Altered regulation of airway epithelial cell chloride channels in cystic fibrosis.

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

Clinical evidence for immunomodulatory effects of probiotic bacteria.

Close, tightly orchestrated interactions between the intestinal epithelium and the mucosa-associated immune system are critical for normal intestinal absorptive and immunological functions. Recent data indicate that commensal intestinal microbiota represents a major modulator of intestinal homeostasis. This review analyzes the process of intestinal colonization and the interaction of microbiota with the intestinal epithelium and mucosal immune system, with special reference to the first years of extrauterine life. Dysregulation of the symbiotic interaction between intestinal microbiota and the mucosa may result in a pathological condition with potential clinical repercussions. Based on the concept that there is a beneficial and symbiotic relation between the host and endogenous microbiota, strategies aimed at directly modulating intestinal microbiota with regard to disease prevention or treatment have been developed. One strategy involves administering viable probiotic bacteria. Clinical evidence for the beneficial effect of probiotics in the prevention and/or treatment of necrotizing enterocolitis, infectious and antibiotic-associated diarrhea, allergic diseases, and inflammatory bowel disorders is reviewed herein.

Isolation of basolateral membranes from columnar cells of the proximal colon of the guinea pig.

A method for an analytical isolation of plasma membranes from columnar cells (colonocytes) of the proximal colon of the guinea pig is described. Isolation of the colonocytes was performed by a mild EDTA-chelation method. After homogenization, two subsequent sucrose gradient centrifugations (isokinetic and isopycnic) yielded a plasma-membrane fraction which was enriched 12-fold in (Na+ + K+)-ATPase activity and 8-fold in adenylate cyclase activity. It is suggested that the purified membrane fraction consists mainly of basolateral membranes of the colonocytes. Due to the lack of suitable marker enzymes, no evidence for enrichment of the brush-border membranes was obtained. Histochemical studies demonstrated that alkaline phosphatase is absent from the luminal membrane of the surface epithelial cells of the proximal colon of the guinea pig.

Radioimmunoassay for circulating human guanylin.

A highly specific and sensitive radioimmunoassay for circulating human guanylin (guanylin-22-115) has been developed. Antibodies were raised against the amino-terminus (positions 4-16) of the peptide. Western blot analysis confirmed that the antibody selected for radioimmunoassay recognizes circulating high molecular weight (10.3 kDa) guanylin. Extraction and purification of guanylin from blood hemofiltrate and from blood plasma showed that circulating guanylin is detectable in corresponding amounts by the radioimmunoassay and by a specific bioassay. In 30 healthy subjects, the mean plasma concentration of immunoreactive (IR) guanylin was 42 +/- 3 fmol/ml. In 22 patients with chronic renal insufficiency, the concentrations of IR-guanylin were significantly enhanced (1,074 +/- 24 fmol/ml), indicating that kidneys metabolize and/or eliminate the circulating hormone.

Functional expression of a human C5a receptor clone in Xenopus oocytes requires additional RNA.

cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes.

Protective role of probiotics and prebiotics in colon cancer.

Ingestion of viable probiotics or prebiotics is associated with anticarcinogenic effects, one mechanism of which is the detoxification of genotoxins in the gut. This mechanism was shown experimentally in animals with use of the rat colon carcinogen 1,2-dimethylhydrazine and by determining endpoints that range from tumorigenesis to induction of DNA damage. Because of the complexity of cancer initiation, cancer progression, and the exposure of cancer in the gut, many types of interactions may be envisaged. Notably, some of our newer studies showed that short-lived metabolite mixtures isolated from milk that was fermented with strains of Lactobacillus bulgaricus and Streptococcus thermophilus are more effective in deactivating etiologic risk factors of colon carcinogenesis than are cellular components of microorganisms. Ingestion of prebiotics results in a different spectrum of fermentation products, including the production of high concentrations of short-chain fatty acids. Gut flora, especially after the ingestion of resistant starch, induces the chemopreventive enzyme glutathione transferase pi in the colon of the rat. Together, these factors lead to a reduced load of genotoxic agents in the gut and to an increased production of agents that deactivate toxic components. Butyrate is one such protective agent and is associated with lowering cancer risk. It was recently shown that buytrate may inhibit the genotoxic activity of nitrosamides and hydrogen peroxide in human colon cells. In humans, the ingestion of probiotics leads to the excretion of urine with low concentrations of components that are genotoxic in human colon cells and high concentrations of components that induce oxidized DNA bases.

Mechanisms by which vegetable consumption reduces genetic damage in humans.

A previous intervention study had shown that consumption of carotenoid-containing vegetable juices reduces oxidative DNA damage in lymphocytes of 23 male subjects. It was the aim of this study to elucidate the potential mechanisms involved. Specifically, we studied the modulation of protein expression and determined susceptibility factors. Cryopreserved lymphocytes from the study were analyzed for genetic polymorphisms of glutathione S-transferase (GSTM1, GSTP1, and GSTT1) using multiplex PCR, GSTP1-protein with an ELISA, total protein by a colorimetric enzyme reaction, and DNA-repair enzymes with the Comet Assay. Analyses of the genotoxicity data revealed a more steady state of protection for GSTM1*+ than for GSTM1*0 (15 and 8 of 23, respectively) genotypes. Increased expression of cytosolic protein was observed in 11 of 23 subjects, increased expression of GSTP1 in 6 of 23 subjects, and capacity of repair of oxidized DNA bases in 9 of 21 subjects. GSTP1 induction was independent of the GSTP1 genotype (GSTP1a or GSTP1b/c alleles). Kinetics of induction of cytosolic protein and of GSTP1 were compared in one GSTM1*+ and one GSTM1*0 subject and showed an efficacy of tomato and carrots, but not of spinach. Reduced genetic DNA damage in lymphocytes may be due to the enhancement of cytosolic GSTP1, and DNA-repair proteins by tomato and carrot juices. Enhancement of cytosolic proteins may be indicative of increased gene expression by vegetable juices, some of which may be associated with protective activities.

Analysis of DNA strand breaks, oxidized bases, and glutathione S-transferase P1 in human colon cells from biopsies.

The balance of genetic damage and deactivating enzymes is decisive for cancer risk. To assess these factors in normal human colon cells, we determined background levels of DNA breaks or oxidized bases and of glutathione S-transferases (GSTs) as potential biomarkers of risk and chemoprevention, respectively. Also, genotoxicity by compounds involved in lipid peroxidation was determined to elucidate possible sources of damage. Cells were isolated from sigmoid biopsies of 51 donors and processed with the comet assay to reveal genetic damage. GST proteins were analyzed immunologically. HT29 clone 19A colon tumor cells, resembling primary cells, were treated with 2-trans-hexenal (400 microM) or hydrogen peroxide (75 microM) and processed for damage. Fifteen percent of primary colon cells contained strand breaks; 22% contained additional oxidized bases, with distinct sex differences. Similar damage was found in HT29 clone cells and is induced by both test compounds. GST levels were similar in both cell types. The comet assay is sufficiently sensitive to detect oxidative genetic damage in small amounts of cells from small amounts of biopsies. Lipid peroxidation is a possible risk factor. Together with GST as a potential biomarker of chemoprevention, the technique may serve as a valuable biomarker to assess exposure to risk factors.

First pass effect of 1-naphthol in the gastric mucosa. Studies with isolated epithelium and cultured mucous cells of guinea pig.

Disposition of 1-[14C]naphthol was investigated in stripped gastric mucosal segments mounted in Ussing chambers. During 2-hr incubations, naphthol was glucuronidated (44-55% of added dose) and sulfated (7-15%). When naphthol was added to the luminal fluid at pH 7.0, conjugates were released with equal velocity to the luminal and to the vascular side, but with a luminal pH of 3, conjugates appeared predominantly on the vascular side. When naphthol was added to the vascular side (both sides pH 7), conjugates appeared predominantly on the vascular side. Cultured gastric mucous cells formed naphthol glucuronide and naphthol sulfate at a ratio of 9:1. These conjugates were transiently accumulated within cells up to 300-fold followed by slow release into the medium. In conclusion, the intact gastric mucosa is able to conjugate 1-naphthol at neutral and acidic luminal pH. The data suggest that ingested phenolic compounds might be modified by a gastric first pass effect.

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells.

INTRODUCTION: Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology. METHODS: We have measured H2O2-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H2O2 and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis ("Comet") Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H2O2-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage. RESULTS: A dose-response relationship was found for the H2O2-induced dye decomposition in NIH3T3 cells (7.8-125 microM H2O2) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 M H2O2). Fluorescence was significantly increased at 62microM H2O2 in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H2O2 than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells. CONCLUSIONS: Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H2O2-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H2O2.

Short-term moderate aflatoxin B1 exposure has only minor effects on the gut-associated lymphoid tissue of Brown Norway rats.

Aflatoxin B1 (AFB1) is toxic to the systemic immune system in various animal species, whereas little is known about its effect on the gut-associated lymphoid tissue (GALT). It may be hypothesized that the toxicity of AFB1 and its locally generated metabolites in the intestinal tissue may result in a disturbed intestinal integrity and, subsequently, in an impaired immune response towards dietary proteins. The objective of our study was to investigate the toxic effect of short-term moderate AFB1 exposure on the intestinal epithelium and on the immune cells associated with the intestinal tract. The toxicological potential of AFB1 and its metabolites to the intestinal epithelium was determined by measuring viability and genotoxic damage in isolated jejunal epithelial cells (comet assay) after 30 min incubation in vitro. In vivo toxicology studies were carried out with Brown Norway (BN) rats, which were exposed orally once a week with AFB1 (1 x 100 microg/kg body weight (b.w.)/week) for 5 consecutive weeks. Viability and genotoxicity were measured in explanted jejunal epithelial cells. For studying the effectiveness of AFB1 on immunological parameters BN rats were treated with a high (study 1: 1 x 1 mg/kg b.w./week) or a low (study 2: 1 x 100 microg/kg b.w./week) AFB1 dose for 5 consecutive weeks with or without ovalbumin (OVA). Mesenteric lymphocytes were isolated and proliferative responsiveness, secretion of interferon-gamma, and changes in lymphocyte subpopulations as well as mucosal mast cell specific protease and anti-OVA specific antibody concentrations were measured. In vitro, AFB1 ( >30 microM) induced genotoxicity in rat jejunal epithelial cells. The oral administration of AFB1 (1 x 100 microg/kg b.w./week) did not induce DNA damage in jejunal epithelial cells. The high AFB1 dose increased the number of CD8+ and CD8/CD71 + cells in mesenteric lymph nodes. The immune response towards OVA was not affected. The low AFB1 dose only reduced the proliferative responsiveness of mesenteric lymphocytes (P < 0.05). Serum concentrations of anti-OVA specific IgE antibody, of RMCPII, and the capacity of mesenteric lymphocytes to produce interferon-gamma were not impaired by AFB1. In conclusion, exposure to moderate doses of AFB1 does not damage the intestinal epithelium and has only minor effects on the GALT. The low exposure, as it may predominantly occur in western countries, does not appear to increase the risk for sensitization to dietary antigens.

Daily moderate amounts of red wine or alcohol have no effect on the immune system of healthy men.

OBJECTIVE: To investigate whether the daily intake of red wine (RW) at a dose which inversely correlates with cardiovascular disease (CVD) risk modulates immune functions in healthy men. DESIGN: Randomized single-blind trial with four intervention periods. SETTING: The Institute of Nutritional Physiology, Federal Research Centre for Nutrition, Karlsruhe, Germany. SUBJECTS: A total of 24 healthy males with moderate alcohol consumption patterns were recruited and all completed the study. INTERVENTION: Participants consumed 500 ml of RW (12% ethanol (ETOH)) or 500 ml of a 12% ETOH dilution per day for a period of 2 weeks. To control the potential effects of RW polyphenols, accordingly 500 ml/day of dealcoholized red wine (DRW) and of red grape juice (RGJ) were given. The following immune parameters were measured before beverage consumption and at 1 and 2 weeks following beverage consumption: phagocytic activity of neutrophils and monocytes, production of tumor necrosis factor-alpha (TNFalpha), interleukin-2 and -4, transforming growth factor-beta, TNFalpha mRNA, lymphocyte proliferation, lytic activity of natural killer cells, and percentage of apoptotic lymphocytes. RESULTS: Consumption of a moderate volume of alcohol with RW and with a 12% ETOH dilution had no effect on immune functions in healthy males. Consumption of polyphenol-rich beverages (DRW and RGJ) did not affect immunity-related parameters. CONCLUSIONS: Daily moderate consumption of alcohol and of RW for 2 weeks at doses which inversely correlate with CVD risk has no adverse effects on human immune cell functions. Polyphenol-rich beverages such as RGJ and DRW further do not suppress immune responses in healthy men.

Contribution of apoptosis to responses in the comet assay.

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.

Enhancement of ovalbumin-induced antibody production and mucosal mast cell response by mercury.

Food contaminants may contribute to the recent increased incidence of food allergies. We have investigated this hypothesis experimentally. It was our objective to determine whether toxicity to the intestinal tissue by orally applied mercury (Hg) could modulate the immune response to food allergens. Effective mechanisms were studied with functional immunological and toxicological parameters. Brown Norway rats were immunized intraperitoneally by ovalbumin (OVA). Before oral challenge with OVA, immunized and non-immunized animals were exposed to HgCl2. Immunological responses were measured by enzyme-linked immunosorbent assays [anti-OVA-IgE and-IgG, rat mast cell protease II (RMCPII), interferon-gamma, interleukin-4, lymphocyte proliferation] and by flow cytometry (lymphocyte subpopulations). Toxicity of Hg to the intestinal barrier was determined by measuring viability, DNA damage and induction of glutathione S-transferase in isolated intestinal epithelial cells and lymph node cells, and by measuring permeability, short-circuit current and tissue conductance of the intact intestinal epithelium. A single high oral dose of HgCl2 enhanced the serum concentrations of anti-OVA-IgE and IgG (P < 0.05) and of RMCPII (P < 0.05) in immunized rats. The treatment resulted in a higher number of CD4/CD25+ T cells in the lymph nodes (P < 0.05). The multiple application of low HgCl2 doses (5 x 0.2 mg/kg body weight) only resulted in an elevated RMCPII serum concentration (P < 0.05). Neither treatment schedules impaired proliferation and cytokine production of lymphocytes. In non-immunized rats only minor immunological changes were observed. Oral HgCl2 induced genotoxic damage in lymph node cells and in jejunal epithelial cells (P < 0.05). Moreover, HgCl2 increased the permeability of intestinal epithelial tissue and of Caco-2 monolayers and was genotoxic and cytotoxic to isolated intestinal epithelial cells in vitro. In conclusion, these studies indicate that the food contaminant Hg can stimulate the immune response to OVA in immunized rats. One possible mechanism could be the toxicity of Hg to the intestinal epithelial and the lymph node cells. Whether humans with allergies respond to high oral doses of Hg in a similar way needs to be investigated in further studies.

Urodilatin secretion in salt-loaded Wistar rats.

The aim of our study was to investigate whether urodilation (URO, INN: ularitide) is present in rat urine and if URO excretion in the rat is influenced by dietary sodium intake. Therefore, three groups of Wistar rats were placed in metabolic cages where they received different sodium diets for 9 days (0.05%, 0.4%, and 8.0% NaCl, respectively). Food and water intake were determined by weight. At days -4, 2, 5, and 8 blood pressure was measured non invasively using the tail cuff method. After nine days rats were anesthetized and blood was drawn for serum electrolyte, plasma A-type natriuretic peptide (CDD/ANP-99-126), and plasma aldosterone concentration measurements. Using a highly specific antibody against URO combined with high performance liquid chromatography and gel chromatography, we were able to show that a URO-like substance of approx. 3.5 kD that is distinct from CDD/ANP-99-126, brain natriuretic peptide, and C-type natriuretic peptide, is present in rat urine. Sodium chloride loaded rats showed significantly increased urinary excretion rates of URO (p < 0.001), chloride (p < 0.001), sodium (p < 0.001), and the fractional excretion of sodium (p < 0.001). In the plasma, sodium (p < 0.01) and chloride (p < 0.001) increased, while potassium, hematocrit, osmolality, plasma CDD/ANP-99-126, as well as glomerular filtration rate (GFR), and systolic blood pressure did not change. Since CDD/ANP-99-126 is believed to be a natriuretic peptide, it is suggested that CDD/ANP-99-126 might participate in the natriuresis due to high dietary sodium intake. In sodium-loaded rats, however, plasma CDD/ANP-99-126 remains unchanged, while URO excretion increases with sodium excretion, independent of GFR and blood pressure. We conclude that URO secretion is stimulated by dietary salt loading and might be involved in the regulation of water and electrolyte metabolism in the rat.

Secretion of a urodilatin-like immunoreactive (URO-like-IR) substance from a human kidney cell line (HEK-293).

The aim of this study was to explore whether sodium chloride is involved in the release of urodilatin from epithelial human embryonic kidney cells (HEK-293). Using a highly specific urodilatin radioimmunoassay combined with HPLC, gel chromatography, and a cyclic GMP generating bioassay, we demonstrate that HEK-293 cells release a biologically active 3.5 kD, urodilatin-like immunoreactive substance. To show the effect of sodium on urodilatin release, HEK-293 cells were incubated with cell culture buffer containing 120, 130, 140, and 150 mmol/l sodium, respectively. Urodilatin secretion from HEK-293 cells is increased to 176% when extracellular sodium is raised to 150 mmol/l (control: 120 mmol/l = 100%). There was no significant difference when exposing the cells to 140 mmol/l or 150 mmol/l sodium. It is suggested, that beside the known extrarenal factors influencing urodilatin secretion, high cephalic sodium and cardiac volume load, urodilatin secretion might also be regulated by an intrarenal sodium sensitive mechanism.

Cultured gastric parietal cells from the guinea pig: adherence, cell growth and stimulus coupling of Ca2+ and cyclic AMP.

The aim of the study was to establish cell culture conditions for responsive guinea pig parietal cells. Parietal cells were isolated by a pronase/collagenase method, enriched by counterflow elutriation and cultured on plastic culture dishes in minimum essential medium. Precoating with gelatine or collagen increased adherence; optimum fetal calf serum concentration was 10%. Parietal cells were cultured for up to 120 h. Intracellular calcium levels in cells cultured for 48 h were 150 nmol/l and increased to 320 nmol/l after stimulation with carbachol and to 250 nmol/l after histamine stimulation as determined by video imaging microscopy. Intracellular cyclic AMP levels were increased 9-fold by histamine in cells cultured for 24 h and more than 30-fold in cells cultured for 48 h. The results show that guinea pig parietal cells grow in primary culture and are suitable for studying second messenger coupling.

Comparative views of electrophysiological parameters of large intestinal segments in pig, sheep, pony, guinea pig and rat.

Short circuit current (ISC) and transepithelial conductance (gt) across sheets of epithelia were measured in the caecum, the proximal and the distal colon of pig, sheep, pony, rat and guinea pig. The electrical parameters underline the basic segmental and species differences. The diversity of ISC demonstrates the different nature of electrogenic transport mechanisms, and data clearly show the heterogeneity with respect to transport mechanisms along the large intestine in the various species. The great differences in amiloride sensitive ISC indicate the variabilities in the electrogenic Na transport. Whereas in the pig, sheep and pony caecum, in the guinea pig proximal colon and in all segments of the rat hindgut no indications for a major electrogenic Na transport was seen, in all other segments amiloride caused a marked decrease in ISC. Electrogenic Na transport seems to be highest in sheep distal colon and in pig proximal and distal colon, somewhat less in guinea pig and in pony distal colon. The epithelium with the lowest Powest transepithelial conductance clearly is that of the pony caecum. Except in sheep, gt-values are not much different from those in pony and also pig and guinea pig. By far the epithelium with the highest conductance is that of the rat proximal colon. gt was similar in the proximal and the distal colon of pig, sheep and pony; in guinea pig values were slightly, in rat much lower.