, basal liver blood flow was significantly decreased in these mice when compared to controls though their blood flow during reperfusion was not significantly reduced from their wild type controls. Treatment of eNOS(-/-) mice with gadolinium chloride, a potent inhibitor of Kupffer cell function, but not superoxide dismutase, significantly reduced post-ischemic hepatocellular injury while either treatment protected the wild type mouse livers. Taken together, these data suggest that NO derived from eNOS may act to protect the post-ischemic liver possibly by suppression of Kupffer cell function and not by modulation of tissue perfusion. Further the data presented here would indicate that the protective effects conferred by SOD are related to its ability to increase the bioavailability of NO rather than by attenuating superoxide-dependent reactions. Data generated from these studies may prove useful in developing new drug therapies to treat the post-ischemic liver.