Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Cleusonite, (Pb,Sr)(U4+,U6+)(Fe2+ Zn)2(Ti,Fe2+ Fe3+)18(O,OH)38, a new mineral species of the crichtonite group from the western Swiss Alps

Cleusonite, (Pb,Sr)(U4+,U6+) (Fe2+,Zn)(2) (Ti,Fe2+,Fe3+)(18) (O,OH)(38), is a new member of the crichtonite group. It was found at two occurrences in greenschist facies metamorphosed gneissic series of the Mont Fort and Siviez-Mischabel Nappes in Valais, Switzerland (Cleuson and Bella Tolla summit), and named after the type locality. It occurs as black opaque cm-sized tabular crystals with a bright sub-metallic lustre. The crystals consist of multiple rhombohedra and hexagonal prisms that are generally twinned. Measured density is 4.74(4) g/cm(3) and can be corrected to 4.93(12) g/cm(3) for macroscopic swelling due to radiation damage; the calculated density varies from 5.02(6) (untreated) to 5.27(5) (heat-treated crystals); the difference is related to the cell swelling due to the metamictisation. The empirical formula for cleusonite from Cleuson is (Pb0.89Sr0.12)(Sigma=1.01) (U0.79+4U0.30+6)(Sigma=1.09) (Fe1.91+2Zn0.09)(Sigma=2.00) (Ti11.80Fe3.44+2Fe2.33+3V0.19+5Mn0.08Al0.07)(Sigma=17.90) [O-35.37(OH)(2.63)](Sigma=38). Cations were measured by electron microprobe, the presence of structural (OH) was confirmed by infrared spectroscopy and the U6+/U4+ and Fe2+/Fe3+ ratios were determined by X-ray photoelectron spectroscopy. Cleusonite is partly metamict, and untreated crystals only show three major X-ray diffraction peaks. Because of this radiation-damaged state, the mineral appears optically isotropic and shows a light-grey to white colour in reflected polarized light. Cleusonite is trigonal, space group R $(3) over bar $, and unit-cell parameters are varying from a = 10.576(3), c = 21.325(5) angstrom (untreated crystal) to a = 10.4188(6), c = 20.942(1) angstrom (800 degrees C treatment) and to a = 10.385(2), c = 20.900(7) angstrom (1000 degrees C treatment). The three cells give a common axial ratio 2.01 (1), which is identical to the measured morphological one 2.04(6). ne name cleusonite also applies to the previously described ``uranium-rich senaite'' from Alinci (Macedonia) and the ``plumbodavidite'' from Huanglongpu (China).

In vitro neuroendocrine effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the AhR-expressing hypothalamic rat GnV-3 cell line.

The aryl hydrocarbon receptor (AhR) is involved in a wide variety of biological and toxicological responses, including neuroendocrine signaling. Due to the complexity of neuroendocrine pathways in e.g. the hypothalamus and pituitary, there are limited in vitro models available despite the strong demand for such systems to study and predict neuroendocrine effects of chemicals. In this study, the applicability of the AhR-expressing rat hypothalamic GnV-3 cell line was investigated as a novel model to screen for neuroendocrine effects of AhR ligands using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as reference compound. The qRT-PCR analyses demonstrated the presence of several sets of neurotransmitter receptors in the GnV-3 cells. TCDD (10nM) altered neurotransmitter signaling by up-regulation of glutamate (Grik2), gamma-amino butyric acid (Gabra2) and serotonin (Ht2C) receptor mRNA levels. However, no significant changes in basal and serotonin-evoked intracellular Ca(2+) concentration ([Ca(2+)]i) or serotonin release were observed. On the other hand, TCDD de-regulated period circadian protein homolog 1 (Per1) and gonadotropin releasing hormone (Gnrh) mRNA levels within a 24-h time period. Both Per1 and Gnrh genes displayed a similar mRNA expression pattern in GnV-3 cells. Moreover, the involvement of AhR in TCDD-induced alteration of Neuropeptide Y (Npy) gene expression was found and confirmed by using siRNA targeted against Ahr in GnV-3 cells. Overall, the combined results demonstrate that GnV-3 cells may be a suitable model to predict some mechanisms of action and effects of AhR ligands in the hypothalamus.

L'autochtonie en pays de nappes: réponse à G. Fuchs

From the detai1ed examination of four key areas of Ladakh and Zanskar (NW Himalaya), the autochtonist hypothesis of the Tibetan zone is absolutely refuted. The nappes and thrusts are reality in Zanskar as well as in other areas to the N of the main Himalayan Range. The new Zangla, Zumlung and Khurna nappes are confirmed. A partir de l'examen de quatre régions clés du Ladakh et du Zanskar (NW Himalaya), la théorie de l'autochtonie de la zone tibétaine est réfutée. La tectonique de nappes est une réalité aussi bien au Zanskar que dans les autres régions au N de l'Himalaya. Les nouvelles nappes de Zangla, Zumlung et Khurna précédemment découvertes (Baud et al., 1983) son confirmées.

Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase.

The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

Detection and Characterization of Rotavirus G and P Types from Children Participating in a Rotavirus Vaccine Trial in Belém, Brazil

This study sought the characterization of rotaviruses in a trial with a tetravalent rhesus-human rotavirus vaccine in Belém, Brazil in children who received three doses of vaccine or placebo in the 1st, 3rd and 5th months of life. Rotavirus electropherotypes, subgroups, G serotypes, G, [P] and [P],G genotypes were determined in 93.3%, 95.9%, 93.3%, 73.3%, 95.5% and 92.2% of isolates, respectively. Serotypes G1, G2 and G4 were detected in 58.9%, 30% and 4.4% of the cases, respectively. Rotavirus genotype G5 was detected for the first time in Northern region in 4.4% of the infections. Rotavirus genotypes P[8], P[4], P[6] and P[8+6] were detected in 54.5%, 26.7%, 12.2%, and 2.2% of the cases, respectively. The predominant genotypes were P[8],G1 and P[4],G2 with 53% and 26.6% of the infections, respectively. Unusual strains accounted for 20.5% including P[4],G1, P[6],G1, P[6],G4, P[6],G5, P[8],G2, P[8],G5. Mixed infections involving P[8+6],G2 and P[8+6],G1 were also noted. The neonatal P[6] strains associated with diarrhea were detected among children aged 9-24 months. To our knowledge, this study represents the first in Brazil to analyse, on molecular basis, rotavirus genotypes from children participating in a rotavirus vaccine trial. These results are of potential importance regarding future rotavirus vaccination strategies in Brazil.

In Vitro and in Vivo Assays of 3,5-Disubstituted-Tetrahydro-2H-1,3,5-Thiadiazin-2-Thione Derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 µg/ml for macrophages and a few of them maintained this cytotoxicity even at 10 µg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1µg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.

Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype-phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Prevalence and genotypes of GB Virus C/Hepatitis G virus among blood donors in Central Brazil

A survey was conducted in a blood donor population of Central Brazil aiming to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection and also to analyze the virus genotypes distribution. A total of 241 voluntary blood donors were interviewed at the State Blood Bank in Goiânia, State of Goiás, Brazil. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Seventeen samples were GBV-C/HGV RNA-positive, resulting in a prevalence of 7.1% (95% CI: 4.2-11.1). A significant trend of GBV-C/HGV RNA positivity in relation to age was observed, with the highest prevalence in donors between 29-39 years old. Ten infected individuals were characterized by reporting parenteral (30%), sexual (18%), both (6%) and intrafamiliar (6%) transmission. However, 7 (40%) GBV-C/HGV RNA-positive donors did not mention any potential transmission route. RFLP analysis revealed the presence of genotypes 1 and 2 of GBV-C/HGV; more precisely, 10 (58.9%) samples were found belonging to the 2b subtype, 4 (23.5%) to the 2a subtype, and 3 (17.6%) to genotype 1. The present data indicate an intermediate endemicity of GBV-C/HGV infection among this blood donor population, and a predominant circulation of genotype 2 (subtype 2b) in Central Brazil.

High antigen concentration inhibits T cell proliferation but not interleukin 2 production: examination of limiting dilution microcultures and T cell clones.

The effect of high antigen dose on the activation of cytochrome c peptide-primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide-specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 production by these clones was not impaired regardless of the antigen concentrations used.

Universal screening and decolonization for control of MRSA in nursing homes : a cluster randomized controlled study

OBJECTIVE The risk of carrying methicillin-resistant Staphylococcus aureus (MRSA) is higher among nursing home (NH) residents than in the general population. However, control strategies are not clearly defined in this setting. In this study, we compared the impact of standard precautions either alone (control) or combined with screening of residents and decolonization of carriers (intervention) to control MRSA in NHs. DESIGN Cluster randomized controlled trial SETTING NHs of the state of Vaud, Switzerland PARTICIPANTS Of 157 total NHs in Vaud, 104 (67%) participated in the study. INTERVENTION Standard precautions were enforced in all participating NHs, and residents underwent MRSA screening at baseline and 12 months thereafter. All carriers identified in intervention NHs, either at study entry or among newly admitted residents, underwent topical decolonization combined with environmental disinfection, except in cases of MRSA infection, MRSA bacteriuria, or deep skin ulcers. RESULTS NHs were randomly allocated to a control group (51 NHs, 2,412 residents) or an intervention group (53 NHs, 2,338 residents). Characteristics of NHs and residents were similar in both groups. The mean screening rates were 86% (range, 27%-100%) in control NHs and 87% (20%-100%) in intervention NHs. Prevalence of MRSA carriage averaged 8.9% in both control NHs (range, 0%-43%) and intervention NHs (range, 0%-38%) at baseline, and this rate significantly declined to 6.6% in control NHs and to 5.8% in intervention NHs after 12 months. However, the decline did not differ between groups (P=.66). CONCLUSION Universal screening followed by decolonization of carriers did not significantly reduce the prevalence of the MRSA carriage rate at 1 year compared with standard precautions

Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains.

BACKGROUND: Two long synthetic peptides representing the dimorphic and constant C-terminal domains of the two allelic families of Plasmodium falciparum merozoite surface proteins 2 are considered promising malaria vaccine candidates. The aim of the current study is to characterize the immune response (epitope mapping) in naturally exposed individuals and relate immune responses to the risk of clinical malaria. METHODS: To optimize their construction, the fine specificity of human serum antibodies from donors of different age, sex and living in four distinct endemic regions was determined in ELISA by using overlapping 20 mer peptides covering the two domains. Immune purified antibodies were used in Western blot and immunofluorescence assay to recognize native parasite derivate proteins. RESULTS: Immunodominant epitopes were characterized, and their distribution was similar irrespective of geographic origin, age group and gender. Acquisition of a 3D7 family and constant region-specific immune response and antibody avidity maturation occur early in life while a longer period is needed for the corresponding FC27 family response. In addition, the antibody response to individual epitopes within the 3D7 family-specific region contributes to protection from malaria infection with different statistical weight. It is also illustrated that affinity-purified antibodies against the dimorphic or constant regions recognized homologous and heterologous parasites in immunofluorescence and homologous and heterologous MSP2 and other polypeptides in Western blot. CONCLUSION: Data from this current study may contribute to a development of MSP2 vaccine candidates based on conserved and dimorphic regions thus bypassing the complexity of vaccine development related to the polymorphism of full-length MSP2.

GB virus C/hepatitis G virus infection in dialysis patients and kidney transplant recipients in central Brazil

In order to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection in dialysis patients and kidney transplant recipients in Central Brazil and also to analyze the virus genotypes distribution, a total of 123 patients including 98 on hemodialysis, 13 on continuous ambulatory peritoneal dialysis treatment, and 12 who received kidney transplantation were interviewed in one unit of dialysis treatment in Goiânia city. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Eighteen samples were GBV-C/HGV RNA-positive, resulting in an overall prevalence of 14.6% (95% CI: 9.2-21.7). A high positivity for GBV-C/HGV RNA was observed in patients who had received kidney transplant (16.7%), followed by those on hemodialysis (15.3%), and peritoneal dialysis (7.7%). RFLP analysis revealed the presence of genotypes 1, 2, and 3 of GBV-C/HGV; more precisely, 9 (50%) samples were found belonging to the 2b subtype, 4 (22%) to the 2a subtype, 3 (17%) to genotype 1, and 2 (11%) to genotype 3. The present data indicate an intermediate prevalence of GBV-C/HGV infection among dialysis patients and kidney transplant recipients in Central Brazil. Genotype 2 (subtype 2b) seems to be the most prevalent GBV-C/HGV genotype in our region.

Current genetic data do not improve the prediction of type 2 diabetes mellitus: the CoLaus study.

CONTEXT: Several genetic risk scores to identify asymptomatic subjects at high risk of developing type 2 diabetes mellitus (T2DM) have been proposed, but it is unclear whether they add extra information to risk scores based on clinical and biological data. OBJECTIVE: The objective of the study was to assess the extra clinical value of genetic risk scores in predicting the occurrence of T2DM. DESIGN: This was a prospective study, with a mean follow-up time of 5 yr. SETTING AND SUBJECTS: The study included 2824 nondiabetic participants (1548 women, 52 ± 10 yr). MAIN OUTCOME MEASURE: Six genetic risk scores for T2DM were tested. Four were derived from the literature and two were created combining all (n = 24) or shared (n = 9) single-nucleotide polymorphisms of the previous scores. A previously validated clinic + biological risk score for T2DM was used as reference. RESULTS: Two hundred seven participants (7.3%) developed T2DM during follow-up. On bivariate analysis, no differences were found for all but one genetic score between nondiabetic and diabetic participants. After adjusting for the validated clinic + biological risk score, none of the genetic scores improved discrimination, as assessed by changes in the area under the receiver-operating characteristic curve (range -0.4 to -0.1%), sensitivity (-2.9 to -1.0%), specificity (0.0-0.1%), and positive (-6.6 to +0.7%) and negative (-0.2 to 0.0%) predictive values. Similarly, no improvement in T2DM risk prediction was found: net reclassification index ranging from -5.3 to -1.6% and nonsignificant (P ≥ 0.49) integrated discrimination improvement. CONCLUSIONS: In this study, adding genetic information to a previously validated clinic + biological score does not seem to improve the prediction of T2DM.