Aquaporin expression is downregulated in a murine model of colitis and in patients with ulcerative colitis, Crohn's disease and infectious colitis.

Colitis is associated with alterations in electrolyte and water transport. These changes give rise to some of the symptoms experienced by patients with colitis. Alterations in fluid flux may also contribute to increased susceptibility to mucosal injury. Recently, endogenous water channel proteins (aquaporins; AQPs), have been identified in colonic tissue. The expression of AQP4, AQP7 and AQP8 was examined, via reverse transcription/polymerase chain reaction, Western blotting and immunohistochemistry, in a murine model of colitis and in patients with inflammatory bowel disease or infectious colitis. Colitis was induced in C57BL/6 mice by the addition of 2.5% dextran sodium sulphate (DSS) to their drinking water. AQP expression in these mice was assessed following 12 h to 7 days of DSS exposure and during the recovery phase from 1 to 15 days following cessation of DSS exposure. Colonic water transport was measured after 1 and 3 days of DSS and following 7 days of recovery. The expression of AQP4 and AQP8 mRNA was significantly decreased after 12-24 h of DSS exposure and remained depressed throughout the treatment period. Expression of AQP7 was more variable. Protein expression followed a similar pattern to that observed for AQP mRNA. Significant alteration in colonic fluid secretion was correlated with reduced expression of AQP isoforms. Significantly, patients with active ulcerative colonic, Crohn's colitis or infectious colitis had similar dramatic reductions in AQP expression that appeared to be correlated with disease activity. Thus, colonic injury in both mouse and man is associated with a downregulation in AQP expression.

Mucosal inflammation in a genetic model of spontaneous type I diabetes mellitus.

The BioBreeding (BB) rat provides a model of spontaneous type I diabetes mellitus that closely resembles the human disease. Diabetes-prone BB rats demonstrate increased intestinal permeability prior to the development of insulinitis. Studies suggest that alterations in intestinal permeability can lead to increased intestinal inflammatory activity. Diabetes-prone (BBdp) and diabetes-resistant (BBdr) BB rats were examined at 45 days and at >70 days of age following the development of clinical disease (BBd). In separate experiments, tissue was assayed for myeloperoxidase (MPO) or fixed for histological assessment and immunohistochemistry. Blood was obtained for leukocyte MPO measurements and morphological assessment of circulating leukocytes. MPO activity was significantly elevated in the distal small intestine of 45-day-old BBdp rats. In contrast, at >70 days of age, MPO activity was significantly increased throughout the small intestine of BBd and non-diabetic BBdp rats. Subsequently, all measurements were performed in >70-day-old rats. An increase in inflammatory infiltrate was noted in the distal small intestine of BBd rats by light microscopy. Infiltrating cells were identified as bands (a maturing cell type of the neutrophil lineage) and mature neutrophils. The findings suggest diabetes susceptibility is associated with an increase in intestinal inflammatory activity.

The effect of epidermal growth factor on the distribution of SGLT-1 in rabbit jejunum.

The effect of epidermal growth factor (EGF) on the cellular and villous distribution of the sugar transporter SGLT-1 was examined. New Zealand White rabbits (1 kg) were anesthetized, and two jejunal blind loops were isolated and exposed to either 0.9% saline or EGF (60 ng/mL saline), for 1 h. In separate experiments, tissue was harvested for brush border membrane vesicles (BBMV), microsomal membranes, or fixed for immunohistochemistry. SGLT-1 was measured in membrane fractions by Western immunoblot or localized along the villus-crypt axis by immunofluorescent microscopy. EGF increased BBMV SGLT-1 content compared with paired controls. EGF stimulation also induced a corresponding decrease in microsomal SGLT-1 levels and induced the expression of additional SGLT-1 immunoreactivity further down the villus axis. The findings suggest that EGF upregulates intestinal glucose transport by stimulating the translocation of SGLT-1 from an internal microsomal pool into the brush border, thereby recruiting more villus enterocytes into the glucose transporting population.

A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus.

In systemic lupus erythematosus, antibodies against double-stranded DNA are a major contributor to renal disease. We have previously demonstrated that the pentapeptide Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly is a molecular mimic of double-stranded DNA. This sequence is also present in the extracellular domain of murine and human NMDA (N-methyl-D-aspartate) receptor subunits NR2a and NR2b. Here we show that the NR2 receptor is recognized by both murine and human anti-DNA antibodies. Moreover, anti-DNA antibodies with this cross-reactivity mediate apoptotic death of neurons in vivo and in vitro. Finally, we show that the cerebrospinal fluid of a patient with systemic lupus erythematosus contains these antibodies and also mediates neuronal death via an apoptotic pathway. These observations indicate that lupus antibodies cross-react with DNA and NMDA receptors, gain access to cerebrospinal fluid and may mediate non-thrombotic and non-vasculitic abnormalities of the central nervous system.

Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes.

Cryopreserved porcine hepatocytes are a ready source of metabolic function for use in a bioartificial liver (BAL). However, cryopreservation is associated with a loss of hepatocyte viability. The mechanism of cell death during cryopreservation is incompletely understood, but may involve apoptosis through caspase activation. This study evaluates the cytoprotective effect of a global caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVAD-fmk) during cryopreservation of porcine hepatocytes. Freshly isolated porcine hepatocytes (viability, 97.4% +/- 0.9%) were cryopreserved in 60 micromol/L ZVAD-fmk (+ZVAD group) or without ZVAD-fmk (-ZVAD group) for 24 to 72 hours. Apoptotic and necrotic death were both observed after thawing and after 24 hours of culture. Caspase 3-like activity was significantly reduced by ZVAD-fmk, and was associated with improved viability and reduced apoptotic death of porcine hepatocytes after cryopreservation. Mitochondrial membrane potential (MMP) was increased in cultures of porcine hepatocytes that were cryopreserved in ZVAD-fmk. These results demonstrate the following: 1) Caspase 3-like protease activation and apoptosis occurs in porcine hepatocytes during cryopreservation; and 2) mitochondrial injury in this process is reduced by caspase inhibition.

A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera.

Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins. One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination. Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex. One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies. To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage. These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera. The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface. The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex.

Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver.

BACKGROUND: This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. METHODS: Isolated rat hepatocytes were entrapped in collagen gel (1.0-10.0 x 10(6) cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (GERH) were determined after anoxic exposure (0-20 hours). Cell functionality (measured by urea production), cell viability (quantitated by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]), and the mechanism of cell death (verified by electron microscopy and DNA fragmentation studies) were determined for each condition. RESULTS: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure to longer periods of anoxia (4, 8, 12, and 20 hours). ZVAD-fmk provided dose-dependent cytoprotection to GERH with an optimum benefit at a concentration of 60 mumol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. CONCLUSIONS: The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.

Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes.

A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro. The PERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL). We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes. Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL. Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested). Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase. Primers targeting the pol gene of PERV were used for PCR. Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene. Levels of PERV sequences were estimated by serial dilution. All positive samples were tested for infectivity in HEK293 cells. Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively. Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA). PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes. The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA. In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera). These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.

Bacterial colonization and healing of gastric ulcers: the effects of epidermal growth factor.

Experimental gastric ulcers are rapidly colonized by various bacteria, resulting in significantly impaired healing. Epidermal growth factor (EGF) is capable of preventing bacterial colonization of the healthy intestinal mucosa. In this study, we examined the possibility that EGF accelerates gastric ulcer healing by reducing bacterial colonization of the ulcer. Gastric ulcers were induced by serosal application of acetic acid. The effect of daily administration of EGF on ulcer healing and bacterial colonization was assessed and compared with the effect of daily treatment with broad-spectrum antibiotics. EGF administration reduced colonization levels and accelerated ulcer healing as effectively as the antibiotic treatment. EGF was without effect on acid secretion or neutrophil infiltration into the ulcer. Bacterial growth was not inhibited in the presence of EGF in vitro. These results demonstrate that EGF reduces bacterial colonization during an established infection of a compromised mucosal surface. This effect may contribute to the ability of EGF to accelerate gastric ulcer healing. This effect is acid independent and not due to an anti-inflammatory effect or to direct bactericidal actions.

The role of the epidermal growth factor receptor in microbial infections of the gastrointestinal tract.

The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein with an intrinsic tyrosine kinase. Ligand-binding to the EGFr activates cell signaling, phosphorylates protein kinases, and rearranges cytoskeletal proteins - responses that resemble those induced by microbial attachment to cell surfaces, a process known to be mediated by host cell receptors in a number of cases. This article critically reviews the possible role played by the EGFr in microbial colonization, and discusses how modulation of the EGF-EGFr axis may affect infection of the gastrointestinal tract.

Human RNA helicase A is a lupus autoantigen that is cleaved during apoptosis.

Proteolytic cleavage by caspases is the central event in cells undergoing apoptosis. Cleaved proteins are often targeted by autoantibodies, suggesting that the cleavage of self Ags enhances immunogenicity and is prone to induce an autoimmune response. We found autoantibodies that immunoprecipitated a 140-kDa RNA-associated protein, provisionally designated Pa, in 11 of 350 patient sera that were positive for antinuclear Abs in an immunofluorescence test. The Pa protein gave rise to three fragments with m.w. ranging from 120-130 kDa during anti-Fas-activated apoptosis. Pure caspase-3 cleaved the Pa protein into a 130-kDa fragment corresponding to the largest of these three products. Peptide sequence analysis of a tryptic digest from immunoaffinity-purified Pa showed 100% identity to human RNA helicase A (RHA). The identity of Pa with RHA was further confirmed by immunoblotting with rabbit anti-RHA Ab using anti-Pa immunoprecipitates as substrates. All 10 anti-RHA-positive patients who were clinically analyzed were diagnosed as having systemic lupus erythematosus, and 7 of them had lupus nephritis. RHA is a multifunctional protein with roles in cellular RNA synthesis and processing. Inactivation of RHA by cleavage may be an important part of the process leading to programmed cell death. The cleaved RHA fragments that are produced during apoptosis may trigger an autoimmune response in systemic lupus erythematosus.

Effect of a dietary nonionic surfactant on small intestinal nutrient transport.

Nonionic surfactants are commonly present in many prepared foods and drug formulations as stabilizing agents. The aim of the current study was to examine the effect of the common nonionic surfactant Tween-80 on jejunal glucose transport. New Zealand White rabbits (800-1200 g) were fasted for 24 hr. Jejunal tissue was stripped and mounted in short-circuited Ussing chambers. Unidirectional 3-O-methyl glucose fluxes were determined during a 15-min basal transport period and a subsequent 15-min experimental period after the mucosal addition of Tween-80 at final concentrations of 0.001%, 0.01%, 0.05%, and 0.1%. Tween-80 at final concentrations of 0.1% and 0.05% significantly increased net 3-O-methyl glucose transport over basal levels. The increase in Jnet was due to a significant increase in the absorptive Jm-s flux. Tween-80 at 0.01% and 0.001% did not significantly alter net glucose transport although Jm-s was significantly increased in the presence of 0.01% Tween-80. Tween-80, in concentrations commonly found in prepared foods, enhances the intestinal absorption of glucose.

Transfer of porcine endogenous retrovirus across hollow fiber membranes: significance to a bioartificial liver.

BACKGROUND: A porcine endogenous retrovirus (PERV) capable of infecting human cells has been identified. This study was designed to determine whether hollow fiber membranes, such as those used in a bioartificial liver, block the transfer of PERV. METHODS: Three hollow fiber cartridges (HFCs) were studied in duplicate: cellulose fibers with 70 kD nominal molecular weight cut-off (MWCO), polysulfone fibers with 400 kD MWCO, and mixed cellulose fibers with 200 nm porosity. PK15 cells (porcine kidney cell line), known to produce PERV, were grown in the intraluminal compartment of HFCs fiber cartridges. Samples of medium were collected from both intraluminal and extraluminal compartments of the HFCs fiber cartridge during 14 days of culture. Samples were screened for PERV using reverse transcription polymerase chain reaction. All positive samples were tested for PERV infectivity in human 293 cells. RESULTS: PERV was detected in all samples from the intraluminal space and all intraluminal samples seemed to infect 293 cells. All extraluminal samples from the fibers of 200 nm porosity tested positive for PERV. Detection of PERV in the extraluminal space was delayed by fibers of 400 kD MWCO and 70 kD MWCO until at least day 3 and day 7, respectively, after inoculation of PK15 cells. Positive extraluminal samples from fibers of 400 kD MWCO and 70 kD MWCO did not infect 293 cells. CONCLUSION: Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.

A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity.

OBJECTIVE: To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. METHODS: Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti-double-stranded DNA (anti-dsDNA), anti-single-stranded DNA, antihistone, anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 (DNA topoisomerase I), anticentromere, and anti-Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. RESULTS: Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anticentromere, and anti-Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. CONCLUSION: No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases.

Role of actin in EGF-induced alterations in enterocyte SGLT1 expression.

Na+-glucose cotransporter (SGLT1) expression and the role of actin in epidermal growth factor (EGF)-induced alterations in glucose transport and brush-border surface area were examined in New Zealand White rabbit jejunal loops. In separate experiments, EGF or EGF concurrent with cytochalasin D, an inhibitor of actin polymerization, was administered to the experimental loop and compared with its vehicle control. SGLT1 expression was measured by Western blot in brush-border membrane vesicles (BBMV) after 5-min and 1-h exposure. Glucose kinetics were determined by a rapid filtration technique, and brush-border surface area was examined by electron microscopy after 1-h exposure. The effect of cytochalasin D alone on BBMV glucose kinetics and brush-border surface area was also assessed. EGF resulted in a significant increase in BBMV SGLT1 expression (P < 0.05), glucose maximal uptake (Vmax; P < 0.001), and absorptive brush-border surface area (P < 0.001). These effects were abolished with concurrent cytochalasin D treatment. Cytochalasin D alone had no effect on glucose transport or brush-border surface area. The findings suggest that EGF acutely upregulates jejunal brush-border surface area and the Vmax for jejunal glucose uptake via the recruitment and insertion of SGLT1 from an internal pool into the brush border by a mechanism that is dependent on actin polymerization.

Macromolecular transport across the rabbit proximal and distal colon.

BACKGROUND: Although many studies have investigated macromolecular uptake in the stomach and small intestine, little is known about macromolecular uptake in the colon. AIMS: To investigate the mechanisms involved in the transport of large antigenically intact macromolecules across the proximal and distal colonic epithelium in the rabbit. METHODS: The mucosal to serosal movement of bovine serum albumin (BSA) was examined in modified Ussing chambers under short circuited conditions. The mucosal surface was exposed to varying concentrations of BSA, and after a 50 minute equilibration period, the mucosal to serosal flux of immunologically intact BSA was determined by ELISA. Total BSA flux was determined by the transport of radiolabelled 125I-BSA. RESULTS: Intact BSA transport in proximal and distal colonic tissue showed saturable kinetics. Intact BSA transport in the proximal and distal segment was 7% and 2% of the total 125I-BSA flux respectively. Immunologically intact BSA transport in the distal segment was significantly less than that in the proximal segment. Intact BSA transport in the proximal colon was significantly reduced following treatment with sodium fluoride, colchicine, and tetrodotoxin. Cholinergic blockade had no effect on the uptake of intact BSA. CONCLUSION: The findings indicate that the transport of intact macromolecules across the proximal and distal large intestine is a saturable process. Further, intact BSA transport in the proximal colon is an energy dependent process that utilises microtubules and is regulated by the enteric nervous system.

The effect of epidermal growth factor on brush border surface area and function in the distal remnant following resection in the rabbit.

BACKGROUND: Epidermal growth factor (EGF) has been shown to increase intestinal absorptive surface area and transport function in normal animals. AIMS: To examine the effect of EGF on absorptive surface area and brush border membrane function in a model of massive small bowel resection. METHODS: New Zealand white rabbits were randomised into two groups: a resected group (60% proximal small bowel resection); and an unmanipulated control group. Distal remnant tissue was examined 10 and 21 days postsurgery. In separate experiments oral EGF (40 g/kg/day) was administered to resected animals from days 3 to 8 and animals were studied on day 10. RESULTS: Ten days postsurgery brush border surface area and total absorptive surface area were significantly increased in remnant tissue while brush border membrane vesicle (BBMV) glucose uptake was significantly decreased compared with controls. By 21 days brush border surface area returned to control levels though BBMV glucose uptake remained depressed. EGF treatment induced a further increase in brush border surface area in remnant intestine but did not alter BBMV glucose uptake. CONCLUSIONS: Surgical resection results in significant elevations in absorptive surface area coupled with a decrease in brush border membrane transport function distal to the site of anastomosis. EGF enhances glucose uptake in remnant intestine via recruitment of additional microvillus membrane into the brush border.

Human autoantibodies recognizing a native macromolecular structure composed of Sm core proteins in U small nuclear RNP particles.

OBJECTIVE: Monoclonal antibody (mAb) F78 recognizes a heat-labile particle composed of Sm core proteins designated F78P. The objective of this study was to identify human autoantibodies recognizing the conformational structure of F78P. METHODS: Immunoblots using HeLa cell extracts without heating prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to identify autoantibodies recognizing F78P. To confirm reactivities with F78P, immunoprecipitates of mAb F78 were used as a substrate for immunoblots. To identify reactivities against the F78P structure in classic anti-Sm-positive sera, autoantibodies to individual Sm core proteins were absorbed with purified U1 small nuclear RNP before immunoblotting. RESULTS: We identified 2 sera that, like F78, recognized only F78P and not its component polypeptides. When classic anti-Sm antibodies were preabsorbed, the presence of F78-like, particle-specific antibodies was revealed in all of the anti-Sm-positive sera tested. CONCLUSION: Autoantibodies against the F78P structure were commonly present in sera from patients with systemic rheumatic diseases, often in combination with4=1998 M autoantibodies.