Gut permeability and mucosal inflammation: bad, good or context dependent.

Inflammatory bowel disease (IBD) is a multifactorial disease. A breach in the mucosal barrier, otherwise known as "leaky gut," is alleged to promote mucosal inflammation by intensifying immune activation. However, interaction between the luminal antigen and mucosal immune system is necessary to maintain mucosal homeostasis. Furthermore, manipulations leading to deregulated gut permeability have resulted in susceptibility in mice to colitis as well as to creating adaptive immunity. These findings implicate a complex but dynamic association between mucosal permeability and immune homeostasis; however, they also emphasize that compromised gut permeability alone may not be sufficient to induce colitis. Emerging evidence further supports the role(s) of proteins associated with the mucosal barrier in epithelial injury and repair: manipulations of associated proteins also modified epithelial differentiation, proliferation, and apoptosis. Taken together, the role of gut permeability and proteins associated in regulating mucosal inflammatory diseases appears to be more complex than previously thought. Herein, we review outcomes from recent mouse models where gut permeability was altered by direct and indirect effects of manipulating mucosal barrier-associated proteins, to highlight the significance of mucosal permeability and the non-barrier-related roles of these proteins in regulating chronic mucosal inflammatory conditions.

NIH consensus development statement on management of hepatitis B.

OBJECTIVE: To provide health care providers, patients, and the general public with a responsible assessment of currently available data on the management of hepatitis B. PARTICIPANTS: A non-DHHS, nonadvocate 12-member panel representing the fields of hepatology and liver transplantation, gastroenterology, public health and epidemiology, infectious diseases, pathology, oncology, family practice, internal medicine, and a public representative. In addition, 22 experts from pertinent fields presented data to the panel and conference audience. EVIDENCE: Presentations by experts and a systematic review of the literature prepared by the Minnesota Evidence-based Practice Center, through the Agency for Healthcare Research and Quality. Scientific evidence was given precedence over anecdotal experience. CONFERENCE PROCESS: The panel drafted its statement based on scientific evidence presented in open forum and on published scientific literature. The draft statement was presented on the final day of the conference and circulated to the audience for comment. The panel released a revised statement later that day at http://consensus.nih.gov. This statement is an independent report of the panel and is not a policy statement of the NIH or the Federal Government. CONCLUSIONS: The most important predictors of cirrhosis or hepatocellular carcinoma in persons who have chronic HBV are persistently elevated HBV DNA and ALT levels in blood. Other risk factors include HBV genotype C infection, male sex, older age, family history of hepatocellular carcinoma, and co-infection with HCV or HIV. The major goals of anti-HBV therapy are to prevent the development of progressive disease, specifically cirrhosis and liver failure, as well as hepatocellular carcinoma development and subsequent death. To date, no RCTs of anti-HBV therapies have demonstrated a beneficial impact on overall mortality, liver-specific mortality, or development of hepatocellular carcinoma. Most published reports of hepatitis therapy use changes in short-term virologic, biochemical, and histologic parameters to infer likelihood of long-term benefit. Approved therapies are associated with improvements in intermediate biomarkers, including HBV DNA, HBeAg loss or seroconversion, decreases in ALT levels, and improvement in liver histology (Table). Although various monitoring practices have been recommended, no clear evidence exists for an optimal approach. The most important research needs include representative prospective cohort studies to define the natural history of the disease and large RCTs of monotherapy and combined therapies, including placebo-controlled trials, that measure the effects on clinical health outcomes. Table. Criteria Useful in Determining for Whom Therapy is Indicated: Patients for whom therapy is indicated: Patients who have acute liver failure, cirrhosis and clinical complications, cirrhosis or advanced fibrosis and HBV DNA in serum, or reactivation of chronic HBV after chemotherapy or immunosuppression; Infants born to women who are HBsAg-positive (immunoglobulin and vaccination). Patients for whom therapy may be indicated: Patients in the immune-active phase who do not have advanced fibrosis or cirrhosis. Patients for whom immediate therapy is not routinely indicated: Patients with chronic hepatitis B in the immune-tolerant phase (with high levels of serum HBV DNA but normal serum ALT levels or little activity on liver biopsy); Patients in the inactive carrier or low replicative phase (with low levels of or no detectable HBV DNA in serum and normal serum ALT levels); Patients who have latent HBV infection (HBV DNA without HBsAg). We recommend routine screening for hepatitis B of newly arrived immigrants to the United States from countries where the HBV prevalence rate is greater than 2%. Screening will facilitate the provision of medical and public health services for infected patients and their families and provide public health data on the burden of disease in immigrant populations. The screening test should not be used to prohibit immigration.

Malondialdehyde-acetaldehyde-protein adducts increase secretion of chemokines by rat hepatic stellate cells.

Findings obtained from our recent studies have demonstrated that malondialdehyde, a product of lipid peroxidation, and acetaldehyde can react together with proteins in a synergistic manner and form hybrid protein conjugates, which have been designated as malondialdehyde-acetaldehyde (MAA)-protein adducts. These adducts have been detected in livers of ethanol-fed rats and are immunogenic because significant increases in circulating antibody titers against MAA-adducted proteins have been observed in ethanol-fed rats and more recently in human alcoholics. Although immunological factors may tend to perpetuate liver injury, little is known about the direct functional consequences of MAA-adducted proteins on the different cellular populations of the liver. Hepatic stellate cells (HSCs) have been shown to be pivotal in the pathogenesis of fibrosis and in the amplification and self-perpetuation of the inflammatory process. The present study was conducted to determine the effects of MAA-adducted proteins on the function of HSCs. Rat HSCs were exposed to various amounts of MAA-protein adducts and their unmodified controls, and the secretion of two chemokines, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, that are involved in the chemotaxis of monocytes/macrophages and neutrophils, respectively, was determined. We observed that bovine serum albumin-MAA induced a dose- and time-dependent increase in the secretion of both of these chemokines. These findings indicate that MAA-adducted proteins may play a role in the modulation of the hepatic inflammatory response and could contribute to the pathogenesis of alcoholic liver disease.

Elucidation of reaction scheme describing malondialdehyde-acetaldehyde-protein adduct formation.

Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed animals. Our previous studies have shown that MAA adducts are comprised of two distinct products. One adduct is composed of two molecules of malondialdehyde and one molecule of acetaldehyde and was identified as the 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group (MDHDC adduct). The other adduct is a 1:1 adduct of malondialdehyde and acetaldehyde and was identified as the 2-formyl-3-(alkylamino)butanal derivative of an amino group (FAAB adduct). In this study, information on the mechanism of MAA adduct formation was obtained, focusing on whether the FAAB adduct serves as a precursor for the MDHDC adduct. Upon the basis of chemical analysis and NMR spectroscopy, two initial reaction steps appear to be a prerequisite for MDHDC formation. One step involves the reaction of one molecule of malondialdehyde and one of acetaldehyde with an amino group of a protein to form the FAAB product, while the other step involves the generation of a malondialdehyde-enamine. It appears that generation of the MDHDC adduct requires the FAAB moiety to be transferred to the nitrogen of the MDA-enamine. For efficient reaction of FAAB with the enamine to take place, additional experiments indicated that these two intermediates likely must be in positions on the protein of close proximity to each other. Further studies showed that the incubation of liver proteins from ethanol-fed rats with MDA resulted in a marked generation of MDHDC adducts, indicating the presence of a pool of FAAB adducts in the liver of ethanol-fed animals. Overall, these findings show that MDHDC-protein adduct formation occurs via the reaction of the FAAB moiety with a malondialdehyde-enamine, and further suggest that a similar mechanism may be operative in vivo in the liver during prolonged ethanol consumption.

Lipoatrophic diabetes and end-stage liver disease secondary to nonalcoholic steatohepatitis with recurrence after liver transplantation.

BACKGROUND: Lipoatrophic diabetes is an insulin resistance syndrome characterized by the complete or partial lack of adipose tissue and disturbances in lipid and glucose metabolism. Nonalcoholic steatohepatitis (NASH) is a well-described change in liver pathology consisting of steatosis, hepatitis, and fibrosis that can be associated with lipoatrophic diabetes. RESULTS: This article describes the first reported case of lipoatrophic diabetes with NASH leading to liver failure and liver transplantation. Before transplantation, the patient required 600-700 U of insulin/day. After transplantation, a dramatic decline in her insulin requirements was observed, despite corticosteroids. Eighteen months after transplantation, her glycemic control worsened, and she developed recurrent NASH on serial liver biopsies. CONCLUSIONS: NASH associated with lipoatrophic diabetes can recur after liver transplantation, and in this case, was accompanied by increased insulin requirements. These results suggest that the development of NASH itself may contribute to the insulin resistance observed in lipoatrophic diabetes.

Expression and cytoskeletal association of integrin subunits is selectively increased in rat perivenous hepatocytes after chronic ethanol administration.

BACKGROUND: For normal function and survival, hepatocytes require proper cell-extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the beta1 integrin subunit, the common beta subunit for two major hepatocyte-ECM receptors, alpha1beta1 and alpha5beta1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of alpha1, alpha5, and beta1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes. METHODS: Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting. RESULTS: Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type. CONCLUSIONS: The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration.

Ethanol feeding selectively impairs the spreading of rat perivenous hepatocytes on extracellular matrix substrates.

BACKGROUND: Hepatocytes require attachment and subsequent spreading on an extracellular matrix for their proper growth, function and survival. Our previous studies have shown that ethanol feeding selectively impairs perivenule hepatocyte attachment to various extracellular matrices. This study was undertaken to determine whether zonal differences in hepatocyte spreading in response to ethanol feeding occurs and to ascertain the influence of ethanol consumption on the zonal expression of the beta1 subunit of integrins, which are the major surface receptors responsible for matrix binding and subsequent interactions. METHODS: Hepatocytes from the perivenous and periportal regions of the liver were isolated by digitonin/collagenase perfusion from rats that were pair-fed for 2 to 3 weeks with a liquid diet containing either ethanol or isocaloric carbohydrate. The ability of perivenous and periportal hepatocytes to spread on plates coated with either type IV collagen, laminin, fibronectin or polylysine was determined. In addition, the isolated cells were used for the analysis of total cellular and surface beta1 integrin expression. RESULTS: With all of the matrix substrates tested, the spreading of perivenous hepatocytes isolated from the ethanol-fed animals was markedly impaired, while the spreading of periportal hepatocytes was essentially unaffected by ethanol feeding. Both the total cellular as well as the surface expression of the beta1 integrin subunit in perivenous cells from the ethanol-fed rats were significantly higher than from the perivenous control cells, whereas the total and surface expression of the beta1 integrin in periportal cells isolated from ethanol-fed and control rats were not significantly different. CONCLUSIONS: The results indicated that in addition to impairing hepatocyte attachment, ethanol feeding also impairs another critical step of the adhesion process, that of hepatocyte spreading on extracellular matrix substrates. This defect occurred preferentially in perivenous cells and not periportal cells and was associated with an increase in beta1 integrin expression, suggesting that a compensatory mechanism occurs as an attempt by the perivenous cells to overcome impaired cell-matrix interactions caused by ethanol. Overall, these alterations in extracellular matrix-hepatocyte interactions could lead to alterations of hepatocyte structure and function and potentially play a role in alcoholic liver injury.

Conversion of acetaldehyde-protein adduct epitopes from a nonreduced to a reduced phenotype by antigen processing cells.

Many investigators have suggested that an immune reaction to acetaldehyde-protein adducts may be involved in the development and/or progression of alcohol liver disease. The most often reported acetaldehyde adduct is the reduced adduct prepared in vitro in the presence of strong reducing agents. However, the production of this adduct in vivo has been difficult to prove. Nevertheless, the detection of serum antibodies to this reduced adduct following alcohol exposure in animals and humans has been used to support the formation of this adduct in vivo. We have recently observed that when acetaldehyde-protein adducts prepared under nonreducing conditions are used to immunize animals, antibody to the reduced protein adduct is detected. Therefore, it was the purpose of this study to demonstrate that nonreduced (NR) adduct epitopes can be modified by intact cells to express reduced (R) adduct epitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to recognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mice were injected intraperitoneally (500 microg/animal) with either keyhole limpet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-NR produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mice were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH proteins, and the cell surface expression of the reduced epitope (RT1.1) and the activated macrophage marker (MAC-3) determined by double immunofluorescent staining. Activated macrophages incubated with KLH-NR expressed the R adduct on 11.5% of the cells, compared with 3.8% following incubation with unmodified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal macrophages in vivo and present an epitope that is detected as a reduced adduct (RT1.1 positive). These observations may explain the presence of circulating antibodies to the reduced adduct that has been reported in human and animal studies.

Hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) for most patients is a terminal complication of chronic inflammatory and fibrotic liver disease. With regrettably few exceptions, treatment is largely palliative, and long-term survival is rare. However, the major causes of HCC worldwide are known and preventable. Hepatitis B and C exist only in man; the viruses have no known non-human reservoirs. Transmission of the viruses can be interrupted by vaccination against hepatitis B virus infection and improvements in medical techniques for hepatitis C, for which no vaccine has yet been developed.

Soluble proteins modified with acetaldehyde and malondialdehyde are immunogenic in the absence of adjuvant.

Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.

Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats.

BACKGROUND & AIMS: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. METHODS: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. RESULTS: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. CONCLUSIONS: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses.

Chronic ethanol ingestion impairs TGF-alpha-stimulated receptor autophosphorylation.

The effects of chronic ethanol feeding on the binding of transforming growth factor-alpha (TGF-alpha) and TGF-alpha-stimulated receptor autophosphorylation were investigated in isolated rat hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 6-8 weeks, these cells exhibited a marked impairment of TGF-alpha-stimulated autophosphorylation of the receptor that binds this growth factor compared with hepatocytes from the pair-fed controls. This impaired autophosphorylation of receptor tyrosine residues was accompanied by significant decreases in the amount of surface-bound TGF-alpha. Immunoanalysis indicated no changes in receptor number, indicating that decreased receptor content was not responsible for decreased TGF-alpha binding in the hepatocytes from the ethanol-fed rats. In conclusion, chronic ethanol feeding reduced TGF-alpha binding to hepatocytes with a concomitant decrease in the ability of the receptor tyrosine kinase to autophosphorylate its tyrosine residues. These changes were not accompanied by decreased receptor protein content. These defects could lead to altered signal transduction and to impaired reparative and regenerative processes in the liver.

Cerebral oedema and increased intracranial pressure in chronic liver disease.

BACKGROUND: Cerebral oedema is a cause of morbidity and mortality in fulminant hepatic failure but has not been well documented as a complication of chronic liver diseases. We report here the development of cerebral oedema and increased intracranial pressure in 12 patients with chronic liver disease. METHODS: Between July 1, 1987, and Dec 31, 1993, we studied 12 patients aged 29-67 years with end-stage chronic liver disease. All the patients had cirrhosis, portal hypertension, hypoprothrombinaemia, hepatic encephalopathy, and decreased serum concentrations of albumin (<25 g/L). During the study, the patients developed signs of increased intracranial pressure and had documented intracranial hypertension, cerebral oedema, or both. Intracranial hypertension was suspected on physical examination and confirmed by epidural catheters. We detected cerebral oedema by computed axial tomography of the head and necropsy of the brain when possible. FINDINGS: All the patients had intracranial hypertension and cerebral oedema. Two patients had successful treatment of cerebral hypertension with improvement of intracranial pressure such that orthotopic liver transplantation was undertaken. Both patients became neurologically normal after transplantation. Eight patients had only a transient response to treatment and died of cerebral oedema before a transplant could be done. INTERPRETATION: Cerebral oedema and increased intracranial pressure can occur in chronic liver disease and presents as neurological deterioration. Treatment guided by monitoring of intracranial pressure can lead to the reversal of intracranial hypertension, but in most patients cerebral oedema contributes to death or places them at too high a risk for liver transplantation.