Institute of Clinical Physiology

Berlin, Germany

Institute of Clinical Physiology

Berlin, Germany

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Scutaru A.M.,Free University of Berlin | Kruger M.,Free University of Berlin | Wenzel M.,Free University of Berlin | Richter J.,Institute of Clinical Physiology | And 2 more authors.
Bioconjugate Chemistry | Year: 2010

Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO2-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used. © 2010 American Chemical Society.


Veshnyakova A.,Leibniz Institute for Molecular Pharmacology | Fromm M.,Institute of Clinical Physiology | Amasheh M.,Charité - Medical University of Berlin | Amasheh S.,Institute of Clinical Physiology
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology | Year: 2010

In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (Repi). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest Repi, followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (Rtrans or TER) overestimated true Repi by ~60%. In colon, strongest expression of "tightening" claudins 1, 3, 4, 5, and 8 was detected. In accordance with Repi the most proximal of the small intestinal segments, duodenum exhibited highest expression of "tightening" claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content. © 2010 Springer-Verlag.


Markov A.G.,Saint Petersburg State University | Kruglova N.M.,Saint Petersburg State University | Fomina Y.A.,Saint Petersburg State University | Fromm M.,Institute of Clinical Physiology | Amasheh S.,Institute of Clinical Physiology
Pflugers Archiv European Journal of Physiology | Year: 2012

Milk production is modulated by the paracellular barrier function of tight junction (TJ) proteins located in the mammary epithelium. The aim of our study was the molecular analysis of TJs in native lactating murine mammary gland epithelium as this process may strongly challenge epithelial barrier properties and regulation. Mammary gland tissue specimens from lactating control mice and animals after a 20-h interruption of suckling were prepared; histological analyses were performed by light and electron microscopy; and expression of TJ proteins was detected by PCR, Western blotting, immunofluorescent staining, and confocal laser scanning microscopy. Discontinuation of suckling resulted in a substantial accumulation of milk in mammary glands, an increase of alveolar size, and a flattening of epithelial cells without effects on inflammatory indicators. In control tissues, PCR and Western blots showed signals for occludin, and claudin-1, -2, -3, -4, -5, -7, -8, -15, and -16. After a 20-h accumulation of milk, expression of two sealing TJ proteins, claudin-1 and -3, was markedly increased, whereas two TJ proteins involved in cation transport, claudin-2 and -16, were reduced. Real-time PCR validated increased transcripts of claudin-1 and claudin-3. During extension of mammary glands in the process of lactation, claudin-1 and -3 are markedly induced and claudin-2 and -16 are decreased. Volume and composition of milk might be strongly dependent on this counter-regulation of sealing claudins with permeability-mediating claudins, indicating a physiological process of a tightening of TJs against a back-leak of solutes and ions from the alveolar lumen. © 2011 Springer-Verlag.


Schumann M.,Infectious Diseases and Rheumatology | Kamel S.,Infectious Diseases and Rheumatology | Pahlitzsch M.-L.,Infectious Diseases and Rheumatology | Lebenheim L.,Infectious Diseases and Rheumatology | And 6 more authors.
Annals of the New York Academy of Sciences | Year: 2012

In celiac disease, the gut-associated immune system is activated in response to the ingestion of gluten, causing an atrophy of the small intestinal mucosa. Although this condition is, in most cases, responsive to a gluten-free diet, celiac disease refractory to treatment occurs in a small percentage of celiacs. An epithelial barrier defect is known to be an integral part of celiac pathophysiology. However, the mucosa in refractory celiac disease underlies a constant inflammatory process. The epithelial barrier has not been addressed in this condition so far. Herein, the tight junction-associated barrier in refractory celiac disease is investigated functionally and structurally. Although normally expressed in celiac disease, claudin-4 is shown to be downregulated in refractory cases, presumably by two mechanisms, reduced protein expression and increased claudin endocytosis. Furthermore, the tightening claudin-5 is downregulated and the pore-forming claudin-2 is upregulated. © 2012 New York Academy of Sciences.


Bruno R.M.,Institute of Clinical Physiology | Faconti L.,University of Pisa | Taddei S.,University of Pisa | Ghiadoni L.,University of Pisa
Current Opinion in Cardiology | Year: 2015

Purpose of review To provide an overview of available evidence of the relationship between birth weight and future hypertension development. Recent findings Fetal programming plays a significant role in future hypertension. Both low and high birth weight are able to influence weight gain during childhood, adult weight and blood pressure values during childhood and adulthood. To date, an increasing amount of evidence is available especially for the relationship between low birth weight and hypertension, supported also by pathophysiological studies. Summary In the era of personalized medicine, the possibility to reduce cardiovascular risk before or soon after birth and intervene on risk factors during childhood is appealing and promising for the future. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.


Schumann M.,Charité - Medical University of Berlin | Gunzel D.,Institute of Clinical Physiology | Buergel N.,Charité - Medical University of Berlin | Richter J.F.,Institute of Clinical Physiology | And 11 more authors.
Gut | Year: 2012

Background: Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. Objective: To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. Methods: Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. Results: A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesionsdfor example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. Conclusions: Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.


Hering N.A.,Campus Benjamin Franklin | Hering N.A.,Charité - Medical University of Berlin | Andres S.,Campus Benjamin Franklin | Fromm A.,Institute of Clinical Physiology | And 6 more authors.
Journal of Nutrition | Year: 2011

TGFb (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFβ-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFβ in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFβ1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFβ1-3 antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFβ. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFβ1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition. © 2011 American Society for Nutrition.


Kirschner N.,University of Hamburg | Rosenthal R.,Institute of Clinical Physiology | Gunzel D.,Institute of Clinical Physiology | Moll I.,University of Hamburg | Brandner J.M.,University of Hamburg
Experimental Dermatology | Year: 2012

Skin barrier function is indispensable to prevent the uncontrolled loss of water and solutes and to protect the body from external assaults. To fulfil this function, keratinocytes undergo a complex pathway of differentiation that terminates in the formation of the stratum corneum. Additionally, tight junctions (TJs), which are cell-cell junctions localized in the stratum granulosum, are involved in the barrier function of the skin. Important biological and clinical roles of TJs are strongly suggested by altered TJ protein levels and distribution in skin diseases like psoriasis, ichthyosis and atopic dermatitis. Because these skin diseases show alterations in differentiation and TJs, it was suggested that changes in TJs might simply be a consequence of altered differentiation. However, in this viewpoint, we like to argue that the situation is not as simple and depends on the specific microenvironment. We discuss three hypotheses regarding the interplay between TJs/TJ proteins and differentiation: (1) TJs/TJ proteins are influenced by differentiation, (2) differentiation is influenced by TJs/TJ proteins, and (3) TJs/TJ proteins and differentiation are independent of each other. In addition, the concept is introduced that both processes are going on at the same time, which means that while one specific TJ protein/barrier component might be influenced by differentiation, the other may influence differentiation. © 2011 John Wiley & Sons A/S.


Georgi M.I.,Free University of Berlin | Rosendahl J.,Free University of Berlin | Ernst F.,Free University of Berlin | Gunzel D.,Institute of Clinical Physiology | And 3 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2014

It has long been established that the absorption of short-chain fatty acids (SCFA) across epithelia stimulates sodium proton exchange. The apically released protons are not available as countercations for the basolateral efflux of SCFA anions and a suitable transport model is lacking. Patch clamp and microelectrode techniques were used to characterize an anion conductance expressed by cultured cells of the sheep and bovine rumen and the sheep omasum and to localize the conductance in the intact tissue. Cells were filled with a Na-gluconate solution and superfused with sodium salts of acetate, propionate, butyrate, or lactate. Reversal potential rose and whole cell current at +100 mV decreased with the size of the anion. Anion-induced currents could be blocked by diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS), NPPB (200μmol l-1), or pCMB (1 mmol l-1). In patches of bovine ruminal cells, single channels were observed with a conductance for chloride (327±11 pS), acetate (115±8 pS), propionate (102±10 pS), butyrate (81±2 pS), and gluconate (44±3 pS). Channels expressed by sheep rumen and omasum were similar. Microelectrode experiments suggest basolateral localization. In conclusion, forestomach epithelia express basolateral maxi-anion channels with a permeability sequence of chloride>acetate>propionate>butyrate. SCFA absorption may resemble functionally coupled transport of NaCl, with the Na+/K +-ATPase driving the basolateral efflux of the anion through a channel. Since protons are apically extruded, the model accurately predicts that influx of buffers with saliva is essential for the pH homeostasis of the ruminant forestomach. © 2013 Springer-Verlag.


Juric M.,Hannover Medical School | Xiao F.,Tongji Hospital | Amasheh S.,Institute of Clinical Physiology | May O.,Hannover Medical School | And 5 more authors.
Inflammatory Bowel Diseases | Year: 2013

Background: Bicarbonate loss into the lumen occurs during intestinal inflammation in different species. However, candidate pathways like CFTR or DRA are inhibited in the inflamed gut. This study addressed the question whether and how inflammation-associated increased intestinal permeability may result in epithelial HCO- 3 loss. Methods: Murine proximal colon was studied because it does not express functional DRA but is inflamed in the tumor necrosis factor a overexpressing mouse model (TNFδARE). Luminal alkalization, 3H-mannitol fluxes, impedance spectroscopy, and dilution potentials were measured in Ussing chambers, whereas expression and localization of tight junction-associated proteins were analyzed by Western blots and immunohistochemistry. Results: Luminal alkalization rates and 3H-mannitol fluxes were increased in TNF+/δARE proximal colon, whereas forskolin-stimulated Isc was not altered. Epithelial resistance was reduced, but subepithelial resistance increased. The epithelial lining was intact, and enterocyte apoptosis rate was not increased despite massively increased Th1 cytokine levels and lymphoplasmacellular infiltration. Measurement of dilution potentials suggested a loss of cation selectivity with increased anion permeability. Western analysis revealed a downregulation of occludin expression and an upregulation of both claudin-2 and claudin-5, with no change in ZO-1, E-cadherin, claudin-4, and claudin-8. Immunohistochemistry suggested correct occludin localization but reduced tight junction density in TNF +/δARE surface epithelium. Conclusions: Inflammation during TNF-α overexpression leads to increased epithelial permeability in murine proximal colon, decreased tight junctional cation selectivity, and increased HCO- 3 loss into the lumen. Inflammation-associated colonic HCO- 3loss may occur through leaky tight junctions rather than through HCO23 secreting ion transporters. Copyright © 2013 Crohn's & Colitis Foundation of America, Inc.

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