Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).

Structured intermittent interruption of chronic HIV infection treatment with highly active antiretroviral therapy: Effects on leptin and TNF-alpha.

The changes in nutritional parameters and adipocytokines after structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection are analyzed. Twenty-seven patients with chronic HIV infection (median CD4+ T cell count/microl: nadir, 394; at the beginning of structured interruptions, 1041; HIV viral load: nadir, 41,521 copies/ml; at the beginning of structured interruptions <50 copies/ml; median time of previous treatment: 60 months) were evaluated during three cycles of intermittent interruptions of therapy (8 weeks on/4 weeks off). CD4+ T cell count, HIV viral load, anthropometric measures, and serum concentrations of triglycerides, cholesterol, leptin, and tumor necrosis factor and its soluble receptors I and II were determined. After the three cycles of intermittent interruptions of therapy, no significant differences in CD4+ T cell count/microl, viral load, or serum concentrations of cholesterol or triglycerides with reference to baseline values were found. A near-significant higher fatty mass (skinfold thicknesses, at the end, 121 mm, at the beginning, 100 mm, p = 0.100), combined with a significant increase of concentration of leptin (1.5 vs. 4.7 ng/ml, p = 0,044), as well as a decrease in serum concentrations of soluble receptors of tumor necrosis factor (TNFRI, 104 vs. 73 pg/ml, p = 0.022; TNFRII 253 vs. 195 pg/ml, p = 0.098) were detected. Structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection induces a valuable positive modification in markers of lipid turnover and adipose tissue mass.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Phylogenetic alpha and beta diversities of butterfly communities correlate with climate in the western Swiss Alps

The observation of non-random phylogenetic distribution of traits in communities provides evidence for niche-based community assembly. Environment may influence the phylogenetic structure of communities because traits determining how species respond to prevailing conditions can be phylogenetically conserved. In this study, we investigate the variation of butterfly species richness and of phylogenetic - and -diversities along temperature and plant species richness gradients. Our study indicates that butterfly richness is independently positively correlated to temperature and plant species richness in the study area. However, the variation of phylogenetic - and -diversities is only correlated to temperature. The significant phylogenetic clustering at high elevation suggests that cold temperature filters butterfly lineages, leading to communities mostly composed of closely related species adapted to those climatic conditions. These results suggest that in colder and more severe conditions at high elevations deterministic processes and not purely stochastic events drive the assemblage of butterfly communities.

Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza.

INTRODUCTION Human host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1. METHODS We profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene. RESULTS Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1beta), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-gamma) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-alpha, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients. CONCLUSIONS While infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.

Acute oxygen sensing in heme oxygenase-2 null mice.

Hemeoxygenase-2 (HO-2) is an antioxidant enzyme that can modulate recombinant maxi-K(+) channels and has been proposed to be the acute O(2) sensor in the carotid body (CB). We have tested the physiological contribution of this enzyme to O(2) sensing using HO-2 null mice. HO-2 deficiency leads to a CB phenotype characterized by organ growth and alteration in the expression of stress-dependent genes, including the maxi-K(+) channel alpha-subunit. However, sensitivity to hypoxia of CB is remarkably similar in HO-2 null animals and their control littermates. Moreover, the response to hypoxia in mouse and rat CB cells was maintained after blockade of maxi-K(+) channels with iberiotoxin. Hypoxia responsiveness of the adrenal medulla (AM) (another acutely responding O(2)-sensitive organ) was also unaltered by HO-2 deficiency. Our data suggest that redox disregulation resulting from HO-2 deficiency affects maxi-K(+) channel gene expression but it does not alter the intrinsic O(2) sensitivity of CB or AM cells. Therefore, HO-2 is not a universally used acute O(2) sensor.

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon gamma enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/10(6) mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen

Allergologie: 3. Intérêts des allergènes recombinants dans la pratique de l'allergologie [Interests of recombinant allergens in the practice of allergy].

Compared to total allergenic extracts, recombinant allergens available for specific IgE measurement represent an important advance in the diagnosis and treatment of IgE-mediated allergies. Recombinant allergens lead to define the sensitization profile of allergic patients, to identify markers of sensitization and to understand better polysensitivities related to cross-reactions and markers of severity of allergic reactions. They also contribute to the decision to establish tolerance induction (allergen specific immunotherapy) and the optimal selection of the allergenic composition of the vaccine.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Marked increase in the secretion of a fully antigenic recombinant carcinoembryonic antigen obtained by deletion of its hydrophobic tail.

Carcinoembryonic antigen (CEA) is a well-known tumor marker, consisting of a single heavily glycosylated polypeptide chain (mol. wt 200 kD), bound to the cell surface by a phosphatidylinositol-glycan anchor. The hydrophobic domain, encoded by the 3' end of the open reading frame of the CEA gene is not present in the mature protein. This domain is assumed to play an important role in the targeting and attachment of CEA to the cell surface. To verify this hypothesis, a recombinant CEA cDNA lacking the 78 b.p. of the 3' region, encoding the 26 a.a. hydrophobic domain, was prepared in a Rc/CMV expression vector containing a neomycin resistance gene. The construct was transfected by the calcium phosphate technique into CEA-negative human and rat colon carcinoma cell lines. Geneticin-resistant transfectants were screened for the presence of CEA in the supernatant and positive clones were isolated. As determined by ELISA, up to 13 micrograms of recombinant CEA per 10(6) cells was secreted within 72 hr by the human transfected cells and about 1 microgram by the rat cells. For comparison, two human carcinoma cell lines, CO112 and LS174T, selected for high CEA expression, shed about 45 and 128 ng per 10(6) cells within 72 hr, respectively. Western blot analysis showed that the size of the recombinant CEA secreted by the transfected human cells is identical to that of reference CEA purified from human colon carcinomas metastases (about 200 kD). The recombinant CEA synthesized by the transfected rat carcinoma cells has a smaller size (about 144 kD, possibly due to incomplete glycosylation), as has already been observed for CEA produced by rat colon carcinoma cells transfected with full-length CEA cDNA. The 100-fold increase in secretion of rCEA encoded by truncated CEA cDNA transfected in human cells confirms the essential role of this domain in the targeting and anchoring of the glycoprotein. These results suggest a new approach for the in vitro production of large amounts of CEA needed in research laboratories and for immunoassay kits.

Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha.

BACKGROUND The human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease. METHODS Breast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis. RESULTS Cancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha. CONCLUSION Breast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

The alpha 1a and alpha 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action.

In this chapter we summarize some aspects of the structure-functional relationship of the alpha 1a and alpha 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the alpha 1a and alpha 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Trial of recombinant follicle-stimulating hormone pretreatment for gnrh-induced fertility in patients with congenital hypogonadotropic hypogonadism.

CONTEXT AND OBJECTIVE: The optimal strategy for inducing fertility in men with congenital hypogonadotropic hypogonadism (CHH) is equivocal. Albeit a biologically plausible approach, pretreatment with recombinant FSH (rFSH) before GnRH/human chorionic gonadotropin administration has not been sufficiently assessed. The objective of the study was to test this method. DESIGN AND SETTING: This was a randomized, open-label treatment protocol at an academic medical center. PATIENTS AND INTERVENTIONS: GnRH-deficient men (CHH) with prepubertal testes (<4 mL), no cryptorchidism, and no prior gonadotropin therapy were randomly assigned to either 24 months of pulsatile GnRH therapy alone (inducing endogenous LH and FSH release) or 4 months of rFSH pretreatment followed by 24 months of GnRH therapy. Patients underwent serial testicular biopsies, ultrasound assessments of testicular volume, serum hormone measurements, and seminal fluid analyses. RESULTS: rFSH treatment increased inhibin B levels into the normal range (from 29 ± 9 to 107 ± 41 pg/mL, P < .05) and doubled testicular volume (from 1.1 ± 0.2 to 2.2 ± 0.3 mL, P < .005). Histological analysis showed proliferation of both Sertoli cells (SCs) and spermatogonia, a decreased SC to germ cell ratio (from 0.74 to 0.35), and SC cytoskeletal rearrangements. With pulsatile GnRH, the groups had similar hormonal responses and exhibited significant testicular growth. All men receiving rFSH pretreatment developed sperm in their ejaculate (7 of 7 vs 4 of 6 in the GnRH-only group) and showed trends toward higher maximal sperm counts. CONCLUSIONS: rFSH pretreatment followed by GnRH is successful in inducing testicular growth and fertility in men with CHH with prepubertal testes. rFSH not only appears to maximize the SC population but also induces morphologic changes, suggesting broader developmental roles.