Johannes Grossmann

Immunomodulatory consequences of oral administration of Lactobacillus rhamnosus strain GG in healthy volunteers

Probiotic microorganisms, especially lactic acid bacteria, are effective in the treatment of infectious diarrhoeal diseases and experimental colitis. Although the mechanisms by which these organisms exert their anti-inflammatory effects are largely unknown, immunomodulating effects are suggested. The objective of this study was to examine the effect of a 5-week oral administration of Lactobacillus rhamnosus subspecies GG (Lb. GG) on the cellular immune response to intestinal microorganisms in ten healthy volunteers. Peripheral blood cells (PB) were stimulated with either self or non-self preparations of faecal samples and isolated Bacteroides fragilis group-organisms (Bfg) or Escherichia coli (Esch. coli), and pro- and anti-inflammatory cytokines (IL-10, IL-4, IL-6, IFN-gamma, TNF-alpha) were measured in the culture supernatant. CD4+ T-lymphocyte activation was determined by measurement of intracellular ATP following lysis of the cells. The activational response of CD4+ T-lymphocytes towards isolated and heat-inactivated intestinal organisms was increased after the probiotic treatment. Additionally, TNF-alpha, IL-6 and in part IFN-gamma cytokine secretion by PB cells following stimulation with whole stool preparations and single members of the flora was significantly decreased, whereas the IL-10 and in part IL-4 cytokine secretion was increased at the end of the study. In contrast, the activational response of CD4+ T-lymphocytes following stimulation with whole non-self intestinal flora was higher than by self intestinal flora, but both responses showed a trend towards a reduction at the end of the study. This study documents a direct effect by Lb. GG on the cellular immune system of healthy volunteers and offers a promising tool to investigate systemic immunomodulation due to oral administration of probiotic microorganisms.

Induction of apoptosis before shedding of human intestinal epithelial cells

OBJECTIVES:Human intestinal epithelial cells (IECs) derive from stem cells at the crypt base and migrate along the so-called crypt-villus axis toward the intestinal lumen. As they reach the luminal surface in the colon or the villus tip in the small intestine, IECs are shed and their life cycle is terminated. The role of apoptosis during IEC migration along the crypt-villus axis has been subject to studies with conflicting results. In this study we use a novel approach to identify the initiation of apoptosis within normal human IECs.METHODS:Normal mucosa from the large and small human intestine was analyzed employing a novel antibody directed against activated caspase-3—an early marker of apoptosis.RESULTS:IECs initiate the apoptotic cascade as they approach the area of shedding before displaying evident morphological features of apoptosis. IECs of the small bowel also show caspase-3 activation in the small intestinal crypt base, whereas IECs of the colononic crypt base rarely show evidence of ongoing apoptosis.CONCLUSIONS:These findings indicate that apoptosis is initiated in human IECs as they reach the luminal surface/villus tip and before shedding. Furthermore, they show that different sections of the intestinal tract vary significantly in the rate of IEC apoptosis, possibly reflecting their difference in susceptibility to epithelial cell neoplasia.

Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC)

Intestinal epithelial cells (IEC) form the largest surface of the human body and are of pivotal importance to digest and absorb nutrients. Furthermore these cells play a critical role shielding the organism against microorganisms and toxins present in the intestinal lumen. It is therefore not surprising that a large group of researchers take great interest in the study of these cells. However, to date it is a challenge to purify viable primary human intestinal epithelial cells and it has been even more fastidious to maintain IEC in culture ex-vivo as IEC undergo apoptosis within hours due to loss of cell anchorage (anoikis) following the isolation process. Over recent years the authors aimed to continuously improve the isolation technique for primary IEC, allowing a simple, effective and rapid isolation of highly purified non-apoptotic human IEC. In this study the newly improved method is presented and applied to establish ex-vivo cultures of highly purified, fully viable primary IEC displaying important functional properties, making these cells amenable for ex-vivo research on primary human intestinal epithelial cells.

Comparison of interobserver agreement for different scoring systems for reflux esophagitis: Impact of level of experience.

BACKGROUNDnThe Savary-Miller, the Los Angeles, and the MUSE (metaplasia, ulcer, stricture, erosion) scoring systems have been developed to assess esophageal lesions related to GERD. Interobserver agreement for these systems was compared, with particular reference to the experience of the endoscopist.nnnMETHODSnBy using videoendoscopes, videotapes were made of the gastroesophageal junction of 60 patients who presented with symptoms suggestive of GERD. The Savary-Miller, the Los Angeles, and the MUSE systems were used to score all video clips by 9 endoscopists who were subgrouped by level of experience (3 levels, 3 endoscopists per level). Agreement was assessed by using weighted kappa statistics (kappa).nnnRESULTSnThe Savary-Miller scoring system revealed moderate agreement for the experienced group (kappa=0.41) but performed poorly when applied by inexperienced raters (kappa=0.16). The Los Angeles system was most reproducible in all subgroups, irrespective of the level of experience (kappa=0.49 to 0.65). The MUSE scoring system was highly similar to the Los Angeles scoring system with respect to erosions and, in addition, allowed assessment of complications of GERD.nnnCONCLUSIONSnThe Los Angeles and the MUSE scoring systems are most reliable for the assessment of erosions caused by GERD. Because of low reliability, use of the Savary-Miller scoring system is not recommended. For all scoring systems, interobserver agreement varies with the level of experience in the performance of upper endoscopy.

TNF-α induces apoptosis of parietal cells

Helicobacter pylori infection can be associated with chronic gastric inflammation and hypochlorhydria with increased levels of the proinflammatory cytokines. The current study investigated the effects of TNF-alpha on programmed death of gastric parietal cells. TNF-alpha induced apoptosis of parietal cells in isolated perfused rat stomachs at 10ng/mL. In isolated and highly enriched rat parietal cells, 10ng/mL TNF-alpha induced a 2.6-fold increase in the apoptotic rate. The 55kDa protein of TNFR-1 but not the 75kDa of TNFR-2 was detected by Western blot analysis. TNF-alpha-induced apoptosis of isolated parietal cells was inhibited by pretreatment with different NF-kappaB-inhibitors, nitric oxide synthase inhibitors and with antisense-oligodeoxynucleotides against the p65 subunit of NF-kappaB. Investigation of downstream signaling pathways of apoptosis revealed that TNF-alpha induced the expression of iNOS, but failed to stimulate the activity of caspase 3. The TNF-alpha effect on gastric parietal cells may contribute to the atrophy and hypochlorhydria of the gastric mucosa observed during chronic H. pylori infection.