Osteoarthritis induced by destabilization of the medial meniscus is reduced in germ-free mice.

OBJECTIVE: To determine the contribution of the gut microbiota to the development of injury-induced osteoarthritis (OA). DESIGN: OA was induced using the destabilized medial meniscus (DMM) model in 20 germ-free (GF) C57BL/6J male mice housed in a gnotobiotic facility and 23 strain-matched specific pathogen free (SPF) mice in 2 age groups -13.5 weeks avg age at DMM (17 SPF and 15 GF) and 43 weeks avg age at DMM (6 SPF and 5 GF). OA severity was measured using scores for articular cartilage structure (ACS), loss of safranin O (SafO) staining, osteophyte size, and synovial hyperplasia. Microbiome analysis by 16S rRNA amplicon sequencing was performed on stool samples and LPS and LPS binding protein (LBP) were measured in plasma. RESULTS: Compared to the SPF DMM mice, the maximum (MAX) ACS score per joint was 28% lower (p = 0.036) in GF DMM mice while the SafO sum score of all sections evaluated per joint was decreased by 31% (p = 0.009). The differences between SPF and GF mice in these scores were greater when only the younger mice were included in the analysis. The younger GF DMM mice also had significant reductions in osteophyte size (36%, P = 0.0119) and LBP (27%, P = 0.007) but not synovial scores or LPS. Differences in relative abundance of a number of Operational Taxonomic Units (OTUs) were noted between SPF mice with high vs low maximum ACS scores. CONCLUSIONS: These results suggest factors related to the gut microbiota promote the development of OA after joint injury.

Murine model of Clostridium difficile infection with aged gnotobiotic C57BL/6 mice and a BI/NAP1 strain.

The increased incidence and severity of Clostridium difficile infection (CDI) in older adults (age, ≥65 years) corresponds with the emergence of the BI/NAP1 strain, making elucidation of the host immune response extremely important. We therefore infected germ-free C57BL/6 mice aged 7-14 months with a BI/NAP1 strain and monitored the mice for response. Infected mice were moribund 48-72 h after infection and developed gross and histological cecitis and colitis and elevated concentrations of keratinocyte chemoattractant, interleukin 1ß, monocyte chemotactic protein 1, and granulocyte colony-stimulating factor and decreased levels of interferon γ, interleukin 12 p40, interleukin 12 p70, and interleukin 10 compared with controls. We conclude that aged, germ-free C57BL/6 mice are susceptible to fulminant CDI from a BI/NAP1 strain and represent a novel model to further elucidate the host immune response to acute CDI.

Lipopolysaccharide induces recurrence of arthritis in rat joints previously injured by peptidoglycan-polysaccharide.

Rat ankle joints injected intraarticularly with 5 micrograms of group A streptococcal peptidoglycan-polysaccharide (PG-APS) developed an acute course of arthritis. Recurrence of arthritis was induced in 100% of these joints by intravenous injection of as little as 10 micrograms of Salmonella typhimurium lipopolysaccharide (LPS) 3 wk after intraarticular injection. This reaction was similar in athymic and euthymic rats. Buffalo rats were less susceptible than Lewis or Sprague-Dawley rats. Neisseria gonorrhoeae, Yersinia enterocolitica, and Escherichia coli LPS, and S. typhimurium Re mutant LPS, were also active. Re mutant LPS activity was greatly reduced by mixing with polymyxin B. E. coli lipid A was weakly active. An acute synovitis of much less incidence, severity, and duration was seen in contralateral joints injected initially with saline, and in ankle joints of naive, previously uninjected rats after intravenous LPS injection. The intravenous injection of the muramidase mutanolysin on day 0 or 7 after intraarticular PG-APS injection prevented LPS-induced recurrence of arthritis. These studies suggest that the phlogistic activities of lipid A and peptidoglycan might interact in an inflammatory disease process, and that LPS may play a role in recurrent episodes of rheumatoid arthritis or reactive arthritis.

The specificity of peptides bound to human histocompatibility leukocyte antigen (HLA)-B27 influences the prevalence of arthritis in HLA-B27 transgenic rats.

Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis and colitis. To investigate whether this disease requires the binding of specific peptides to B27, we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391 (SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27(+)NP1(+) rats was confirmed immunologically and by mass spectrometry. The NP1 product displaced approximately 90% of the 3H-Arg-labeled endogenous peptide fraction in B27(+)NP1(+) spleen cells. Male B27(+)NP1(+) rats had a significantly reduced prevalence of arthritis, compared with B27(+)NP- males or B27(+) males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method for efficient transgenic targeting of peptides to the ER.

Multimodal optical and Gd-based nanoparticles for imaging in inflammatory arthritis.

OBJECTIVES: This report documents a multimodal nanoparticle (MNP) contrast agent, containing embedded luminophores and surface-immobilized gadolinium chelates, as a contrast agent of inflamed synovium in a collagen induced arthritis (CIA) model. METHODS: DBA-1J mice were immunized for CIA and imaged after disease onset by two independent modalities. After intravenous administration of MNP contrast, optical and magnetic resonance images were obtained and clinical disease was scored, which was followed by processing of hindlimbs for immunofluorescence and confocal microscopy. RESULTS: We show a correlation between disease severity and MNP optical luminescence that is dose dependent. Immunofluorescence of hindlimb sections reveal that MNP-labeled cells are monocytes/macrophages within the inflamed synovium. Magnetic resonance (MR) relaxation time maps, which determine the quantitative measure of T1 and T2 values at each imaging voxel, demonstrated a decreasing T2 signal in actively inflamed joints that was more pronounced earlier rather than later during disease. CONCLUSIONS: MNPs containing surface-immobilized gadolinium chelates and embedded luminophores are potential dual-modality contrast agents in inflammatory arthritis and localize to monocytes/macrophages within inflamed synovium.

Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in the pathogenesis of inflammatory bowel diseases.

Enteric microbiota can contribute to Crohn's disease and ulcerative colitis in several ways. Pathogenic or functionally altered commensal bacteria with increased mucosal adherence, invasion and intracellular persistence can activate pathogenic T cells and chronic intestinal inflammation. Compositional changes in intestinal microbiota can lead to decreased protective and increased aggressive species. Genetic polymorphisms resulting in increased mucosal permeability, decreased microbial killing, ineffective clearance of bacteria, biased TH1 and TH17 immune responses and loss of immunological tolerance are probably key contributors to IBD. Future therapies for these heterogeneous diseases should be individualized based on the patient-specific subset.

MDR1 deficiency impairs mitochondrial homeostasis and promotes intestinal inflammation.

The multidrug resistance-1 (MDR1) gene encodes an ATP-dependent efflux transporter that is highly expressed in the colon. In mice, loss of MDR1 function results in colitis with similarities to human inflammatory bowel diseases (IBD). Here, we show that MDR1 has an unexpected protective role for the mitochondria where MDR1 deficiency results in mitochondrial dysfunction with increased mitochondrial reactive oxygen species (mROS) driving the development of colitis. Exogenous induction of mROS accelerates, while inhibition attenuates colitis in vivo; these effects are amplified in MDR1 deficiency. In human IBD, MDR1 is negatively correlated to SOD2 gene expression required for mROS detoxification. To provide direct evidential support, we deleted intestinal SOD2 gene in mice and showed an increased susceptibility to colitis. We exploited the genome-wide association data sets and found many (∼5%) of IBD susceptibility genes with direct roles in regulating mitochondria homeostasis. As MDR1 primarily protects against xenotoxins via its efflux function, our findings implicate a distinct mitochondrial toxin+genetic susceptibility interaction leading to mitochondrial dysfunction, a novel pathogenic mechanism that could offer many new therapeutic opportunities for IBD.

Degradation of endogenous bacterial cell wall polymers by the muralytic enzyme mutanolysin prevents hepatobiliary injury in genetically susceptible rats with experimental intestinal bacterial overgrowth.

Jejunal self-filling blind loops with subsequent small bowel bacterial overgrowth (SBBO) induce hepatobiliary injury in genetically susceptible Lewis rats. Lesions consist of portal tract inflammation, bile duct proliferation, and destruction. To determine the pathogenesis of SBBO-induced hepatobiliary injury, we treated Lewis rats with SBBO by using several agents with different mechanisms of activity. Buffer treatment, ursodeoxycholic acid, prednisone, methotrexate, and cyclosporin A failed to prevent SBBO-induced injury as demonstrated by increased plasma aspartate aminotransferase (AST) and elevated histology scores. However, hepatic injury was prevented by mutanolysin, a muralytic enzyme whose only known activity is to split the beta 1-4 N-acetylmuramyl-N-acetylglucosamine linkage of peptidoglycan-polysaccharide (PG-PS), a bacterial cell wall polymer with potent inflammatory and immunoregulatory properties. Mutanolysin therapy started on the day blind loops were surgically created and continued for 8 wk significantly diminished AST (101 +/- 37 U/liter) and liver histology scores (2.2 +/- 2.7) compared to buffer-treated rats (228 +/- 146 U/liter, P < 0.05, 8.2 +/- 1.9, P < 0.001 respectively). Mutanolysin treatment started during the early phase of hepatic injury, 16-21 d after surgery, decreased AST in 7 of 11 rats from 142 +/- 80 to 103 +/- 24 U/liter contrasted to increased AST in 9 of 11 buffer-treated rats from 108 +/- 52 to 247 +/- 142 U/liter, P < 0.05. Mutanolysin did not change total bacterial numbers within the loop, eliminate Bacteroides sp., have in vitro antibiotic effects, or diminish mucosal PG-PS transport. However, mutanolysin treatment prevented elevation of plasma anti-PG antibodies and tumor necrosis factor-alpha (TNF alpha) levels which occurred in buffer treated rats with SBBO and decreased TNF alpha production in isolated Kupffer cells stimulated in vitro with PG-PS. Based on the preventive and therapeutic activity of this highly specific muralytic enzyme, we conclude that systemic uptake of PG-PS derived from endogenous enteric bacteria contributes to hepatobiliary injury induced by SBBO in susceptible rat strains.

Impaired mucosal defense to acute colonic injury in mice lacking cyclooxygenase-1 or cyclooxygenase-2.

To investigate roles in intestinal inflammation for the 2 cyclooxygenase (COX) isoforms, we determined susceptibility to spontaneous and induced acute colitis in mice lacking either the COX-1 or COX-2 isoform. We treated wild-type, COX-1(-/-), COX-2(-/-), and heterozygous mice with dextran sodium sulfate (DSS) to provoke acute colonic inflammation, and we quantified tissue damage, prostaglandin (PG) E(2), and interleukin-1beta. No spontaneous gastrointestinal inflammation was detected in mice homozygous for either mutation, despite almost undetectable basal intestinal PGE(2) production in COX-1(-/-) mice. Both COX-1(-/-) and COX-2(-/-) mice showed increased susceptibility to a low-dose of DSS that caused mild colonic epithelial injury in wild-type mice. COX-2(-/-) mice were more susceptible than COX-1(-/-) mice, and selective pharmacologic blockade of COX-2 potentiated injury in COX-1(-/-) mice. At a high dose, DSS treatment was fatal to 50% of the animals in each mutant group, but all wild-type mice survived. DSS treatment increased PGE(2) intestinal secretion in all groups except COX-2(-/-) mice. These results demonstrate that COX-1 and COX-2 share a crucial role in the defense of the intestinal mucosa (with inducible COX-2 being perhaps more active during inflammation) and that neither isoform is essential in maintaining mucosal homeostasis in the absence of injurious stimuli.

Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.

Genetic and environmental factors are important in the pathogenesis of clinical and experimental chronic intestinal inflammation. We investigated the influence of normal luminal bacteria and several groups of selected bacterial strains on spontaneous gastrointestinal and systemic inflammation in HLA-B27 transgenic rats. Rats maintained germfree for 3-9 mo were compared with littermates conventionalized with specific pathogen-free bacteria. Subsequently, germfree transgenic rats were colonized with groups of five to eight bacteria that were either facultative or strictly anaerobic. Transgenic germfree rats had no gastroduodenitis, colitis, or arthritis, but developed epididymitis and dermatitis to the same degree as conventionalized rats. Colonic proinflammatory cytokine expression was increased in transgenic conventionalized rats but was undetectable in germfree and nontransgenic rats. Colitis progressively increased over the first 4 wk of bacterial exposure, then plateaued. Only transgenic rats colonized with defined bacterial cocktails which contained Bacteroides spp. had colitis and gastritis. Normal luminal bacteria predictably and uniformly induce chronic colonic, gastric and systemic inflammation in B27 transgenic F344 rats, but all bacterial species do not have equal activities.

Review article: the potential mechanisms of action of rifaximin in the management of inflammatory bowel diseases.

BACKGROUND: Gut microbiota dysbiosis contributes to the pathogenesis of inflammatory bowel diseases (IBD). Although the microbiota's role in IBD pathogenesis, specifically Crohn's disease (CD), provides a rationale for antibiotic treatment, antibiotic use in CD remains controversial. Rifaximin, traditionally identified as a nonsystemic bactericidal antibiotic, may be therapeutically beneficial for inducing CD remission. AIM: To examine the role of rifaximin in the management of IBD and its potential mechanisms of action. METHODS: A literature search using the following strategy: ('inflammatory bowel disease' OR 'Crohn's' OR 'ulcerative'), 'rifaximin' AND ('barrier' OR 'translocation' OR 'adhesion' OR 'internalization' OR 'pregnane X'), AND 'pregnane X' AND ('Crohn's' OR 'ulcerative colitis' OR 'inflammatory bowel disease'). RESULTS: In vitro data suggest rifaximin mediates changes in epithelial cell physiology and reduces bacterial attachment and internalisation. In experimental colitis models, rifaximin antagonised the effects of tumour necrosis factor-α on intestinal epithelial cells by activating pregnane X receptor, which inhibits nuclear factor-κB-mediated proinflammatory mediators and induces detoxification genes (e.g. multidrug resistance 1 and cytochrome P450 3A4). Rifaximin also inhibits bacterial translocation into the mesenteric lymph nodes. CONCLUSION: Accumulating evidence suggests that mechanisms of action of rifaximin in IBD may not be limited to direct bactericidal activity; therefore, rifaximin could potentially be redefined as a gut environment modulator.

Rifaximin Exerts Beneficial Effects Independent of its Ability to Alter Microbiota Composition.

OBJECTIVES: Rifaximin has clinical benefits in minimal hepatic encephalopathy (MHE) but the mechanism of action is unclear. The antibiotic-dependent and -independent effects of rifaximin need to be elucidated in the setting of MHE-associated microbiota. To assess the action of rifaximin on intestinal barrier, inflammatory milieu and ammonia generation independent of microbiota using rifaximin. METHODS: Four germ-free (GF) mice groups were used (1) GF, (2) GF+rifaximin, (3) Humanized with stools from an MHE patient, and (4) Humanized+rifaximin. Mice were followed for 30 days while rifaximin was administered in chow at 100 mg/kg from days 16-30. We tested for ammonia generation (small-intestinal glutaminase, serum ammonia, and cecal glutamine/amino-acid moieties), systemic inflammation (serum IL-1ß, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1ß, IL-6, MCP-1, e-cadherin and zonulin) along with microbiota composition (colonic and fecal multi-tagged sequencing) and function (endotoxemia, fecal bile acid deconjugation and de-hydroxylation). RESULTS: All mice survived until day 30. In the GF setting, rifaximin decreased intestinal ammonia generation (lower serum ammonia, increased small-intestinal glutaminase, and cecal glutamine content) without changing inflammation or intestinal barrier function. Humanized microbiota increased systemic/intestinal inflammation and endotoxemia without hyperammonemia. Rifaximin therapy significantly ameliorated these inflammatory cytokines. Rifaximin also favorably impacted microbiota function (reduced endotoxin and decreased deconjugation and formation of potentially toxic secondary bile acids), but not microbial composition in humanized mice. CONCLUSIONS: Rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression independent of gut microbiota. MHE-associated fecal colonization results in intestinal and systemic inflammation in GF mice, which is also ameliorated with rifaximin.

Colitis in HLA-B27/beta 2 microglobulin transgenic rats.

Rats of susceptible genetic backgrounds expressing high copy numbers of the transgene encoding HLA-B27 and human beta 2 mu develop chronic colitis complicated in the advanced stage by adenomatous polyps progressing to adenocarcinoma. Unique features of this model include a spectrum of extraintestinal manifestations resembling to some extent human spondyloarthropathy, with peripheral and axial joint, dermatologic and male genital inflammation. Inflammation is T lymphocyte mediated, although surprisingly CD4+ cells are more active in transferring disease than CD8+ cells, which would be expected to be preferentially activated by Class I MHC peptides. Inflammation is dependent on a nonlymphoid bone marrow-derived cell, expressing high copy numbers of B27, probably APCs. In vitro function of transgenic dendritic cells is deficient, and in vivo competition for peptide binding in the antigen binding site of B27 attenuates arthritis. Normal bacteria are required for disease expression, with B. vulgatus preferentially able to induce colitis, whereas other bacteria such as E. coli stimulate no inflammatory response. Inflammation and resulted complications are modulated by non-MHC genes and are amenable to treatment by bone marrow transplant from normal donors. These results support the hypothesis that gastrointestinal and systemic inflammation in B27 transgenic rats is the result of loss of tolerance to enteric bacteria, as a consequence of defective APC (? dendritic cells) function. Whether disease is the result of selective MHC binding of enteric antigens uniquely capable of inducing disease, lack of appropriate induction of a CD8+ suppressor cell population, or skewed cytokine (IL-12, IL-18) secretion by APCs remains to be determined.

A rat model of ileal pouch-rectal anastomosis.

After colectomy and ileal pouch-rectal anastomosis, pouchitis may occur. Pouchitis is a poorly defined condition with unknown etiology. The aim of this study was to develop an animal model of pouchitis. Ileal pouch-rectal anastomosis was created in Lewis and Sprague-Dawley rats. Rats were studied 4 and 8 weeks after surgery, and pouchitis was assessed by stool output, histology, and tissue myeloperoxidase (MPO) levels. Some rats were treated with allopurinol or metronidazole beginning the day of surgery. Rats with pouches demonstrated inflammation with a monocytic infiltration, luminal exudate, mucosal ulcerations, and serosal inflammation. Rats with pouches had increased anaerobic bacterial flora compared with normal ileum. After creation of pouches, Lewis rats (histology score = 8.4 +/- 1.6; MPO = 17.3 +/- 3.6, mean +/- SD) developed more severe inflammation than Sprague-Dawley rats did (histology score = 4.3 +/- 1.8; MPO = 5.5 +/- 3.6) within 4 weeks, p < 0.001 and 8 weeks after surgery, p < 0.05. Stool output was also greater in Lewis (55 +/- 7 g/kg/day) compared with Sprague-Dawley rats with pouches (43 +/- 5 g/kg/day), p < 0.05. Metronidazole treatment reduced histology score (6.0 +/- 0.5) p < 0.05 and MPO (5.9 +/- 1.6) p < 0.001 in rats with pouches compared with rats with pouches that had no treatment. Allopurinol treatment in rats with pouches reduced histology score (4.0 +/- 1.7) and MPO (3.9 +/- 1.6), p < 0.001, compared with rats with pouches that had no treatment. Ileal pouch-rectal anastomosis in rats induced inflammation within 4 weeks, demonstrated differential host genetic susceptibility, and was associated with increased number of pouch bacteria. Anaerobes, especially bacteroides sp. and free radical, may mediate inflammation. Ileal pouch-rectal anastomosis surgery in rats may be a useful animal model for the study pouchitis.

Cytokine regulation of experimental intestinal inflammation in genetically engineered and T-lymphocyte reconstituted rodents.

Phenotypically similar intestinal inflammation and systemic wasting develops following overexpression or targeted deletion of several key immunoregulatory molecules and transfer of lymphocyte subsets into immunodeficient recipients. Although many of these recently described models of intestinal inflammation have not been thoroughly investigated, several consistent features are present which provide important insights into the role of cytokines in the pathogenesis of intestinal inflammation: (i) entirely distinct genetic alterations of cytokine expression and T-lymphocyte activity can lead to phenotypically similar intestinal inflammation, suggesting that human inflammatory bowel disease could have marked genetic heterogeneity; (ii) dysregulation of any of a number to immunoregulatory molecules can result in intestinal inflammation, illustrating the complexity of the mucosal immune response; (iii) active immunosuppression is critical to maintaining mucosal homeostasis; (iv) interferon-gamma and CD4+ lymphocytes, probably of the TH1 phenotype, are required for progression of chronic intestinal inflammation; (v) monokines are consistently upregulated, but tumour necrosis factor blockade is only partially protective. These models represent powerful new tools to understand better the basic mechanisms of mucosal immunoregulation and to develop novel therapeutic approaches of enhancing endogenous immunosuppressive pathways and blocking key regulatory cytokines and cells.

Review article: Role of the enteric microflora in the pathogenesis of intestinal inflammation and arthritis.

Strong associations exist between intestinal inflammation and arthritis, ranging from infections with enteric pathogens to idiopathic inflammatory bowel disease. Increased exposure of the lamina propia and systemic circulation to enteric microflora and their products are a result of increased proliferation of the luminal bacteria, pathogenic invasion or enhanced mucosal permeability. Data suggest that anaerobic bacteria and other constituents of the normal luminal microbial flora induce and sustain chronic intestinal inflammation and arthritis. However, the normal host develops a tolerance to such bacteria and maintains homeostasis through a controlled inflammatory response and an almost impermeable mucosal barrier.

Review article: How relevant to human inflammatory bowel disease are current animal models of intestinal inflammation?

New rodent models of chronic intestinal inflammation are mediated by a TH1-cell and macrophage dominated immune response to luminal bacterial constituents. The pathology of these spontaneous and induced models differ widely and caution is needed when assessing the comparative aspects of such animal models and human inflammatory bowel diseases (IBD). Considerable immunological and therapeutic evidence suggests that chronic and immune-mediated models are relevant in human IBD and that pathogenic principles are similar. However, animal models have not been able to duplicate exactly the pathological characteristics of ulcerative colitis or Crohn's disease, indicating a need for caution in extrapolating data from experimental models to human IBD.

Identification and characterization of rat intestinal lamina propria cells: consequences of microbial colonization.

Germ-free (GF) animals exhibit an abnormally diminished, cell-mediated immune response which can be rapidly normalized by bacterial colonization of the intestine. This conventionalization suggests that the development and/or regulation of the immune system is dependent upon intestinal bacteria or their products. Here we consider the ontogeny of gut-associated lymphoid tissue (GALT) immunocytes by isolating and characterizing the intestinal lamina propria cells (LPC) of GF rats responding to bacterial colonization or an irrelevant protein antigen, and compared to LPC of specific pathogen-free (SPF) rats which were conventionalized (CV) from birth. Isolation of cells was accomplished by successive EDTA washings of small intestine to remove the epithelium, and enzymatic digestion of the tissue generating single-cell suspensions. Resulting cell suspensions were characterized by monoclonal antibodies directed against leukocyte epitopes using flow cytometry. Functional characterization was measured by the tritiated thymidine proliferation assay with concanavalin A (Con A) and lipopolysaccharide (LPS) as co-stimulators. Germ-free and SPF rats had fewer total LPC than CV rats. Antibody staining revealed that GF rats had fewer total leukocytes than CV and SPF rats, and that CV rats had a greater percentage of T-cells and cells positive for the C3 receptor than GF rats. Co-stimulation of LPC with mitogens only increased proliferation of cells from CV rats compared to GF and SPF rats. In addition, spleen cells from CV rats demonstrated significantly enhanced proliferative responses compared to spleen cells from GF rat and were more analogous to spleen cells from SPF rats in their ability to proliferate in vitro, with and without mitogens. We conclude that T-cells and CD35-positive (C3BR+) cells are recruited and/or proliferate in response to intestinal bacteria and/or their products, and that this results in the induction of immune competency.

Mechanisms of acute and chronic intestinal inflammation induced by indomethacin.

The objective of this study was to characterize the mechanisms of acute and chronic intestinal mucosal injury and inflammation induced by subcutaneously injected indomethacin (Indo). One injection of Indo (7.5 mg/kg) produced acute injury and inflammation in the distal jejunum and proximal ileum that were maximal at three days and completely resolved within one week. Two daily subcutaneous injections of Indo produced a more extensive and chronic inflammation that lasted in an active form in more than 75% of the rats for at least two weeks. Epithelial injury, as measured by enhanced mucosal permeability, was significantly elevated only at one day in the acute model (one injection) but was persistently elevated in the chronic model (two injections). Bile duct ligation completely attenuated increased mucosal permeability in the acute model, however, depletion of circulating neutrophils had no effect. Neither Indo (0-0.1 mg/ml) nor normal bile was cytotoxic to cultured rat intestinal epithelial cells; however, they synergistically promoted significant cytotoxicity. Bile collected from rats treated with Indo was cytotoxic towards the epithelial cells in a dose-dependent manner. Sulfasalazine and metronidazole (100 mg/kg/day, both) attenuated enhanced mucosal permeability in the chronic model. Massive bacterial translocation into the mesenteric lymph nodes, liver, and spleen following two injections of Indo was significantly attenuated by metronidazole. We conclude that: (1) a single injection of Indo produces acute intestinal mucosal injury and inflammation that resolve completely within three to seven days, whereas two daily injections of Indo produce both acute and chronic injury and inflammation, (2) enterohepatic circulation of Indo is important in promoting the acute phases of injury and inflammation, (3) circulating neutrophils do not play a role in the pathogenesis of this model, and (4) endogenous bacteria play an important role in exacerbating and/or perpetuating the chronic phases of injury and inflammation.

Development of a scoring system to predict mortality from upper gastrointestinal bleeding.

Despite the widespread application of endoscopy in acute upper gastrointestinal bleeding, there is little evidence of improved survival among those who undergo the procedure. To select high-risk patients who might benefit most from diagnostic and therapeutic endoscopy, the authors developed and validated a scoring system based on prognostic indicators of increased mortality. The scoring system was developed from the best clinical predictors of mortality, determined in a prospective study of consecutive bleeding patients. The model was then tested in a prospective validation phase at three hospitals. Three main factors in the model predict mortality: bleeding, including hematochezia, drop in hematocrit of 5%, short duration of bleeding, absence of melena, and hypotension; liver disease, manifested by prolonged prothrombin time and encephalopathy; and renal disease. Patients determined to be at high risk for death using the scoring system might be candidates for aggressive management and for therapeutic endoscopy.