Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor alpha, and gamma interferon but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.     

APO-1(CD95)-dependent and -independent antigen receptor-induced apoptosis in human T and B cell lines

Certain B and T cell lines respond to activation signals, e.g.through the antigen receptor, by undergoing apoptotlc cell death.In T cells it has been recently shown that TCR-mediated apoptosisinvolves APO-1/Fas (CD95) receptor-ligand interaction. To investigatewhether the TCR-CD3 complex can trigger alternative apoptosispathways we generated subclones of the T cell line Jurkat whichwere completely resistant towards APO-1-mediated apoptosis.These Jurkat^R cells differed phenotypically from sensitive parentalJurkat^S cells only by the lack of APO-1 protein expression.Although Jurkat^R cells responded normally to anti-CD3 stimulationby expression of APO-1 ligand they failed to undergo anti-CD3-inducedapoptosis. Thus, in Jurkat cells APO-1 -mediated apoptosis wasthe main, and might be the only, mechanism for anti-CD3-inducedcell death. However, BL-60 B cells, highly sensitive to anti-IgM-inducedapoptosis, did not use the APO-1 receptor-ligand system becausethey failed to express APO-1 ligand mRNA. Taken together, ourresults suggest that malignant T and B cell lines may use APO-1receptor-ligand-dependent and -independent antigen receptor-inducedapoptosis pathways respectively. Similarly, differential pathwaysmay be used by T and B cell subsets.     

The Lipopolysaccharide of Bordetella bronchiseptica Acts as a Protective Shield against Antimicrobial Peptides

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.     

Trisomy 3 Is Not a Common Feature in Malignant Lymphomas of Mucosa-Associated Lymphoid Tissue Type

The genetic background of extranodal marginal zone B-cell non-Hodgkin’s lymphoma (NHL) of mucosa-associated lymphoid tissue (MALT) type is poorly understood. In contrast to most entities of primary nodal lymphomas, few cytogenetic data are available, and gene rearrangements frequently encountered in and highly characteristic of certain entities of systemic NHL are absent in this type of lymphoma. Recently, it was suggested that MALT-type NHLs are associated with certain numerical chromosome aberrations and especially with trisomy 3. We performed an extensive study using a sensitive double (bicolor) fluorescence in situ hybridization technique for the analysis of trisomies for chromosomes 3, 7, 12, and 18 in 60 samples of low-grade and 45 high-grade MALT-type tumors. In the low-grade cases, trisomy 3 was found in a frequency of only 20%. High-grade lymphomas of MALT type were associated with trisomies 3, 7, 12, and 18 in 36, 20, 18, and 13% of the cases, respectively. Whereas no difference was encountered for trisomy 3 in primary and secondary/simultaneous high-grade lymphomas, +7 and +12 were associated with primary lymphomas, and a +18 was predominantly found in secondary/simultaneous high-grade NHL. These results challenge earlier reports describing a high frequency of +3 in low-grade MALT-type NHL and indicate a possibly different genetic evolution pattern of primary and secondary/simultaneous high-grade lymphomas of primary mucosal origin.     

A pattern search method for putative anchor residues in T cell epitopes

The binding affinity between an antigenic peptide and its particular major histocompatibility complex (MHC) molecule seems to be largely determined by only a few residues. These residues have been called “anchors” because of their property of fitting into “pockets” inside the groove of the MHC molecule. To predict natural antigenic epitopes within a longer sequence, it therefore appears to be important to know the motif or pattern describing the anchors, i.e. the anchors amino acid residue preference and the distance between anchor residues. A large set of MHC class I-restricted peptides has been described. Peptide sequences vary in length and lack an obvious common sequence motif. For a list of peptides belonging to one type of MHC class I molecule, we describe a method to find the most prominent sequence motif with at least two anchor residues. Briefly, antigenic sequences are aligned, and two anchor positions are searched for, where all anchor residues share a high similarity. The alignments are scored according to the similarity of their anchor residues. We show that the motifs predicted for the MHC alleles A2.1, B27, K^b, K^d, D^b are in substantial agreement with experimental data. We derive binding motifs for the MHC class I alleles HLA-A1, All, B8, B14, H-2L^d and for the MHC class II alleles I-A^b and I-A^s. In some cases, higher scores were obtained by allowing a slight variation in the number of residues between anchors. Therefore, we support the view that the length of epitopes belonging to a particular class I MHC is not uniform. This method can be used to predict the natural short epitope inside longer antigenic peptides and to predict the epitopes anchor residues. Anchor motifs can be used to search for antigenic regions in sequences of infectious viruses, bacteria and parasites.     

Antibiotic susceptibility of Helicobacter pylori in Germany: stable primary resistance from 1995 to 2000

The issue of antibiotic resistance in Helicobacter pylori is of particular concern and has become an important factor leading to eradication failure. This paper reports the prevalence of primary resistance to clarithromycin, amoxicillin, metronidazole and tetracycline among H. pylori isolates in the north-eastern part of Germany. A total of 1644 clinical H. pylori isolates was investigated over a period of 6 years from 1995 to 2000. The MICs were determined by the Etest. The overall rate of primary resistance was 26.2% for metronidazole and 2.2% for clarithromycin. No significant changes in the resistance rates during the period of investigation were observed. No isolate was resistant to amoxicillin or tetracycline. PCR-RFLP analysis for the detection of point mutations associated with clarithromycin resistance was performed with 36 H. pylori isolates. The A --> G transition mutation at position 2143 was detected in 19 H. pylori isolates (52.8%), whereas the mutation at position 2142 was found in 13 isolates (36.1%).     

A modified Sindbis vector for prolonged gene expression in neurons

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.     

Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants

The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA, the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA, ratB, sivI, and sivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genus SALMONELLA: Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA, sivI, and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.     

Cerebro-osseous-digital syndrome: four new cases of a lethal skeletal dysplasia--distinct from Neu-Laxova Syndrome

Neu-Laxova Syndrome (NLS) is a severe disorder with intrauterine growth retardation, edema, and characteristic face (including microcephaly with receding forehead, protuberant eyes, a flattened nose, deformed ears, cleft palate, and micrognathia). Ichthyosis is often present. Limb anomalies include hypoplastic fingers and syndactyly of fingers and toes. Patients are usually stillborn or die shortly after birth. We report five unrelated patients--four with atypical NLS and one with typical NLS. All five patients were stillbirths. Clinically, the atypical NLS patients showed a large skull; rhizo-, meso-, and acromelia; and hypoplasia of the metacarpals and phalanges. The feet were similarly affected. Radiographically, the atypical patients showed interpediculate narrowing and hypoplastic vertebral bodies. The long bones were stick-like, showing diaphyseal widening that spared the metaphyses and was more pronounced in the lower extremities. The ilia had a half-moon configuration with widening of the sacrosciatic notches. The ischia were vertical and the pubic bone was absent. The typical NLS patient showed microcephaly, normal vertebral body, and long bone ossification, but a pelvic configuration similar to that of the atypical NLS patients. The common and distinguishing clinical and radiographic features are reviewed. Scott et al. [1981: Am J Med Genet 9:165-175] described two patients with NLS with radiographic and clinical findings similar to patients 1-4 reported here. Patients 1-4 of this report lack the typical findings of NLS and likely represent a distinct lethal skeletal dysplasia.