Henrik Köhler

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram‐negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype‐specific mechanisms, which targets not only expression, but also cellular distri‐bution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction‐associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin‐1 from Triton X‐insoluble protein fractions upon apical exposure to T‐84 cell monolayers. S. flexneri serotype 2a and 5, but not the non‐invasive Escherichia coli strain F‐18, share the ability to regulate expression of ZO‐1, ZO‐2, E‐cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat‐stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction‐associated proteins.

Bacterial-enterocyte crosstalk: cellular mechanisms in health and disease.

The human colon maintains a microbial density approaching 10 organisms per gram of feces, representing a perfectly balanced ecosystem. The commensal microflora, derived from the Latin “commensalis” meaning “at table together”, consists of more than 400 species and lives in perfect harmony with the human intestine (1). Recently the term probiotics was defined by Fuller (2) as a “live microbial food supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Commercial probiotics have been used extensively in the prevention and treatment of human conditions from diarrhea of the infant to inflammatory bowel disease (3). However, the mechanisms by which these intestinal bacteria prevent an inflammatory response in reaction to the plethora of foreign antigens available in the intestinal lumen have just begun to come under investigation. In contrast to the myriad of indigenous microorganisms in symbiosis it takes just 10 to 100 single organisms of the pathogen Shigella to destroy this peaceful coexistence and result in bloody mucoid diarrhea associated with fever, nausea, and abdominal cramps. This infectious disease still costs more than half a million people their lives every year and most of them are children under 5 years of age. Even in a developed country such as the U.S.A., bacterial food-borne pathogens may cause an estimated 4.1 million illnesses, 45,000 hospitalizations, and 1,500 deaths every year (4). This continuing health threat has resulted in the development of the Foodborne Diseases Active Surveillance Network (Foodnet) by the Centers for Disease Control and Prevention (CDC). To maintain health and cause disease, which are both active processes, bacteria and the intestinal epithelium must actively interact. This interaction is referred to as “crosstalk” and this research area has become one of the most active in gastroenterology today. Commensal microflora together with enterocytes are hypothesized to act as a gatekeeper to protect the human organism from penetration and disease. On the other hand, bacterial pathogens themselves have developed in their two and a half billion years on earth (5) sophisticated mechanisms, like the type III secretion system, to interact with host cells to promote their own survival. An understanding of the molecular mechanisms by which these microorganisms interact with the intestinal epithelium to promote health or cause disease is expanding rapidly. It is becoming more and more obvious that prokaryotic organisms manipulate the human enterocyte functions in many ways for their own benefit. In contrast commensal microflora and probiotics can provide benefits to the host beyond a local effect in the intestine. These organisms use similar mechanisms to that of the pathogen but these mechanisms are largely undefined because of a lack of sufficient basic research. The interaction of the bacterium with the intestinal epithelial cell (IEC) has, somewhat artificially, been divided into the following categories: i) attachment and invasion, ii) altered epithelial barrier function, and iii) inflammation. All these phenomena are constantly both activated and suppressed at the same time in the human gut by different microorganisms in a highly sophisticated interaction with IEC. In this review we will not comprehensively cover these topics but will rather highlight some of the more recent findings to illustrate the cellular Received October 21, 2002; accepted November 21, 2002. Address correspondence to W. Allan Walker, Mucosal Immunology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 114 16 Street (114-3503), Charlestown, MA 02129, U.S.A. (e-mail: wwalker@partners.org). This work was supported by National Institutes of Health grants (RO1-HD31852, R37-HD12437; P30-DK40561, PO1-DK33506) and by the Deutsche Forschungsgemeinschaft (German Research Foundation). Journal of Pediatric Gastroenterology and Nutrition 36:175–185

Shigella flexneri Interactions with the Basolateral Membrane Domain of Polarized Model Intestinal Epithelium: Role of Lipopolysaccharide in Cell Invasion and in Activation of the Mitogen-Activated Protein Kinase ERK.

ABSTRACT An early step governing Shigella flexneri pathogenesis is the invasion of the colonic epithelium from the basolateral surface followed by disruption of the colonic epithelial barrier. Despite recent insight into S. flexneri-host interactions, much remains to be determined regarding the nature of the initial contact between S. flexneri and the host epithelial basolateral membrane domain. Since the lipopolysaccharide (LPS) is located at the outermost part of the bacterial membrane, we considered that this component might be used by S. flexneri to attach to the basolateral surface of the intestinal epithelium and promote a proinflammatory response. Therefore, polarized human T84 intestinal epithelial cells were infected from the basolateral surface with either wild-type S. flexneri or one of its isogenic LPS-defective strains with mutations in either rfc, rfaL, or galU. We found that both adherence to and internalization into the basolateral surface of a polarized intestinal epithelium with S. flexneri were highly dependent on the length of the LPS (i.e., rfc > rfaL > galU). Furthermore, the addition of the anti-inflammatory LPS (RsDPLA) considerably decreased the invasion profile of wild-type S. flexneri by nearly 50%. Since LPS is associated with host inflammation, we further examined whether this molecule was involved in Shigella-induced inflammatory events. We found that S. flexneri LPS plays an important role in mediating epithelial-derived signaling, which leads to directed migration of polymorphonuclear leukocytes across model intestinal epithelium. This signaling most likely involves the activation of the mitogen-activated protein kinase extracellular regulated kinase. Thus, our findings have important implications on the understanding of the mechanisms by which S. flexneri can elicit mucosal inflammation.

Inhibition of Salmonella typhimurium Enteropathogenicity by Piperidine, a Metabolite of the Polyamine Cadaverine

Piperidine is a 1-ring heterocyclic compound formed from the polyamine cadaverine in the human intestine. Because heterocyclic compounds are routinely used in the promotion of antimicrobial treatment strategies, it was considered whether piperidine could be used against infection with enteric pathogens. This study demonstrates that piperidine treatment prevented the invasion of Salmonella typhimurium into model intestinal epithelium by nearly 95%. In vivo studies also revealed that it increased mouse survival and reduced S. typhimurium translocation into and colonization of various organs and tissues. Initial evaluations demonstrated that piperidine reduced the S. typhimurium-induced polymorphonuclear leukocyte transepithelial migration response in vitro by inhibiting activation of protein kinase C. Piperidine did not affect the ability of S. typhimurium to elicit interleukin-8 secretion by epithelial cells or to activate extracellular-regulated kinase signal transduction pathways. These results show that piperidine does not exhibit paninhibitory activity and suggest that piperidine may be useful in down-regulating active inflammation at mucosal surfaces.

Inter- and intraobserver agreement in 24-hour combined multiple intraluminal impedance and pH measurement in children.

Objectives: Assessment of intra- and interobserver agreement in multiple intraluminal impedance (MII) measurement between investigators from different institutions. Methods: Twenty-four 18- to 24-hour MII tracings were randomly chosen from 4 different institutions (6 per center). Software-aided automatic analysis was performed. Each result was validated by 2 independent investigators from the 4 different centers (4 investigator combinations). For intraobserver agreement, 6 measurements were analyzed twice by the same investigator. Agreement between investigators was calculated using the Cohen kappa coefficient. Results: Interobserver agreement: 13 measurements showed a perfect agreement (kappa >0.8); 9 had a substantial (kappa 0.61–0.8), 1 a moderate (kappa coefficient 0.41 to 0.6), and 1 a fair agreement (kappa coefficient 0.11–0.4). Median kappa value was 0.83. Intraobserver agreement: 5 tracings showed perfect and 1 showed a substantial agreement. The median kappa value was 0.88. Conclusions: Most measurements showed substantial to perfect intra- and interobserver agreement. Still, we found a few outliers presumably caused by poorer signal quality in some tracings rather than being observer dependent. An improvement of analysis results may be achieved by using a standard analysis protocol, a standardized method for judging tracing quality, better training options for method users, and more interaction between investigators from different institutions.

Influence of Gestational Age, Cesarean Section, and Type of Feeding on Fecal Human β-Defensin 2 and Tumor Necrosis Factor-α

Objective: Development of the mucosal immune system is essential for controlling antigenic response. External factors are known to influence the immune system, such as breast-feeding or the mode of delivery. The aim of the present study was to investigate maturation of the enteric immune system. Patients and Methods: In stool samples of 59 preterm and term-born infants we measured the concentration of human β-defensin 2 (HBD 2), an endogenous antimicrobial peptide, and tumor necrosis factor-α (TNF-α), a cytokine playing a central role in mucosal inflammation, by enzyme-linked immunosorbent assay. Results: Mode of delivery as well as nutrition (breast-feeding or formula) had no influence on the fecal concentration of HBD-2 or TNF-α, but there was a significant increase in the concentration of HBD-2 in correlation with gestational age. TNF-α showed no change in concentration. Conclusions: Low fecal HBD-2 may be a risk factor in preterm infants to develop neonatal enteric disease, such as necrotizing enterocolitis.

Superoxide Anion Generation in Human Milk Macrophages: Opsonin-Dependent Versus Opsonin-Independent Stimulation Compared with Blood Monocytes

Macrophages are believed to play an important role within the immunoprotective effects of human breast milk. It was the purpose of this study to evaluate the capability of human milk macrophages (MMΦ) to generate superoxide anions (O2-) in comparison with peripheral blood monocytes (BMo) after stimulation with opsonized and unopsonized zymosan. Potential inhibitors of attachment and phagocytosis such as mannose and cytochalasin B were used. Expression of the mannose receptor on MMΦ was demonstrated by staining with MAb. BMo generated more O2- than MMΦ (417 ± 79 versus 216 ± 15 nmol O2-/mg protein, p < 0.05) after stimulation with opsonized zymosan. When unopsonized zymosan was used as a serum-independent stimulus, BMo generated slightly less O2- in comparison with MMΦ (150 ± 34 versus 176 ± 18 nmol O2-/mg protein, p < 0.05). These findings imply a higher proportion of opsonin-independent phagocytosis in MMΦ than in BMo (82 versus 36%). Preincubation with mannose resulted in a significantly higher reduction of O2- generation in MMΦ compared with BMo stimulated with opsonized zymosan, whereas no difference was found when unopsonized zymosan was used. After addition of cytochalasin B, equal inhibition of O2- generation was observed regardless of the cell type or stimulus used. Thus, MMΦ are stimulated to a greater extent by serum-independent mechanisms than BMo. As opsonins like complement or IgG are rare in the colostrum and the neonatal intestinal environment, such a differentiation toward serum-independent phagocytic abilities could play an important role for protective functions of human MMΦ. Possible involvement of the mannose receptor and the β-glucan receptor in this specialization are discussed.

Aplastic Anemia Following Hepatitis Associated With Human Herpesvirus 6

JPGN Volume 51, N ant of aplastic anemi ths after an acute epis H recognized vari a (AA). AA typically develops within 3 mon ode of hepatitis and is reported to occur at a rate of 2% to 5% in pediatric acute hepatitis (1,2). The median survival of untreated patients with AA is 3 to 6 months (3); hence, immediate treatment including allogeneic bone marrow transplantation or immunosuppressive therapy, if there is no human leukocyte antigen-identical sibling donor, is indicated (4–6). Although the pathophysiology of HAA is believed to be immune mediated, the causative agent associated with the syndrome remains unknown (1). Although viral infections have been discussed, none of the known hepatitis viruses has been convincingly associated with HAA (7). We report the case of a 3-year-old boy who developed AA 10 weeks after the onset of acute hepatitis associated with human herpesvirus 6 (HHV-6).

Erkrankungen des oberen Gastrointestinaltrakts

Die Häufigkeit der verschiedenen Formen der Ösophagusatresie in der Durchschnittsbevölkerung liegt bei etwa einer Atresie auf 4000 Lebendgeburten. Meist tritt die Ösophagusatresie sporadisch auf, aber auch familiäre Häufungen sind beschrieben. Vorläufige genetische Studien haben einen Defekt auf dem Arm des Chromosoms 2p23-p24 beschrieben.