Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production.

In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5(-)PD-1(-)Bcl-6(-), CXCR5(+)PD-1(-)Bcl-6(-), CXCR5(-)PD-1(+)Bcl-6(-), and CXCR5(+)PD-1(+)Bcl-6(+). On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5(+)PD-1(+)Bcl-6(+) cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5(-)PD-1(+) cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (<1,000 HIV RNA copies). However, only the percentage of Tfh cells correlated with the levels of plasma viremia. These results demonstrate that Tfh cells serve as the major CD4 T cell compartment for HIV infection, replication, and production.

Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.

This study demonstrates that endogenously produced interferon gamma (IFN-gamma) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-gamma (i.e., IFN-gamma receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-gamma-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-gamma-insensitive p53(-/-) mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-gamma-insensitive mice, we found IFN-gamma's actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-gamma. Thus, IFN-gamma forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.

A novel Th cell epitope of Candida albicans mediates protection from fungal infection.

Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.

Virus-like particles induce robust human T-helper cell responses.

Among synthetic vaccines, virus-like particles (VLPs) are used for their ability to induce strong humoral responses. Very little is reported on VLP-based-vaccine-induced CD4(+) T-cell responses, despite the requirement of helper T cells for antibody isotype switching. Further knowledge on helper T cells is also needed for optimization of CD8(+) T-cell vaccination. Here, we analysed human CD4(+) T-cell responses to vaccination with MelQbG10, which is a Qβ-VLP covalently linked to a long peptide derived from the melanoma self-antigen Melan-A. In all analysed patients, we found strong antibody responses of mainly IgG1 and IgG3 isotypes, and concomitant Th1-biased CD4(+) T-cell responses specific for Qβ. Although less strong, comparable B- and CD4(+) T-cell responses were also found specific for the Melan-A cargo peptide. Further optimization is required to shift the response more towards the cargo peptide. Nevertheless, the data demonstrate the high potential of VLPs for inducing humoral and cellular immune responses by mounting powerful CD4(+) T-cell help.

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.

Effects of thyromimetic drugs on aldosterone-dependent sodium transport in the toad bladder.

Aldosterone increases transepithelial Na+ transport in the urinary bladder of Bufo marinus. The response is characterized by 3 distinct phases: 1) a lag period of about 60 min, ii) an initial phase (early response) of about 2 hr during which Na+ transport increases rapidly and transepithelial electrical resistance falls, and iii) a late phase (late response) of about 4 to 6 hr during which Na+ transport still increases significantly but with very little change in resistance. Triiodothyronine (T3, 6 nM) added either 2 or 18 hr before aldosterone selectively antagonizes the late response. T3 per se (up to 6 nM) has no effect on base-line Na+ transport. The antagonist activity of T3 is only apparent after a latent period of about 6 to 8 hr. It is not rapidly reversible after a 4-hr washout of the hormone. The effects appear to be selective for thyromimetic drugs since reverse T3 (rT3) is inactive and isopropyldiiodothyronine (isoT2) is more active than T3. The relative activity of these analogs corresponds to their relative affinity for T3 nuclear binding sites which we have previously described. Our data suggest that T3 might control the expression of aldosterone by regulating gene expression, e.g. by the induction of specific proteins, which in turn will inhibit the late mineralocorticoid response, without interaction with the early response.

Focal dependent pleural thickening at MDCT: pleural lesion or functional abnormality?

PURPOSE: To describe the characteristics of reversible focal pleural thickenings (PTs) mimicking real plaques, that firstly suggest asbestos exposure or pleural metastasis; to propose an imaging strategy and propose an explanation for their mechanism of formation. PATIENTS AND METHODS: Retrospective review of data from 19 patients with PTs fitting the description of pleural plaques at chest computed tomography (CT) and presenting modifications (clearance or appearance) of at least one PT at an additional chest examination in prone position. RESULTS: A total of 152 PTs were recorded on the first chest CT examinations with a range of two to 19 pleural opacities per patient. All PTs had a posterior distribution in the lower lobes. On the additional acquisitions, 144 PTs disappeared. Seventeen patients presented complete regression of PTs and two patients presented persistence of eight PTs. CONCLUSION: Additional low dose acquisition in prone position should be performed in all patients presenting with focal PT in a dependent and basal location. This may allow to exclude a pleural plaque in case of asbestos exposure but also a pleural metastasis in oncologic patients. These reversible dependent PTs could be related to physiological focal accumulation of lymphatic fluid in subpleural area.

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Inverse association between circulating vitamin D and mortality-dependent on sex and cause of death?

BACKGROUND AND AIMS: In various populations, vitamin D deficiency is associated with chronic diseases and mortality. We examined the association between concentration of circulating 25-hydroxyvitamin D [25(OH)D], a marker of vitamin D status, and all-cause as well as cause-specific mortality. METHODS AND RESULTS: The study included 3404 participants of the general adult Swiss population, who were recruited between November 1988 and June 1989 and followed-up until the end of 2008. Circulating 25(OH)D was measured by protein-bound assay. Cox proportional hazards regression was used to examine the association between 25(OH)D concentration and all-cause and cause-specific mortality adjusting for sex, age, season, diet, nationality, blood pressure, and smoking status. Per 10 ng/mL increase in 25(OH)D concentration, all-cause mortality decreased by 20% (HR = 0.83; 95% CI 0.74-0.92). 25(OH)D concentration was inversely associated with cardiovascular mortality in women (HR = 0.68, 95% CI 0.46-1.00 per 10 ng/mL increase), but not in men (HR = 0.97; 95% CI 0.77-1.23). In contrast, 25(OH)D concentration was inversely associated with cancer mortality in men (HR = 0.72, 95% CI 0.57-0.91 per 10 ng/mL increase), but not in women (HR = 1.14, 95% CI 0.93-1.39). Multivariate adjustment only slightly modified the 25(OH)D-mortality association. CONCLUSION: 25(OH)D was similarly inversely related to all-cause mortality in men and women. However, we observed opposite effects in women and men with respect to cardiovascular and cancer mortality.

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

Lentiviral vectors: a powerful tool to target astrocytes in vivo.

The morphological and functional diversity of astrocytes, and their essential contribution in physiological and pathological conditions, are starting to emerge. However, experimental systems to investigate neuron-glia interactions and develop innovative approaches for the treatment of central nervous system (CNS) disorders are still very limited. Fluorescent reporter genes have been used to visualize populations of astrocytes and produce an atlas of gene expression in the brain. Knock-down or knock-out of astrocytic proteins using transgenesis have also been developed, but these techniques remain complex and time-consuming. Viral vectors have been developed to overexpress or silence genes of interest as they can be used for both in vitro and in vivo studies in adult mammalian species. In most cases, high transduction efficiency and long-term transgene expression are observed in neurons but there is limited expression in astrocytes. Several strategies have been developed to shift the tropism of lentiviral vectors (LV) and allow local and controlled gene expression in glial cells. In this review, we describe how modifications of the interaction between the LV envelope glycoprotein and the surface receptor molecules on target cells, or the integration of cell-specific promoters and miRNA post-transcriptional regulatory elements have been used to selectively express transgenes in astrocytes.

Parent perceived quality of life is age-dependent in children with food allergy.

Food allergy in children significantly affects their quality of life. Its impact can be analyzed by quality of life questionnaires. The aim of our study was to validate the French version of disease-specific questionnaires and to evaluate the quality of life in children with IgE-mediated food allergy. Two validated food allergy-specific questionnaires for quality of life, the parent's and children's forms (FAQLQ-PF and FAQLQ-CF), were translated from English to French and submitted to children with food allergy and their parents. Questionnaires were analyzed in terms of emotional impact, food anxiety, and social and food limitations. NCT 01480427. Sixty-two parents of children aged 0-12 yrs answered the FAQLQ-PF, and 32 children aged 8-12 yrs the FAQLQ-CF. Construct validity of both questionnaires was assessed by correlation between the FAQLQs and FAIM (r = 0.85 and 0.84, respectively). Both FAQLQs had good internal consistency (Cronbach's α = 0.748 and 0.67, respectively). Young children (0-3 yrs old) showed better quality of life scores than older children (FAQLQ-PF global score: p = 0.02). Worse scores were also shown among children with previous severe systemic reactions (FAQLQ-PF global score: p = 0.039), the ones with an allergic mother (FAQLQ-PF global score: p = 0.002), or allergic siblings (FAQLQ-PF emotional impact score: p = 0.034), the ones with multiple food allergy (more than 1 food) (FAQLQ-PF anxiety score: p = 0.04) and among the girls (FAQLQ-CF global score: p = 0.031). Older children, the ones with severe systemic reactions, or with mothers or siblings also affected by allergies, as well as girls, and children with multiple food allergies show worse quality of life scores.

Functional interactions between the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter.

Two distinct, TATA box-containing promoters regulate the transcriptional activity of the Xenopus vitellogenin A1 gene. These two promoters are of different strength and are separated by 1.8 kilobase pairs of untranslated sequence. Estrogen receptor (ER) and its ligand, 17beta-estradiol, induce the activity of both promoters. The estrogen response elements (EREs) are located proximal to the downstream i promoter while no ERE-like sequences have been identified in the vicinity of the upstream io promoter. We show here, that transcriptional activity of the upstream io promoter is Sp1-dependent. Moreover, we demonstrate that estrogen inducibility of the io promoter results from functional interactions between the io bound Sp1 and the ER bound at the proximity of i. Functional interactions between Sp1 and ER do not require the presence of a TATA box for transcriptional activation, as is demonstrated using the acyl-CoA oxidase promoter. The relative positions that ER and Sp1 occupy with respect to the initiation site determines whether these two transcription activators can synergize for transcription initiation.

Charge dependent substrate activity of C3' and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).