Translocation Of Toxins Research Articles

Prä- und Probiotika

Nowadays, the regular consumption of pre- and probiotics is recommended to provide various positive health benefits. The in vitro and in vivo demonstrated actions on the intestinal microflora, the mucosal barrier and the immunological system are very interesting to propose beneficial health effects, but the scientific proof in humans is not demonstrated yet. Pre- and probiotics are very active in the intestinal tract (mainly in the colon) by maintaining a healthy gut microflora and influencing metabolic, trophic and protective mechanism. Prebiotics stimulates the growth of apathogen bacteria and increase the short chain fatty acid concentration by fermentation. Short chain fatty acids are necessary substrates for a healthy gut. Probiotics inhibit the growth of pathogen bacteria, reduce the translocation of bacteria and toxins and modulate the intestinal immune system. For some specific clinical diseases (ulcerative colitis, pouchitis, diarrhoea) a therapeutic and prophylactic effect with pre- and probiotics was shown. In the near future more indications for pre- and probiotics (used as a single strain or as in a combination) will be added. Promising results are already shown in irritable bowel syndrome, prevention of antibiotic induced diarrhoea, in surgical and in intensive care patients. Future studies should focus to determine the characteristics of a healthy gut and the evaluation of specific health benefits by well-designed, controlled human studies of adequate duration.

Differential requirement for the translocation of clostridial binary toxins: Iota toxin requires a membrane potential gradient

Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2-II, respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I, respectively) with ADP-ribosyltransferase activity that depolymerizes the actin cytoskeleton. After internalization, it was found that C2 and Iota toxins were not routed to the Golgi apparatus and exhibited differential sensitivity to inhibitors of endosome acidification. While the C2-I component of C2 toxin was translocated into the cytosol from early endosomes, translocation of the Ia component of Iota toxin occurred between early and late endosomes, was dependent on more acidic conditions, and uniquely required a membrane potential gradient.

Barrera intestinal del paciente crítico: realidades y tendencias

The disturbances of gut barrier in critically ill patients may influence their outcome and prognosis. Experiments in animals show that fasting and stress collaborate to produce intestinal atrophy and translocation of microorganisms and toxins. This fact is one of the main arguments to promote the use of early enteral feeding in critically ill patients. However, the intestinal barrier behaves differently in humans than in animals. The human enteral cells have a good tolerance to fasting and stress, mucosal atrophy is mild and it is not always associated with changes in intestinal permeability. Moreover, the relationship between intestinal permeability with sepsis and bacterial translocation is controversial. This last phenomenon also happens in normal subjects and may be a mechanism to build immunological memory. One of the most important factors that influence bacterial translocation is the microorganism, that under stress conditions can adhere to the intestinal cell and penetrate the intestinal barrier. Splanchnic ischemia and reperfusion is one of the main pathogenic factors in the failure of intestinal barrier. Finally, the fact that the small bowel is an inflammatory target of extra intestinal injuries, explains several clinical situations. The pathophysiology of the intestinal barrier definitely requires more research.

THALIDOMIDE TREATMENT REDUCES THE ALTERATION OF PARACELLULAR BARRIER FUNCTION IN MICE ILEUM DURING EXPERIMENTAL COLITIS

Small intestine permeability is frequently altered in inflammatory bowel diseases and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJs) and adherence. Thus, the aim of the present study was to examine the effects of thalidomide treatment on the permeability and structure of small intestine TJs in an animal model of experimental colitis induced by dinitrobenzene sulfonic acid (DNBS). Four days after colitis induction with DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin and zonula occludens 1. When compared with DNBS-treated mice, thalidomide-treated (200 mg/kg orally starting 30 min after the administration of DNBS) mice subjected to DNBS-induced colitis experienced a significantly reduced rate of the extent and severity of the histological signs of colon injury associated with a significant reduction of plasma and colon tumor necrosis factor alpha levels. After administration of DNBS to the mice induced a significant increase of ileal permeability was observed. Distal colitis in mice induced an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. In particular, we have observed that thalidomide treatment resulted in a significant reduction of the following: (1) the degree of colon injury, (2) the alteration of zonula occludens 1 and occludin localization (immunohistochemistry), and (3) intestinal permeability caused by DNBS in the colon. Taken together, our results clearly show that thalidomide treatment reduced small intestinal permeability in experimental colitis through the regulation of TJ protein.

5-LIPOXYGENASE MODULATES THE ALTERATION OF PARACELLULAR BARRIER FUNCTION IN MICE ILEUM DURING EXPERIMENTAL COLITIS

Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJs) and adherence. Recently, it has been shown that 5-lipoxygenase (5-LO) plays an important role in the development of various inflammatory conditions like inflammatory bowel disease. In the present study, by comparing the responses in wild-type mice (5-LOWT) with those of mice lacking the 5-lipoxygenase (5-LOKO), we investigated the role played by this enzyme in the permeability and structure of small intestine TJs in an animal model of experimental colitis. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). Four days after colitis induction by DNBS, the ileal TJs were studied by means of transmission electron microscopy using lanthanum nitrate and immunohistochemistry of occludin and ZO-1. When compared with DNBS-treated 5-LOWT mice, DNBS-treated 5-LOKO mice experienced a reduced rate of the extent and severity of the histological signs of colon injury. After administration of DNBS, 5-LOWT mice showed a significant increase of ileal permeability (88.3% +/- 1.2%) compared with sham (5.6% +/- 0.5%). In colitis, the percentage of "leaky" junctions in terminal ilea correlated positively with the macroscopic colon damage score. Distal colitis in 5-LOWT mice induces an increase of TJ permeability throughout the entire small intestine, and the extent of alterations correlates with colonic damage. On the contrary, a significant reduction of (1) the degree of colon injury, (2) the alteration of ZO-1 and occludin localization (immunohistochemistry), and (3) ileal permeability (8.1% +/- 0.7%) caused by DNBS in the colon was observed in 5-LOKO mice. Similarly, the treatment of 5-LOWT with zileuton (50 mg/kg per oral gavage twice a day), a 5-LO inhibitor, resulted in a significant reduction of all the previously described parameters. Taken together, our results clearly demonstrate that 5-LO modulates small intestinal permeability in experimental colitis through the regulation of TJ protein.

Tissue Bioenergetics and Microvascular Perfusion Are Impaired in Rat Ileal Mucosa in Normotensive Sepsis

Sepsis is a systemic inflammatory response to a bacterial infection. Inflammation may result in injury to the small bowel and an increase in translocation of bacteria and toxins across the mucosal barrier, which may contribute to the progression of sepsis. Microcirculatory perfusion or cytopathic hypoxia may cause impairment of tissue bioenergetics and injury in sepsis. The objective of this study was to determine if sepsis is associated with microcirculatory hypoperfusion and impaired tissue bioenergetics in the ileal mucosa. Sprague-Dawley rats were randomized to cecal ligation and perforation (sepsis group, n = 12) or control group (n = 14) and received arterial and venous catheters and fluid resuscitation. Following 24 h, rats were anesthetized with isoflurane and the ileum was prepared for intravital microscopy. Images of NADH fluorescence, which is an index of tissue bioenergetics, central arterial diameter, red cell velocity, red cell flux, and average intercapillary area, were recorded in 6-9 villi in each rat. Central arterial red cell flux (control 277 +/- 30 cell/s, sepsis 108 +/- 13 cells/s, p < .05), diameter (control 10.4 +/- 0.4 microm, sepsis 8.2 +/- 0.3 microm, p < .05) and red cell velocity (control 590 +/- 47 microm/s, sepsis 449 +/- 63 microm/s, p < .05) were decreased while average intercapillary area (control 815 +/- 171 microm(2), sepsis 1412 +/- 364 microm(2), p < .05) and NADH fluorescence (control 116 +/- 6 AIU, sepsis 154 +/- 9 AIU, p < .05) were increased at the villus tip in the sepsis group. Sepsis is associated with bioenergetic impairment and capillary hypoperfusion at the villus tip and a decrease in red cell flux in the central arteriole.

Differential effect of imipenem treatment on wild-type and NK cell-deficient CD8 knockout mice during acute intra-abdominal injury

CD8 knockout mice depleted of natural killer (NK) cells by treatment with anti-asialoGM1 (CD8KO/alphaAsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential sources of injury include bacterial dissemination, cecal ischemia, and translocation of bacterial toxins. We treated wild-type and CD8KO/alphaAsGM1 mice with imipenem after CLP to decrease bacterial dissemination. Additional mice were subjected to cecal ligation without puncture of the cecal wall or cecal ligation and removal of cecal contents. Imipenem treatment decreased bacterial counts by at least two orders of magnitude. However, all wild-type mice, whether treated with saline or imipenem, died by 42 h after CLP and exhibited significant hypothermia, metabolic acidosis, and high plasma cytokine concentrations. Wild-type mice subjected to cecal ligation without puncture also died, despite very low bacterial counts in blood, but wild-type mice subjected to cecal ligation and washout of cecal contents survived. In CD8KO/alphaAsGM1 mice subjected to CLP, imipenem treatment increased survival from 50% to 100%. After cecal ligation without puncture, long-term survival was 80-90% in CD8KO/alphaAsGM1 mice. Hypothermia, metabolic acidosis, and cytokine production were attenuated in CD8KO/alphaAsGM1 mice compared with wild-type controls. These results indicate that bacterial dissemination is not a major source of injury in wild-type mice after CLP, but the presence of gut flora in the cecal lumen is required for induction of systemic inflammation after cecal injury. CD8KO/alphaAsGM1 mice are resistant to the systemic manifestations of cecal injury.

Activation of the Innate Immune System and Alcoholic Liver Disease: Effects of Ethanol per se or Enhanced Intestinal Translocation of Bacterial Toxins Induced by Ethanol?

It is generally accepted that activation of the innate immune system and increased release of pro-inflammatory cytokines and other mediators plays an important role in the development of alcoholic liver disease (ALD). The mechanisms involved in the ethanol-induced activation of monocytes/macrophages (including Kupffer cells) are however, still a matter of debate. The brief review will summarize the published data from the literature on the two main pathomechanisms discussed until now: I) Gut-derived bacterial toxins, specially endotoxin; and II) metabolic changes induced by alcohol oxidation (independent of mechanism I). For pathomechanism I, clear evidence has been published from numerous groups: Alcohol induces mucosal injury in the upper gastrointestinal tract and leads to marked increase in the permeability of the gut mucosa to macromolecules such as endotoxin. The resulting endotoxemia then leads to activation of Kupffer cells and other macrophages. The increased release of pro-inflammatory mediators (e.g., TNF-alpha, Il-1, reacting oxygen species) and infiltration of other inflammatory cells (e.g., neutrophils) finally causes liver damage. Regarding the second pathomechanism it has repeatedly been argued that the metabolic alterations which are induced by chronic administration of ethanol to rats or mice might increase the sensitivity of monocytes/macrophages to secrete TNF-alpha and other pro-inflammatory mediators thereby increasing the susceptibility to ethanol-induced liver injury. However, in all feeding experiments the effect of ethanol on intestinal permeability and enhanced translocation of bacterial toxins (endotoxin) is likely to occur (or at least cannot be excluded). The latter holds true also for experiments using isolated macrophages/Kupffer cells from ethanol fed animals. Therefore, to clarify whether or not alterations related to ethanol metabolism ("direct" effects of ethanol) contribute to the activation of the innate immune system studies using germ-free animals are needed to exclude the "indirect" effect of ethanol via gut-derived bacterial toxins.

Differential effect of imipenem treatment on injury caused by cecal ligation and puncture in wild-type and NK cell-deficient β2-microgloblin knockout mice

Our previous studies showed that beta(2)-microglobulin knockout mice treated with anti-asialoGM1 (beta2MKO/alphaAsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential mechanisms of injury include systemic infection, cecal ischemia, and translocation of bacterial toxins such as endotoxin and superantigens. Currently, it is unclear which of these mechanisms of injury contributes to mortality in wild-type mice and whether beta2MKO/alphaAsGM1 mice are resistant to any particular mechanisms of injury. In the present study, we hypothesized that systemic infection is the major cause of injury after CLP in wild-type mice and that beta2MKO/alphaAsGM1 mice are resistant to infection-induced injury. To test this hypothesis, wild-type and beta2MKO/alphaAsGM1 mice were treated with the broad-spectrum antibiotic imipenem immediately after CLP to decrease the impact of systemic infection in our model. Treatment of wild-type and beta2MKO/alphaAsGM1 mice with imipenem decreased bacterial counts by at least two orders of magnitude. However, all wild-type mice, whether treated with saline or imipenem, died by 42 h after CLP and had significant hypothermia, metabolic acidosis, and high plasma concentrations of the cytokines interleukin-6, macrophage inflammatory protein-2, and keratinocyte-derived chemokine. beta2MKO/alphaAsGM1 mice showed 40% long-term survival, which was increased to 90% by imipenem treatment. beta2MKO/alphaAsGM1 mice had less hypothermia, decreased metabolic acidosis, and lower cytokine concentrations at 18 h after CLP compared with wild-type mice. These results suggest that infection is not the major cause of mortality for wild-type mice in our model of CLP. Other mechanisms of injury such as cecal ischemia or translocation of microbial toxins may be more important. beta2MKO/alphaAsGM1 mice appear resistant to these early, non-infection-related causes of CLP-induced injury but showed delayed mortality associated with bacterial dissemination, which was ablated by treatment with imipenem.

Alcoholic Liver Disease: From Pathophysiology to Therapy

This article presents the proceedings of a symposium held at the meeting of the International Society for Biomedical Research on Alcoholism in Mannheim, Germany, in October 2004. A salient feature of this symposium was to demonstrate how the striking advances made in our understanding of the pathophysiology of alcoholic liver disease are now raising prospects for better treatment as well. Genetic factors are now being elucidated, and F. Stickel (Germany) has summarized his own studies as well as those of others. M. Salaspuro (Finland) updated the possible role of gut bacteria in the pathogenesis of alcoholic liver disease, bringing us closer to antibacterial therapy as part of the treatment of alcoholic liver disease. Specifically, the gut bacterial flora may be important via the in situ production of acetaldehyde and the associated intestinal injury, which may favor the translocation of toxins from the gut lumen to the systemic circulation and the liver. The analytical progress in the assessment of alterations of phospholipid composition in liver membranes described by H. P. Schlemmer (Germany) may eventually give us an objective method to recognize patients in whom phospholipid therapy might be indicated. Other novel treatment modalities for severe alcoholic hepatitis were described by Y. Horie and H. Ishii (Japan), including plasma exchange. Finally, the pros and cons of nutraceutical therapy were analyzed by C. S. Lieber, with a demonstration that although some nutraceuticals may have toxicity exacerbated by alcohol and must be administered very carefully within a narrow therapeutic window, others are actually beneficial as demonstrated under controlled conditions.

Intestinal permeability and systemic infections in critically ill patients: Effect of glutamine*

This article provides a critical review of the evidence indicating that an increase in intestinal permeability is associated with the installation of bacteremia, sepsis, and the multiple organ failure syndrome and that glutamine in pharmacologic doses reduces the acute increase of intestinal permeability and the infection frequency in critically ill patients. All studies published until December 2004 about intestinal permeability, bacterial translocation, and glutamine were located by search of PubMed and Web of Science. The reference lists of review articles and primary publications were also examined to identify references not detected in the computer search. Clinical and experimental studies investigating the correlation between intestinal permeability, bacterial translocation, and frequency of infections, associated or not with the effect of glutamine administration. Information regarding patient population, experimental design, glutamine doses and routes of administration, nutritional therapy prescribed, methods used to assess intestinal permeability, metabolic variables, and the frequency of infections were obtained from the primary literature. Intestinal permeability is increased in critically ill patients. The results have not always been consistent, but the studies whose results support the association between intestinal permeability and systemic infections have had better design and more appropriate controls. The administration of glutamine by the intravenous or oral route and at the doses recommended before or immediately after surgery, burns, or the administration of parenteral nutrition has a protective effect that prevents or reduces the intensity of the increase in intestinal permeability. Glutamine reduces the frequency of systemic infections and may also reduce the translocation of intestinal bacteria and toxins, but this has not been demonstrated. Glutamine administration improves the prognosis of critically ill patients presumably by maintaining the physiologic intestinal barrier and by reducing the frequency of infections.

Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.

The Binary Clostridium botulinum C2 Toxin as a Protein Delivery System: IDENTIFICATION OF THE MINIMAL PROTEIN REGION NECESSARY FOR INTERACTION OF TOXIN COMPONENTS

The binary Clostridium botulinum C2 toxin is composed of the enzyme component C2I and the binding component C2II, which are individual and non-linked proteins. Activated C2IIa mediates cell binding and translocation of C2I into the cytoplasm. C2I ADP-ribosylates G-actin at Arg-177 to depolymerize actin filaments. A fusion toxin containing the N-terminal domain of C2I (residues 1-225) transports C3 ADP-ribosyltransferase from Clostridium limosum into cells (Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K. (1998) Infect. Immun. 66, 1364-1369). We characterized the adaptor function of C2I and its interaction with C2IIa. The fusion toxin GST-C2I(1-225)-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I(1-225) adaptor was positioned in the middle of a fusion protein. Amino acid residues 1-87 of C2I were sufficient for interaction with C2IIa and for translocation of C2I fusion toxins into HeLa cells. Residues 1-87 were the minimal part of C2I to bind to C2IIa on the cell surface, as detected by fluorescence-activated cytometry. An excess of C2I(1-87) (but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa. Also, the fragment C2I(30-431) and the fusion toxin C2I(30-225)-C3 competed with C2I-Alexa488 for binding to C2IIa. C2I(30-225)-C3 did not induce cytotoxic effects on cells when applied together with C2IIa, indicating that amino acid residues 1-29 are involved in translocation of C2I but are not absolutely essential for binding to C2IIa.

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8 + T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.

Synthesis and characterization of membrane-active GALA-OKT9 conjugates.

Cellular processing of immunotoxins is inefficient, limiting the overall effectiveness of current immunotoxin therapies. Specifically, translocation of ribosome-inactivating toxins across intracellular membranes is agonizingly slow. In one strategy to improve immunotoxin efficacy, membrane-active peptides are attached to immunotoxins to facilitate transfer of the toxic moiety across a cellular membrane to the cytosol. pH-sensitive peptides are of particular interest, as the membrane activity can be localized to the endosomal/lysosomal pathway, reducing nonspecific interactions at the cell surface. In this study, GALA, a pH-sensitive peptide that forms multimeric pores in membranes, was chemically attached to OKT9, an anti-transferrin receptor mAb. Conjugates were tested by measuring release of encapsulated dyes from liposomes to determine the extent to which the membrane-lytic properties of GALA were retained. The most significant feature affecting the lytic properties of GALA-OKT9 conjugates was the number of attached GALA per OKT9. Conjugates with a single GALA per OKT9 caused almost no leakage while conjugates with two or three GALA per OKT9 caused significant leakage in a concentration-dependent manner. Invariably, GALA-OKT9 conjugates were significantly less active than unconjugated GALA, attributable to a decrease both in partitioning and in surface aggregation. No improvement in membrane-lytic activity was achieved by using a longer, more flexible poly(ethylene glycol) cross-linker. Attachment of GALA via C- versus N-terminal linkage had no effect on membrane-lytic properties. Size-selective release of high molecular weight dextrans was almost identical for conjugated and unconjugated GALA, suggesting that GALA forms the same pore structure regardless of conjugation state.

Role of Free Radicals and Poly(ADP-ribose) Synthetase in Intestinal Tight Junction Permeability

Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with permeable TJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or strand depth in the ilea from control or treated animals. In addition, a significant disrupted immunofluorescence signal for occludin, ZO-1 and beta-catenin was observed in the terminal ilea of zymosan-treated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular distribution patterns of occludin, ZO-1 and beta-catenin. Similarly NAC or 3-AB treatments were able to prevent zymosan-induced damage of junctional proteins in iNOSWT mice. In conclusion, this study demonstrates that the alteration of permselectivity is most likely induced by ROS and PARS activation.

Fish oil-supplemented parenteral diets normalize splanchnic blood flow and improve killing of translocated bacteria in a low-dose endotoxin rat model.

Intestinal ischemia decreases barrier function of the gut and enhances translocation of bacteria and toxins. Several studies indicate that fish oil can modulate prostaglandin formation and thus, regional blood flow and immune function. This study was performed to determine the effects of parenteral diets with omega-3 fatty acids on microcirculation and barrier function of the gut.0 Prospective, randomized, controlled animal study. University laboratory. A total of 64 male Sprague-Dawley CD rats. For 48 hrs, eight groups of eight rats each received total parenteral nutrition with four different types of lipids. The source of fat in group L was soybean oil only and in group L-M a mixture of soybean oil and medium-chain triglycerides. In groups FO-20 and FO-40, 20% or 40%, respectively, of the soybean oil in group L-M was replaced by fish oil. The other four groups received an additional continuous infusion of endotoxin (0.1 mg/100 g body weight per day) for the last 24 hrs. Blood flow was measured with microspheres, and translocation was determined by microbiological methods and instillation of radioactive-marked bacteria into the gut. In the animals without fish oil, the endotoxin application reduced the blood flow to the intestine approximately 25%. Animals with fish oil in their diets showed normal values. Translocation of gut bacteria was increased significantly in all endotoxin groups. However, less-viable bacteria could be detected in the animals with fish oil diets in their mesenteric lymph nodes and livers. In this model, diets enriched with fish oil abolish the endotoxin-induced decrease of nutritive blood flow to the gut and ameliorate the bactericidal defense of the splanchnic region. The lower count of viable bacteria in the fish oil groups is more related to an improved killing of translocated bacteria than a reduction of the translocation rate.

Increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease

Background/Aims: No information is yet available about the influence of alcohol abuse on the translocation of larger molecules (M r>1200) through the intestinal mucosa in man. The present study aimed to determine the intestinal permeability to macromolecules in patients with chronic alcohol abuse and mild to more advanced stages of liver disease, and to measure the concentration of endotoxins in the plasma, as these compounds derive from the intestinal flora and are suspected to contribute to the development of alcoholic liver disease (ALD). Methods: The permeability to polyethylene glycol M r 400, M r 1500, M r 4000, and M r 10 000 and endotoxin plasma concentrations were measured in 54 patients with alcoholic liver disease, 19 of them with cirrhosis, and in 30 non-alcoholic healthy controls. Results: Permeability to polyethylene glycol M r 400 was found to be unchanged in patients with ALD in comparison to healthy controls, whereas polyethylene glycol M r 1500 and M r 4000 were recovered in about twice as high concentrations in the urine of ALD patients ( p<0.01). Polyethylene glycol M r 10 000 was detected significantly less frequently in urine from healthy controls (0/30) than in urine of patients with alcoholic liver disease (20/54, p<0.01). Endotoxin concentrations in the plasma of alcoholics were increased more than 5-fold compared to healthy controls ( p<0.01). Conclusions: The results of this study indicate that alcohol abuse impairs the function of the intestinal barrier, which might enhance the translocation of bacterial toxins, thereby contributing to inflammatory processes in alcoholic liver disease.

The effects of antibiotic use on gastrointestinal function

The bacterial flora of the gastrointestinal (GI) tract plays an important role in maintaining the integrity of the enterocyte, modulating metabolic and immunologic processes, and protecting against colonization by invasive pathogens. Disruption of this finely tuned and stable gut flora by antibiotics, infection, chemotherapy, or radiation has profound effects on the protective barrier and results in overgrowth by pathogens, invasion and translocation of toxins, and life-threatening infections. Use of antibiotics promotes the emergence of resistant organisms, and multiple-antibiotic resistance has become a major public health issue. Preservation of protective species or recolonization with nonpathogenic yeasts or lactobacilli during periods of stress (infections, drugs) has begun to show promise in the management of patients receiving multiple antibiotics, particularly in hospital-acquired infections.