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PubMed2006

Intraperitoneal administration of hyperbarically oxygenated perfluorochemical enhances preservation of intestinal mucosa against ischemia/reperfusion injury.

Perfluorochemicals (PFCs) have a high solubility for oxygen. We have previously demonstrated the effect of peritoneal lavage with oxygenated PFC (O2-PFC) on ameliorating ischemia/reperfusion (I/R)-induced intestinal ischemic damage in an animal model. In this study, we applied hyperbarically O2-PFC (HBO-PFC) to investigate whether a larger amount of oxygen carried by PFC could enhance the protective effect of O2-PFC during intestinal malperfusion. Rats were subjected to ischemia by clamping the superior mesenteric artery (SMA) for 90 min. The SMA was then declamped. Rats were divided into four groups. In group A, only anesthesia and abdominal incision were performed. In group B, SMA was clamped without O2-PFC. In group C, during the SMA clamp, 1 atm O2-PFC was injected into the abdominal cavity. In group D, 5 atm O2-PFC (HBO-PFC) was prepared using a custom-made hyperbaric oxygen tank and administered to the abdominal cavity during the SMA clamp. Ileal tissue adenosine triphosphate (ATP) levels 90 min after SMA declamping were determined using luciferase assay. To assess intestinal mucosal barrier function at 90 min after release of the SMA clip, everted gut sacs were prepared to measure the mucosal-to-serosal passage of fluorescein-conjugated dextran (FD4, molecular weight = 4 kDa). Thirty minutes after i.p. administration, partial pressure of oxygen in HBO-PFC remained around 1000 mmHg, whereas partial pressure of oxygen in 1 atm O2-PFC decreased to around 400 mmHg. The intestinal tissue ATP was significantly preserved in group D. Moreover, the mucosal hyperpermeability of the gut sac after I/R was significantly ameliorated in group D. Hyperbarically oxygenated perfluorochemical might supply a larger amount of oxygen to ischemic tissue during SMA clamp, which protected the small intestine from I/R injury, possibly caused by the maintenance of tissue ATP levels during ischemia.

Adenosine Triphosphate↗
PubMed1998

IL-6 rescues enterocytes from hemorrhage induced apoptosis in vivo and in vitro by a bcl-2 mediated mechanism.

Following a hemorrhagic event, damage to the highly metabolic intestinal tissue induces loss of barrier function leading to bacterial escape and LPS contamination of the host. Orally administered IL-6 restores intestinal barrier function following hemorrhage in both rat and mouse models. IL-6 prevents apoptosis in a variety of lymphoid cells and lines, through the activation of the proto-oncogene bcl-2. This communication elucidates the role of the IL-6-bcl-2 interaction in intestinal apoptosis following hemorrhagic shock. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) and p53 immunohistochemical staining were used to examine intestines from mice hemorrhaged and fed saline or IL-6 and enterocytes (IEC-6) exposed to hypoxia and LPS alone or LPS and IL-6 in vitro. In situ hybridization for bcl-2 expression was performed on intestines or enterocytes. Intestinal sections from mice hemorrhaged and fed IL-6 showed reduction in apoptosis and increases in bcl-2 gene expression relative to sections taken from mice hemorrhaged and fed saline. IEC-6 cells exposed to hypoxia and LPS had high numbers of TUNEL staining cells. Subsequent exposure to IL-6 after hypoxia and LPS reduced apoptotic cell numbers and increased bcl-2 gene expression. The data show that exposure of intestinal epithelial cells to IL-6 either by oral administration in hemorrhaged mice or by coculture following hypoxia and LPS treatment results in increased bcl-2 gene expression and reduced damage from apoptosis.

Animals↗
PubMed1998

Specific inhibition of iNOS decreases the intestinal mucosal peroxynitrite level and improves the barrier function after thermal injury.

Failure of GI tract mucosa to act as a barrier against bacterial translocation (BT) has been proposed as a potential source of sepsis and subsequent multiple organ failure post thermal injury. Nitric oxide (NO) is an inorganic radical produced by NO synthase (NOS) from L-arginine. Gut mucosal constitutive NOS (cNOS) provides protection for itself. In contrast to cNOS, inducible NOS (iNOS) releases far greater amounts of NO, promotes oxidative reactions and is responsible for tissue injury. Peroxynitrite formed by the rapid reaction between superoxide and NO, is a toxic substance that contributes to tissue injury in a number of biological systems. This study was designed to investigate the effect of iNOS specific inhibitor S-methylisothiourea (SMT) on the postburn intestinal mucosal barrier function and the possible mechanism of SMT's action. Female SPF Sprague Dawley rats underwent 35% total body surface area (TBSA) or sham burn. Either SMT or the same volume of saline was given (5 mg/kg, i.p. q 12 h) for 2 days to assess the effect of iNOS inhibition. On postburn day 2, the intestinal mucosal cNOS and iNOS activity were assayed by using Griess' reagent, the mesenteric lymph node (MLN), spleen and liver were collected and cultured for BT assay and the cellular localization of nitrotyrosine, a marker for peroxynitrite activity, was examined by immunostaining. After thermal injury in rats, administration of SMT for 2 days decreased the intestinal mucosal iNOS activity/ tNOS activity ratio and the BT incidence. Nitrotyrosine immunostaining of the intestinal mucosa showed a decrease in the SMT-treated group. These findings suggest that SMT, a specific inhibitor for iNOS improves the barrier function after burn by suppression of the intestinal mucosal iNOS activity. The decrease in NO production resulted in decreased formation of peroxynitrite and subsequently decreased damage of mucosal tissue.

Animals↗
PubMed1993

Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion.

Neutrophil, or polymorphonuclear leukocyte (PMN), migration across intestinal epithelial barriers, such as occurs in many disease states, appears to result in modifications of epithelial barrier and ion transport functions (Nash, S., J. Stafford, and J. L. Madara. 1987. J. Clin. Invest. 80:1104-1113; Madara, J. L., C. A. Parkos, S. P. Colgan, R. J. MacLeod, S. Nash, J. B. Matthews, C. Delp, and W. I. Lencer. 1992. J. Clin. Invest. 89:1938-1944). Here we investigate the effects of epithelial exposure to IFN-gamma on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was initially assessed in the apical-to-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to-basolateral and in the basolateral-to-apical directions. In the apical-to-basolateral direction, epithelial exposure to IFN-gamma markedly upregulated transepithelial migration of PMN in a dose- and time-dependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-gamma-elicited effect on transmigration was specifically due to a IFN-gamma effect on epithelial cells and was not secondary to IFN-gamma effects on epithelial tight junction permeability. Moreover, this IFN-gamma effect was dependent on epithelial protein synthesis, and involved a pathway in which CD11b/18, but not ICAM-1 or CD11a/18, appeared to play a crucial role in PMN-epithelial adhesion. IFN-gamma also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-gamma effect on naturally directed transmigration was also specifically due to an IFN-gamma effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CD11b/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-gamma affected transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-gamma markedly downregulated transepithelial migration of PMN in the natural direction. This downregulation of PMN migration in the natural direction, however, was not due to failure of PMN to move across filters and into monolayers. Indeed, IFN-gamma exposure to epithelia increased the number of PMN which had moved into the basolateral space of the epithelium in naturally directed transmigration. These results represent the first detailed report of influences on PMN transepithelial migration by a cytokine, define conditions under which a qualitative difference in PMN transepithelial migration exists, and suggest that migration of PMN across epithelia in the natural direction may involve multiple steps which can be differentially regulated by cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1998

Relationship between enteric microecologic dysbiosis and bacterial translocation in acute necrotizing pancreatitis.

AIM:To investigate the potential role of intestinal microflora barrier in the pathogenesis of pancreatic infection.METHODS:Fifteen dogs were colonized with a strain of E.coli JM109 bearing ampicillin resistance plasmid PUC18.The animals were divided into two groups. In experimental group (n = 8), acute necrotizing pancreatitis (ANP) was induced by injection of 0.5ml/kg of sodium tarocholate with 3000U/kg trypsin into the pancreatic duct. The control group (n = 7) underwent laparotomy only. All animals were sacrificed 7 days later. Mucosal and luminal microflora of intestine were analyzed quantitatively, and various organs were harvested for culturing, blood samples were obtained for determination of serum amylase activities and plasma lipopolysaccharide (LPS) concentrations.RESULTS:In the experimental group, the number of E. coli in the intestine was much higher than those of the controls, while bifidobacterium and lactobacillus were decreased significantly (Jejunum, 1.75 ± 0.95 vs 2.35 ± 0.79,P <0.05; 1.13 ± 0.8 vs 1.83 ± 0.64,P <0.05; ileum, 2.89 ± 0.86 vs 3.87 ± 1.05,P <0.05; 1.78 ± 0.79 vs 3.79 ± 1.11,P <0.01;cecum, 2.70 ± 0.88 vs 4.89 ± 0.87,P <001; 2.81 ± 0.73 vs 3.24 ± 0.84,P <0.05. Content of Cecum, 3.06 ± 0.87 vs 5.15 ± 1.44,P <0.01; 2.67 ± 0.61 vs 4.25 ± 0.81,P <0.01), resulting in reversal of bifido-bacterium/E. coli ratio as compared with the control group (jejunum,0.51 ± 0.76 vs 1.23 ± 0.53, P <0.05; ileum, 0.62 ± 0.68 vs 1.16 ± 0.32,P <0.05; cecum,0.46 ± 0.44 vs 1.03 ± 0.64, P < 0.05). In addition, intestinal bacteria were isolated from organs of all animals in the experimental group, and JM109 was also detected in most cases. Positive blood culture was 75.0% and 62.5% on day 1 and 2 after induction of ANP, respectively, but no bacterium was found in the controls. As compared with the control group, blood LPS levels and serum amylase activities increased 1-3 times and 3-8 times respectively.CONCLUSION: Microecological disturbance could occur in ANP, and overgrowth of intestinal gram-negative bacteria may lead to translocation to the pancreas and other organs, becoming the source of pancreatic and peripancreatic infection.

Journal Article↗
PubMed1996

Effects of native and hydrolyzed whey protein on intestinal repair of severely starved rats at weaning.

The aim of this study was to evaluate the effect of two sources of dietary nitrogen (isolated whey protein and hydrolyzed whey protein) on the intestinal repair of malnourished rats at weaning. The malnutrition was achieved by a 3 days' starvation period. Normally fed male Wistar rats were used as controls. Intestinal repair was studied after a refeeding period of 4 days. The parameters studied included nitrogen balance, lactase, sucrase, isomaltase, and maltase activities of the jejunum; liver acetylcholinesterase and glutamate dehydrogenase activities; and the serum amino acid profile. In addition, tests of intestinal permeability to macromolecules were performed by measurement of ovalbumin and beta-lactoglobulin in serum. Both diets of led to the recovery of the severely starved rats, in terms of the values of all the parameters evaluated. The serum beta-lactoglobulin was the only exception, because its concentration was significantly lower in the normally fed animals. This study suggests that the intestinal mucosal barrier is not completely repaired, even after a 4-day refeeding period, to the point of being suitable to accept an increase in the uptake of antigens.

Acetylcholinesterase↗
PubMed2000

Interaction of Listeria monocytogenes with the intestinal epithelium.

Listeria monocytogenes is a food-borne pathogen that must cross the intestinal epithelial barrier to reach its target organs. We have investigated the importance of M cells in translocation using an experimental mouse model and a novel, recently described in vitro coculture system that mimics the follicle-associated epithelium (FAE). Our data demonstrate that L. monocytogenes does not require, nor specifically use, M cells of the FAE to cross the gut. We also show that bacterial translocation is rapid and L. monocytogenes can attach very efficiently to exposed basal lamina of the small intestine indicating an important role for extracellular matrix proteins.

Animals↗
PubMed1994

Bacterial translocation in the neonate.

Bacterial translocation has been a major topic of investigation for the past two decades. Despite recent evidence that bacterial translocation may play a significant role in the morbidity and mortality of adults faced with multiple types of stress, very little is known about the effect of bacterial translocation on the neonate. Recently more and more evidence has suggested that normal as well as premature or ill neonates experience spontaneous bacterial translocation quite commonly. This article reviews the recent literature on bacterial translocation in stressed adults, the development of an intact intestinal mucosal barrier in the newborn as a protection against bacterial translocation, and the role of spontaneous bacterial translocation in the development of systemic sepsis and its accompanying morbidity and mortality.

Adult↗
PubMed1984

Intestinal permeability in healthy and allergic children before and after sodium-cromoglycate treatment assessed with different-sized polyethyleneglycols (PEG 400 and PEG 1000).

Gastrointestinal permeability was investigated in twenty-two children on two occasions, before and after treatment with sodium cromoglycate. The children were between 8 and 10 years old; half of them were classified as allergic according to history and laboratory tests, and half of them as healthy. The 6-hr urinary recovery of different-sized polyethyleneglycols (PEG 400 and PEG 1000) in combination with a mathematical model was used to assess the intestinal permeability barrier. No significant differences were seen in the first PEG test between healthy and allergic children, although those with gastrointestinal allergy showed a slightly lower, and those with other allergies a slightly higher recovery of the smaller PEGs than seen in the healthy individuals. After treatment with sodium cromoglycate, however, there was a significant decrease in uptake by allergic children, which could indicate that the permeability properties had returned to normal. The PEG method offers a simple, harmless and reproducible method to measure intestinal permeability properties. The change in permeability observed after sodium cromoglycate corresponds well with the clinical experience of usefulness of the drug in some children with food allergy.

Cell Membrane Permeability↗
PubMed2003

NAD+ ameliorates inflammation-induced epithelial barrier dysfunction in cultured enterocytes and mouse ileal mucosa.

In the course of other experiments, we serendipitously observed that extracellular nicotinamide adenine dinucleotide (NAD+) ameliorated the development of epithelial hyperpermeability when monolayers of Caco-2 enterocyte-like cells were incubated with cytomix, a mixture containing interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha. We sought to characterize the effects of NAD+ on inflammation-induced epithelial barrier dysfunction using Caco-2 monolayers that were exposed to cytomix in the absence or presence of NAD+ or other purine-containing molecules. Paracellular barrier function measured as the apical-to-basolateral passage of fluorescein isothiocyanate-conjugated dextran (mol. wt. approximately 4000) was preserved in a concentration-dependent manner when immunostimulated Caco-2 cells were exposed to extracellular NAD+. Incubation with NAD+ prevented cytomix-induced derangements in the expression and localization of the tight junction proteins occludin and zonula occludens-1 in Caco-2 cells. Treatment of cytomix-stimulated cells with NAD+ also blocked nuclear factor-kappaB (NF-kappaB) activation, inducible nitric-oxide synthase induction, and increased production of nitric oxide (NO.). Ileal mucosal permeability to fluorescein isothiocyanate-dextran mol. wt. approximately 4000 was increased in mice 18 h after lipopolysaccharide (endotoxin) injection, but treatment of endotoxemic mice with NAD+ ameliorated the development of gut mucosal hyperpermeability. Thus, extracellular NAD+ seems to ameliorate inflammation-induced intestinal epithelial barrier dysfunction by inhibiting NF-kappaB activation and increased NO. production.

Animals↗
PubMed2003

Proinflammatory cytokines cause NO*-dependent and -independent changes in expression and localization of tight junction proteins in intestinal epithelial cells.

Intestinal epithelial barrier function is impaired after the exposure of enterocytes to proinflammatory cytokines. The mechanism(s) responsible for this phenomenon remain incompletely understood. We used cultured monolayers of Caco-2 enterocyte-like cells to characterize the effect of cytomix, a mixture of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta, on the expression and localization of several tight junctions proteins. Cells were stimulated with cytomix in the presence or absence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1 -oxyl-3-oxide (cPTIO), an NO* scavenger. Some cells were treated with (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate] (DETA-NONOate), an NO* donor. Tight junction protein expression was measured in cellular extracts by Western blotting and localized in cells using immunofluorescence. Steady-state mRNA levels were determined using semi-quantitative reverse-transcription polymerase chain reaction. Incubation of cells with DETA-NONOate or cytomix decreased epithelial barrier function, decreased expression of ZO-1 mRNA, decreased expression of ZO-1, ZO-3, and occludin protein, and increased expression of claudin-1 protein. The effects of cytomix on barrier function and tight junction protein expression were modulated by cPTIO. Cytomix caused incorrect subcellular localization of ZO-1, occludin, and claudin-1, and this was modulated by co-incubation with cPTIO. DETA-NONOate caused similar protein mislocalization as observed with cytomix. The effectiveness of cPTIO in maintaining tight junction protein expression and correct subcellular localization was less apparent at early time points (12 h) compared with later points, suggesting an NO*-independent effect of cytokines on barrier function. Thus, cytomix appears to increase the permeability of Caco-2 monolayers through NO*-dependent and -independent mechanisms that are associated with changes in the expression and/or targeting of proteins involved in tight junction function.

Base Sequence↗
PubMed2005

Innate immune relationship between commensal flora and the mammalian intestinal epithelium.

Commensal bacteria in the lumen of the intestine exist in a mutually advantageous relationship with the mammalian host, providing benefits such as increased metabolic/digestive capabilities and exclusion of harmful microbes, and in turn receiving a nutrient-rich environment. However, in the context of a dysfunctional intestinal epithelial barrier, commensal bacteria may elicit an immune inflammatory response similar to what occurs during infection by a pathogen. Recent work has established that most eukaryotic cells possess families of receptors that can detect the structural signatures of prokaryotic life. Cells may respond to the perception of microbes by activating distinct cytoplasmic signaling cascades that ultimately result in the transcriptional activation of genes needed for proinflammatory and anti-apoptotic functions, as well as for a pro-apoptotic response. Collectively, these responses generally suffice to eliminate microbial threats and may be integral to normal intestinal homeostasis. An understanding of these mechanisms, as well as those by which microbes themselves influence intestinal epithelial responses, may help provide a new perspective on the pathogenesis of intestinal diseases.

Animals↗
PubMed2005

AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins.

Antrum mucosal protein (AMP)-18 and a synthetic peptide of amino acids 77-97 have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco-2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking whether AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Observations using laser scanning confocal (CF) microscopy supported the capacity of AMP peptide to enhance accumulation of occludin and zonula occludens-1 in TJ domains of C2 cell monolayers and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin.

Actins↗
PubMedDate not supplied

The effects of sodium deoxycholate, lactulose and glutamine on bacterial translocation in common bile duct ligated rats.

BACKGROUND/AIMS: Sepsis is a major cause of post-operative morbidity and mortality in obstructive jaundice as a result of bacterial translocation from the gut. This study was conducted to investigate the effects of glutamine, lactulose, and the bile salt Na deoxycholate in preventing bacterial translocation in an animal model where obstructive jaundice was developed by common bile duct ligation. METHODOLOGY: Fifty Wistar albino rats were divided into 5 groups of 10 animals each. The animals in groups I-IV underwent common bile duct ligation and received, respectively, either saline, Na deoxycholate, lactulose or glutamine, orally. Group V had sham ligation and received saline orally. The animals were sacrificed at the end of the 7th day, and serum concentrations of bilirubin, aspartate aminotransferase (ALT), alanine aminotransferase (ALT), and alkaline phosphatase (AP) were measured. In addition, mesenteric lymph nodes were removed and cultured together with cecal content. Histopathologic examination of terminal ileum specimens was made. RESULTS: Na deoxycholate, lactulose and glutamine all reduced bacterial translocation rates to mesenteric lymph nodes (p<0.05), with glutamine causing the greatest effect. Na deoxycholate and lactulose prevented bacterial translocation by causing a decrease in cecal intraluminal bacterial content (p<0.001), while glutamine exerted its effect by preserving intestinal mucosal integrity. CONCLUSIONS: The integrity of the intestinal mucosal barrier is of paramount importance in preventing bacterial translocation, and the measures taken to protect mucosal integrity reduce bacterial translocation to a greater extent than those taken to decrease the number of bacteria in the gut.

Animals↗
PubMed1996

Radionuclide studies in intestinal transplantation. Diagnosis of rejection and assessment of permeability.

Three patients who received intestinal allografts were studied using two distinct radionuclide investigations. In the first, 111In or 99mTc-labeled leukocyte scanning was performed to assist in the diagnosis of rejection. It was able to demonstrate the occurrence of rejection in the transplanted intestine, and the response to antirejection therapy. In 1 case, the abnormality on the scan preceded the histological confirmation of rejection. The second technique studied mucosal integrity by serial 51Cr-EDTA/14C-mannitol permeability tests. These studies demonstrated the initial marked impairment and the slow return to normal function of the intestinal mucosal barrier. In 1 patient, this occurred by 91 days; in another, it took 232 days. A single assay performed in the third patient at the time of allograft rejection was also abnormal. Both radionuclide tests were helpful in the care of these complicated cases.

Adult↗
PubMed1999

Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers.

PURPOSE: Previous studies demonstrated that inhibition of P glycoprotein (P-gp) by Pluronic P85 (P85) block copolymer increases apical (AP) to basolateral (BL) transport of rhodamine 123 (R123) in the polarized monolayers of bovine brain microvessel endothelial cells (BBMEC) and Caco-2 cells. The present work examines the effects of P85 on the transport of fluorescein (Flu), doxorubicin (Dox), etoposide (Et), taxol (Tax), 3'-azido-3'-deoxythymidine (AZT), valproic acid (VPA) and loperamide (Lo) using BBMEC and Caco-2 monolayers as in vitro models of the blood brain barrier and intestinal epithelium respectively. METHODS: Drug permeability studies were performed on the confluent BBMEC and Caco-2 cell monolayers mounted in Side-Bi-Side diffusion cells. RESULTS: Exposure of the cells to P85 significantly enhanced AP to BL permeability coefficients of Flu, Tax, Dox and AZT in both cell models. Further, P85 enhanced AP to BL transport of Et, VPA and Lo in Caco-2 monolayers. No changes in the permeability coefficients of the paracellular marker mannitol were observed in the presence of the copolymer. CONCLUSIONS: P85 increases AP to BL permeability in BBMEC and Caco-2 monolayers with respect to a broad panel of structurally diverse compounds, that were previously shown to be affected by P-gp and/ or multidrug resistance associated protein (MRP) efflux systems. Broad specificity of the block copolymer effects with respect to drugs and efflux systems appears to be a valuable property in view of developing pharmaceutical formulations to increase drug accumulation in selected organs and overcome both acquired and intrinsic drug resistance that limits the effectiveness of many chemotherapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Transport of proteolytic enzymes across Caco-2 cell monolayers.

PURPOSE: To investigate the mechanisms by which proteolytic enzymes, such as trypsin, chymotrypsin, papain, and bromelain, are able to cross the intestinal mucosal barrier after oral administration to man. METHODS: Filter-grown Caco-2 cell monolayers were incubated with proteolytic enzymes and then the transepithelial electrical resistance (TEER) and the transport of the paracellular marker fluorescein were monitored. The effects of the enzymes on the cells were investigated by light microscopy and by biochemical assays. Transport of intact proteases across the cells was verified by monitoring the proteolytic activity and MALDI-TOF mass spectroscopic identification of undegraded trypsin. RESULTS: Depending on time, concentration, and side of exposure to Caco-2 cell monolayers, all proteases decreased the TEER and increased the transport of fluorescein. Some morphological and metabolic changes were observed. The effects were reversible, but until 24 hours after removal of the proteases. Under the conditions of this in-vitro model, approximately 10% of the apically applied dose reached the basolateral compartment as biologically active, non-degraded molecules. CONCLUSIONS: Proteolytic enzymes were found to exert considerable effects on the barrier function of Caco-2 monolayers, facilitating the transport of normally non-absorbable compounds. This suggests the also reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may occur by self-enhanced paracellular transport.

Biological Transport↗
PubMed2002

Supernatants of HIV-infected immune cells affect the barrier function of human HT-29/B6 intestinal epithelial cells.

OBJECTIVES: Characterization of the diarrhoea-inducing effect of altered cytokine production in HIV infection. METHODS: Monocyte-derived macrophages (MDM) were infected with macrophagetropic (SF162) and lymphocytotropic (IIIB) HIV-1 strains and cocultured with autologous peripheral blood mononuclear cells (PBMC). After 24 h the supernatants were collected and tested for their immunoreactive levels of cytokines by enzyme-linked immunosorbent assay. The effects of the supernatants and the respective recombinant human cytokines on barrier function of HT-29/B6 cells were determined. RESULTS: Infection of MDM with HIV-1 SF162 or IIIB led to increased production of tumour necrosis factor-alpha (TNFalpha), interleukin-1-beta, interferon-alpha and interferon-gamma after cell-cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (R(t)), with higher effects on R(t) in HIV IIIB infection, which was due to higher cytokine concentrations. The effect was not due to cytotoxicity (negative LDH assay) or epithelial monolayer disruption [zonula occludens protein-1 (ZO-1) immunofluorescence staining]. The effect of HIV-1 IIIB coculture supernatants could be mimicked by the respective recombinant human cytokines. TNFalpha is an effector cytokine, because inhibition of TNFalpha by its soluble receptor decreased the effect of the supernatants on transepithelial resistance. Conductance scanning indicated the cytokine-induced barrier defect to be due to both, induction of epithelial apoptoses and tight junction alterations. CONCLUSIONS: Cell-cell interaction of HIV-infected macrophages with PBMC leads to a release of cytokines sufficient to alter intestinal epithelial barrier function. The main effect was mediated by TNFalpha inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).

Apoptosis↗