Thomas A. Churchill

Hepatitis C virus replication in mice with chimeric human livers

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Reduction of colitis by prebiotics in HLA-B27 transgenic rats is associated with microflora changes and immunomodulation.

&NA; HLA‐B27 transgenic rats develop spontaneous colitis under specific pathogen‐free conditions (SPF) but germ‐free rats remain disease‐free, emphasizing a role for intestinal bacteria in the pathogenesis of chronic intestinal inflammation. Prebiotics are dietary substances that affect the host by stimulating growth and/or activity of potentially health promoting bacteria. The aims of this study were to investigate whether prebiotics can prevent colitis in SPF HLA‐B27 rats, and secondly, to explore mechanisms of protection. SPF HLA‐B27 transgenic rats received orally the prebiotic combination long‐chain inulin and oligofructose (Synergy 1), or not, prior to the development of clinically detectable colitis. After seven weeks, cecal and colonic tissues were collected for gross cecal scores (GCS), histologic inflammatory scores (scale 0‐4), and mucosal cytokine measurement. Cecal and colonic contents were collected for analysis of the gut microbiota by PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE) and fluorescent in‐situ hybridization (FISH), and analysis of short‐chain fatty acid composition. Prebiotic treatment significantly decreased GCS and inflammatory histologic scores in the cecum and colon. Prebiotic treatment also decreased cecal IL‐1&bgr;, but increased cecal TGF‐&bgr; concentrations. Inulin/oligofructose altered the cecal and colonic PCR‐DGGE profiles, and FISH analysis showed significant increases in cecal Lactobacillus and Bifidobacterium populations after prebiotic treatment compared with water‐treated rats. In conclusion, the prebiotic combination Synergy 1 reduced colitis in HLA‐B27 transgenic rats, which effect was associated with alterations to the gut microbiota, decreased tissue proinflammatory cytokines and increased immunomodulatory molecules. These results show promise for prebiotics as primary or adjuvant maintenance therapy for chronic inflammatory bowel diseases.

Probiotic bacteria prevent hepatic damage and maintain colonic barrier function in a mouse model of sepsis

A breakdown in intestinal barrier function and increased bacterial translocation are key events in the pathogenesis of sepsis and liver disease. Altering gut microflora with noninvasive and immunomodulatory probiotic organisms has been proposed as an adjunctive therapy to reduce the level of bacterial translocation and prevent the onset of sepsis. The purpose of this study was to determine the efficacy of a probiotic compound in attenuating hepatic and intestinal injury in a mouse model of sepsis. Wild‐type and interleukin‐10 (IL‐10) gene–deficient 129 Sv/Ev mice were fed the probiotic compound VSL#3 for 7 days. To induce sepsis, the mice were injected with lipopolysaccharide (LPS) and D‐galactosamine (GalN) in the presence and absence of the peroxisome proliferator‐activated receptor gamma (PPARγ) inhibitor GW9662. The mice were killed after 6 hours, and their colons were removed for the measurement of the cytokine production and epithelial function. The functional permeability was assessed by the mannitol movement and cyclic adenosine monophosphate–dependent chloride secretion in tissue mounted in Ussing chambers. The livers were analyzed for bacterial translocation, cytokine production, histological injury, and PPARγ levels. The tissue levels of tumor necrosis factor alpha, interferon gamma, IL‐6, and IL‐12p35 ribonucleic acid were measured by semiquantitative reverse transcription polymerase chain reaction. Mice injected with LPS/GalN demonstrated a breakdown in colonic barrier function, which correlated with enhanced proinflammatory cytokine secretion, bacterial translocation, and significant hepatic injury. A pretreatment with oral probiotics prevented the breakdown in intestinal barrier function, reduced bacterial translocation, and significantly attenuated liver injury. The inhibition of PPARγ with GW9662 abrogated the protection induced by probiotics. Conclusion: Orally administered probiotics prevented liver and intestinal damage in a mouse model of sepsis through a PPARγ‐dependent mechanism. (HEPATOLOGY 2007.)

Surface Expression of Toll-Like Receptor 9 Is Upregulated on Intestinal Epithelial Cells in Response to Pathogenic Bacterial DNA

ABSTRACT Colonic epithelial cells are constantly exposed to high levels of bacterial DNA in the intestinal lumen and must recognize and respond appropriately to pathogens, while they maintain a tolerance to nonpathogenic commensal bacterial strains. Bacterial DNA is recognized by Toll-like receptor 9 (TLR9). The aim of this study was to investigate TLR9 expression and localization in colonic epithelial cells under basal conditions and in response to bacterial DNA. HT-29 cells were exposed to DNA from various strains of commensal and pathogenic microbes. TLR9 mRNA expression was determined by real-time reverse transcription-PCR, and interleukin-8 (IL-8) secretion was measured by an enzyme-linked immunosorbent assay. Localization of TLR9 was determined by flow cytometry in HT-29 cells and by immunofluorescence in HT-29 cells and mouse colonic tissue. Immunofluorescence and flow cytometric analyses demonstrated that there was intracellular and surface expression of TLR9 in HT-29 cells under basal conditions. Exposure of cells to DNA from pathogenic strains of Salmonella and Escherichia coli resulted in a significant increase in TLR9 mRNA expression. Salmonella enterica serovar Dublin DNA increased surface TLR9 protein and IL-8 secretion. There was no change in mRNA levels or localization of TLR9 in response to Bifidobacterium breve. Chloroquine did not block IL-8 secretion in response to S. enterica serovar Dublin DNA. TLR9 was expressed on the colonic apical surface in wild-type mice but not in germfree mice. These results demonstrate that intestinal epithelial cells recognize pathogenic bacterial DNA and respond by increasing surface localization and expression of TLR9, suggesting that the epithelial inflammatory response to pathogenic DNA is mediated at least in part by increased TLR9 expression.

Glutaraldehyde-Fixed Bioprosthetic Heart Valve Conduits Calcify and Fail From Xenograft Rejection

Background— Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification. Methods and Results— Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification. Conclusions— G-F xenografts have cellular/humoral rejection and calcify secondarily.

5-Aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent jejunal glucose transport: a possible role for AMPK

AMPK (AMP-activated protein kinase) is a key sensor of energy status within the cell. Activated by an increase in the AMP/ATP ratio, AMPK acts to limit cellular energy depletion by down-regulating selective ATP-dependent processes. The purpose of the present study was to determine the role of AMPK in regulating intestinal glucose transport. [3H]3-O-methyl glucose fluxes were measured in murine jejunum in the presence and absence of the AMPK activators AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin and the p38 inhibitor, SB203580. To differentiate between a sodium-coupled (SGLT1) and diffusive (GLUT2) route of entry, fluxes were measured in the presence of the SGLT1 and GLUT2 inhibitors phloridzin and phloretin. Glucose transporter mRNA levels were measured by reverse transcriptase-PCR, and localization by Western blotting. Surface-expressed GLUT2 was assessed by luminal biotinylation. Activation of p38 mitogen-activated protein kinase was analysed by Western blotting. We found that treatment of jejunal tissue with AICAR resulted in enhanced net glucose uptake and was associated with phosphorylation of p38 mitogen-activated protein kinase. Inhibition of p38 abrogated the stimulation of AICAR-stimulated glucose uptake. Phloretin abolished the AICAR-mediated increase in glucose flux, whereas phloridzin had no effect, suggesting the involvement of GLUT2. In addition, AICAR decreased total protein levels of SGLT1, concurrently increasing levels of GLUT2 in the brush-border membrane. The anti-diabetic drug metformin, a known activator of AMPK, also induced the localization of GLUT2 to the luminal surface. We conclude that the activation of AMPK results in an up-regulation of non-energy requiring glucose uptake by GLUT2 and a concurrent down-regulation of sodium-dependent glucose transport.

Intermediary Energy Metabolism during Dormancy and Anoxia in the Land Snail Otala lactea

Metabolic responses to dormancy and anoxia were assessed in foot muscle and he-patopancreas of the land snail Otala lactea (Pulmonata, Helicidae). In both states cellular energetics (arginine phosphate content, adenylate energy charge) were maintained at a high level, fueled during estivation by aerobic metabolism and during anoxia by carbohydrate fermentation. D-lactate was the major product of anaerobiosis in hepatopancreas, but in foot muscle D-lactate, L-alanine, and succi-nate accumulated (in net amounts of 13.5, 3.5, and 1.8 μmolg⁻¹ wet weight, respec-tively). Changes in the concentrations of glycolytic intermediates were compared for both short-and long-term stress: 2 and 14 h of exposure to N₂ gas atmosphere, 3 and 22 d of dormancy at 22° C. Both stresses appeared to include glycolytic activation in the short term in foot muscle, with crossover analyses indicating regulatory control at the phosphofructokinase, aldolase, and pyruvate kinase loci. Over the long term, however, this was reversed and a glycolytic rate depression was observed aspart of the overall metabolic rate depression of these states. In foot muscle, inhibition at phosphofructokinase and pyruvate kinase was apparent during anoxia, whereas aldolase and pyruvate kinase were the key sites of inhibitory control during estivation. In hepatopancreas, phosphofructokinase was the primary locus for inhibitory control during both prolonged anoxia and estivation. These data suggest that common molecular mechanisms underlie glycolytic rate depression during dormancy and anaerobiosis.

Short-term storage of the ischemically damaged human pancreas by the two-layer method prior to islet isolation.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 ± 0.5 h after 11.1 ± 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 ± 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.

Novel approaches toward early diagnosis of islet allograft rejection.

Background. The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. Methods. (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (&bgr;-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. Results. (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57±0.2 vs. 2.95±0.3 for control islets, P =ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet “sentinel signal” could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58±0.3 vs. 1.63±0.2%/min, respectively, P <0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. Conclusions. Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

IFN-gamma is an absolute requirement for spontaneous acceptance of liver allografts.

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon‐γ (IFN‐γ) in the spontaneous acceptance of mouse liver allografts. Strain of mouse (CBA) liver allografts transplanted into normal BALB/c mice developed histologic changes typical of rejection that spontaneously regressed, permitting long‐term survival of these allografts similar to that of syngeneic grafts. In contrast, CBA liver allografts in IFN‐γ‐deficient hosts manifested not only infiltration but also hemorrhage and necrosis, with no survival beyond 14 days. Despite differences in survival, local expression of cytotoxic T‐cell genes in the transplant was not increased in IFN‐γ‐deficient hosts, but livers in interferon‐γ‐deficient mice (GKO) hosts displayed much less induction of major histocompatibility complex (MHC) class I and II expression. To determine whether the difference in survival was secondary to the direct effects of IFN‐γ on the liver, we transplanted livers from IFN‐γ‐receptor‐deficient mice into normal hosts. Liver allografts lacking IFN‐γ receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN‐γ mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.