H. J. Epple

Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum

Background:Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. Aim: To characterise the inherent pathomechanisms of G lamblia infection. Methods: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short-circuit current (ISC) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining. Results: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Ω cm2; p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm2). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal ISC increased from 191 (20) in control to 261 (12) µA/h/cm2 in giardiasis. The bumetanide-sensitive portion of ISC in giardiasis was also increased (51 (5) vs 20 (9) µA/h/cm2 in control; p<0.05). Finally, phlorizin-sensitive Na+–glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) µA/h/cm2). Conclusions:G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na+-dependent d-glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components.

Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients

Background and aims: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. Methods: Epithelial barrier function was characterised by impedance spectroscopy and [3H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. Results: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) Ω cm2, p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor α (TNFα) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFα and IL13 reduced the transepithelial resistance of rat jejunal mucosa. Conclusions: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

Structural and functional changes of the duodenum in human norovirus infection

Background: Norovirus infection is the most frequent cause of infectious diarrhoea in the western world. This study aimed to characterise functionally and histomorphologically the diseased duodenum in human biopsies. Methods: Norovirus infection was diagnosed by the Kaplan criteria and confirmed by PCR of stool samples. Duodenal biopsies were obtained endoscopically. In miniaturised Ussing chambers, short circuit current, flux measurements and impedance spectroscopy were performed. Histological analysis including apoptosis staining and characterisation of intraepithelial lymphocytes was performed. Tight junction proteins were quantified by immunoblotting. Results: In norovirus infection, epithelial resistance decreased from (mean (SEM)) 24 (2) Ω cm2 in controls to 10 (1) Ω cm2. Mannitol flux increased from 113 (24) nmol h−1 cm−2 in controls to 242 (29) nmol h−1 cm−2. Microdissection revealed a villus surface area reduced by 47% (6.6%). Intraepithelial lymphocytes were increased to 63 (7) per 100 enterocytes, with an increased rate of perforin-positive cytotoxic T cells. Expression of tight junctional proteins occludin, claudin-4 and claudin-5 was reduced. The epithelial apoptotic ratio was doubled in norovirus infection. Furthermore, the basal short circuit current was increased in norovirus infection and could be reduced by bumetanide and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Conclusions: Norovirus infection leads to epithelial barrier dysfunction paralleled by a reduction of sealing tight junctional proteins and an increase in epithelial apoptosis, which may partly be mediated by increased cytotoxic intraepithelial lymphocytes. Furthermore, active anion secretion is markedly stimulated. Thus, the diarrhoea in norovirus infection is driven by both a leak flux and a secretory component.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines

Background: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. Methods: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2×105 cagPAI− H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. Results: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. Conclusion: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Antisecretory Effect of Loperamide in Colon Epithelial Cells by Inhibition of Basolateral K+ Conductance

Background: The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). Methods: Chloride secretion was stimulated via cAMP by forskolin (FSK, 10-5 M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10-4 M), and via protein kinase C by the phorbol ester PMA (5·10-9 M). Stimulated Cl- secretion was quantified as short circuit current (ISC) of HT-29/B6 monolayers mounted in Ussing-type chambers. Results: Loperamide (5·10-5 M) inhibited ISC stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10-5 M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10-4 M) inhibited ISC induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5·10-5 M), on the other hand, inhibited only PMA-stimulated ISC, but had no effect on FSK or CCh-induced ISC. Conclusions: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.BACKGROUND The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). METHODS Chloride secretion was stimulated via cAMP by forskolin (FSK, 10(-5) M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10(-4) M), and via protein kinase C by the phorbol ester PMA (5 x 10(-9) M). Stimulated Cl- secretion was quantified as short circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. RESULTS Loperamide (5 x 10(-5) M) inhibited I(SC) stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10(-5) M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10(-4) M) inhibited I(SC) induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5 x 10(-5) M), on the other hand, inhibited only PMA-stimulated I(SC),but had no effect on FSK or CCh-induced I(SC) CONCLUSIONS: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.

Aeromonas hydrophila Beta-Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells (HT-29/B6)

ABSTRACT The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (ISC) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (Rt) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant ISC (39 ± 3 μA/cm2) and decreased the Rt to ∼10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant ISC in totally stripped human colon. Tracer flux and ion replacement studies revealed the ISC to be mainly accounted for by electrogenic Cl− secretion. Supernatant applied serosally completely abolished basal ISC. The supernatant-induced ISC was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca2+ chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatants active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical ISC and Rt responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl− secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.

Prostaglandin I2 sensory input into the enteric nervous system during distension‐induced colonic chloride secretion in rat colon

Aim:  Intestinal pressure differences or experimental distension induce ion secretion via the enteric nervous system, the sensorial origin of which is only poorly understood. This study aimed to investigate sensorial inputs and the role of afferent and interneurones in mechanically activated submucosal secretory reflex circuits.

Microbial Translocation and the Effects of HIV/SIV Infection on Mucosal Barrier Function

Human immunodeficiency virus (HIV) infection leads to complex alterations within the network of mucosal immune cells, which collectively cause mucosal immune activation. Mucosal immune activation in turn causes damage to the epithelial barrier via cytotoxic effects of CD8 T cells, inflammatory cytokines, and downregulation of cytokines and T cell subsets related to epithelial repair. Structural correlates of the ensuing epithelial barrier defect are increased epithelial apoptosis and altered expression of tight-junction proteins. The mucosal barrier defect leads to increased microbial translocation, which is a major driving force for the systemic immune activation. Systemic immune activation is considered a central pathomechanism in chronic HIV infection contributing to disease progression and to HIV-associated noninfectious comorbidity such as coronary heart disease, neurocognitive disorders, and liver disease. Therefore, there is a causal link between the HIV-induced intestinal mucosal barrier defect and disease progression, morbidity, and mortality of HIV-infected patients.