SummaryAPS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

Spores from a number of different Bacillus species are currently being used as human and animal probiotics, although their mechanisms of action remain poorly understood. Here we describe the isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. Thirty-one representative isolates were characterized according to their morphological, physiological, and biochemical properties as well as partial 16S rRNA gene sequences and screening for the presence of plasmid DNA. The Bacillus species identified included B. subtilis, B. pumilus, B. licheniformis, B. clausii, B. megaterium, B. firmus, and species of the B. cereus group, whereas a number of our isolates could not be classified. Intrinsic properties of potential importance for survival in the gut that could be advantageous for spore-forming probiotics were further investigated for seven isolates belonging to five different species. All isolates sporulated efficiently in the laboratory, and the resulting spores were tolerant to simulated gastrointestinal tract conditions. They also exhibited antimicrobial activity against a broad spectrum of bacteria, including food spoilage and pathogenic organisms such as Bacillus spp., Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes. Importantly, the isolates were susceptible to most of the antibiotics tested, arguing that they would not act as donors for resistance determinants if introduced in the form of probiotic preparations. Together, our results suggest that some of the sporeformers isolated in this study have the potential to persist in or transiently associate with the complex gut ecosystem.The widespread use of antibiotics as therapeutic and prophylactic agents and as growth promoters in animal husbandry has led to a worldwide increase in antibiotic resistance and the emergence of untreatable multidrug-resistant strains of bacteria (4, 33). This has prompted the European Union to phase out the use of these compounds as animal feed additives by 2006 (47). The use of competitive exclusion agents and probiotic feed additives in the livestock industry is therefore attracting increased attention as a cost-effective alternative to controlling animal disease and improving breeding performance (54).The genus Bacillus comprises a diverse collection of aerobic endospore-forming bacteria whose spores consist of several protective layers surrounding the nucleoid in the spore core (20, 51). This structural organization makes the spores extremely resistant to external physical and chemical insults and in part determines their exceptional longevity in the environment (20, 38). The resilient and ubiquitous nature of these bacteria results in considerable daily intake of these organisms by humans and animals. This ingestion, which is often in the form of spores, occurs primarily through contaminated food and water.Bacillus spores are being used as human and animal probiotics...

SummaryThe ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB , acrD , acrF and tolC , to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro . The tolC and acrB knockout mutants were hyper-susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages . In vivo , both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB-TolC system is important for the colonization of chickens by S . Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.

Salmonella enterica isolates (n ‫؍‬ 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.The four most common nontyphoidal Salmonella enterica serovars isolated from humans in England and Wales in 1999 were Enteritidis (57%), Typhimurium (18%), Virchow, and Hadar (14). In the last decade, the numbers of isolates of these serovars with decreased susceptibility to ciprofloxacin (MIC, Ն1 g/ml) have increased (12). In 2000, 52% of the S. enterica serovar Virchow isolates tested exhibited decreased susceptibility to ciprofloxacin (12). It is postulated that many of these isolates have arisen in animals after fluoroquinolone exposure and have been transferred to humans via the food chain (11).In salmonellae, where DNA gyrase is the primary target of quinolone action, a single point mutation in the quinolone resistance-determining region (QRDR) of gyrA can mediate resistance to the nonfluorinated quinolone nalidixic acid and reduced susceptibility to fluoroquinolones such as ciprofloxacin, e.g., an MIC of 0.25 g/ml (10). Mutations in the gyrB and topoisomerase IV genes parC and parE are considered rare in salmonellae (6,7,9,11). It was hypothesized that those isolates with decreased susceptibility harbored a single mutation in gyrA, whereas resistant isolates would contain two or more mutations in gyrA and/or gyrB and/or parC and/or parE.S. enterica isolates (n ϭ 182; 156 from animals, 18 from humans, and 18 from the environment) representative of the 25 serotypes typically isolated between 1997 and 2000 were obtained from the Enteric Bacteria Reference Laboratory of the Veterinary Laboratories Agency. Because of the recent debate about the breakpoint concentration of ciprofloxacin (1,8), isolates resistant to Ն0.12 g of ciprofloxacin per ml were investigated. Isolates were serotyped by a microagglutination method (13) and, where appropriate, were phage typed (15). All isolates were from geographically and temporally distinct samples. Isolates were also analyzed by pulsed-field gel electrophoresis, which confirmed that no clones were examined.

αβ T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1 + dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1 + dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.γδ | T-cell receptor | skin immunology | thymus | development