New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

Background  

Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H₂O₂ from 0 to 130 ppm.     

Egg capsules, eggs and embryos of the southwestern Atlantic gastropod Coronium coronatum (Gastropoda: Muricidae)

The egg capsules, eggs and embryos of the muricid gastropodCoronium coronatum are described for the first time. Capsulesare sessile, bulliform, semi-circular, with a plug in the dorsalcenter. Sutures split the capsule into two asymmetrical halves.Recently laid capsules showed the presence of 3639 (n = 2) uncleavednurse eggs with a diameter of 180–210 µm (mean= 197.4 ± 8.9). The number of early embryos was 9–11.The size of the embryos was 320 x 320 to 820–880 µm.Nine pre-hatching embryos of 3.94 mm (n = 8, SD = 0.32)were found inside the older capsule. SEM illustrations of embryosand radulae are provided. Comparison of shell and radula ofembryos with the protoconch and radulae of adults of C. coronatumrevealed that the capsule belongs to this species. (Received 18 March 2006; accepted 10 October 2006)     

SYBR green real-time PCR method to detect Clostridium botulinum type A

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).     

Central memory Vgamma9Vdelta2 T lymphocytes primed and expanded by bacillus Calmette-Guérin-infected dendritic cells kill mycobacterial-infected monocytes

In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), is a feature of cells as dendritic cells (DC) and gammadelta T cells. In this study, we show that BCG infection of human monocyte-derived DC induces a rapid activation of Vgamma9Vdelta2 T cells (the major subset of gammadelta T cell pool in human peripheral blood). Indeed, in the presence of BCG-infected DC, Vgamma9Vdelta2 T cells increase both their expression of CD69 and CD25 and the production of TNF-alpha and IFN-gamma, in contrast to DC treated with Vgamma9Vdelta2 T cell-specific Ags. Without further exogenous stimuli, BCG-infected DC expand a functionally cytotoxic central memory Vgamma9Vdelta2 T cell population. This subset does not display lymph node homing receptors, but express a high amount of perforin. They are highly efficient in the killing of mycobacterial-infected primary monocytes or human monocytic THP-1 cells preserving the viability of cocultured, infected DC. This study provides further evidences about the complex relationship between important players of innate immunity and suggests an immunoregulatory role of Vgamma9Vdelta2 T cells in the control of mycobacterial infection.     

CD1-restricted recognition of exogenous and self-lipid antigens by duodenal gammadelta+ T lymphocytes

Gammadelta T cells are present in the mucosal intestinal epithelia and secrete factors necessary to maintain tissue integrity. Ags recognized by these cells are poorly defined, although in mice non-classical MHC class I molecules have been implicated. Since MHC class I-like CD1 receptors are widely expressed at the surface of epithelial and dendritic intestinal cells and have the capacity to present lipid Ags to T cells, we hypothesized that these molecules might present autologous and/or exogenous phospholipids to intestinal gammadelta T lymphocytes. Intraepithelial T lymphocytes from normal human duodenal mucosal biopsies were cloned and exposed to natural and synthetic phospholipids using CD1a-, CD1b-, CD1c- or CD1d-transfected C1R lymphoblastoid or HeLa cell lines as APCs. Their cytolytic properties and regulatory cytokine secretion were also examined. Most clones obtained from duodenal mucosa (up to 70%) were TCRalphabeta+, and either CD4+ or CD8+, whereas 20% were CD4-CD8- (6 clones) or TCRgammadelta+ (12 clones). A relevant percentage (up to 66%) of TCRgammadelta+ but few (<5%) TCRalphabeta+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [(3)H]thymidine incorporation and IL-4 release assays. A Th1-like cytolytic and functional activity along with the ability to secrete regulatory cytokines was observed in most phospholipid-specific gammadelta T cell clones. Thus, a substantial percentage of TCRgammadelta+ but few TCRalphabeta+ from human duodenal mucosa recognize exogenous phospholipids in a CD1-restricted fashion. This adaptive response could contribute to mucosal homeostasis, but could also favor the emergence of inflammatory or allergic intestinal diseases.     

Discovery of cyclopentane- and cyclohexane-trans-1,3-diamines as potent melanin-concentrating hormone receptor 1 antagonists

We herein report the optimization of cyclopentane- and cyclohexane-1,3-diamine derivatives as novel and potent MCH-R1 antagonists. Structural modifications of the 2-amino-quinoline and thiophene moieties found in the initial lead compound served to improve its metabolic stability profile and MCH-R1 affinity, and revealed unprecedented SAR when compared to other 2-amino-quinoline-containing MCH-R1 antagonists.     

Biodiversity gradients in the Alps: the overriding importance of elevation

Land abandonment is causing woodland expansion and loss of open habitats in the Alps, coupled with a shift in forestry practices from coppice management to high forest. Despite such rapid large-scale changes, there has been very little investigation of the environmental predictors of biodiversity in the Alpine landscape. We assessed the richness of amphibians, reptiles and breeding birds (n = 189 species), used as a surrogate of biodiversity, in 58 quadrats of 100 km², located within a well surveyed area of the province of Trento (central-eastern Italian Alps). The surrogates were then related to a series of environmental variables by means of stepwise multiple regression. Depending on the surrogate analysed, species richness declined linearly or quadratically with elevation, and increased with habitat heterogeneity and the availability of grassland and arid-rocky habitats. The same results were obtained when incorporating a measure of species threat into the biodiversity estimates. Different surrogates were positively inter-correlated, probably because of a common response to the same factor, namely elevation, which was the only variable to enter all models. Such elevational gradient produced a clear biodiversity peak in low-elevation areas, generating potential conflict between efficient biodiversity conservation and economic interests linked to human development, a scenario which probably applies to many mountain regions worldwide. The current network of protected areas was quite satisfactory in terms of area covered but biased towards high-elevation areas, of high scenic beauty but relatively low in animal biodiversity value. Low-elevation reserves were small and isolated. Proposed conservation targets include the establishment of corridors increasing the connectivity of low-elevation reserves and the promotion of incentives for the extensive management of grassland, an agro-ecosystem of high historical and biological value.     

Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation

T(H)17 lymphocytes appear to be essential in the pathogenesis of numerous inflammatory diseases. We demonstrate here the expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells (BBB-ECs) in multiple sclerosis lesions, and show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo. Furthermore, T(H)17 lymphocytes transmigrate efficiently across BBB-ECs, highly express granzyme B, kill human neurons and promote central nervous system inflammation through CD4+ lymphocyte recruitment.