HIV-1 drug resistance transmission networks in southwest Switzerland.

To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

Impact of clinical practice guidelines on priorisation for intensive care beds allocation in high-risk acute coronary syndrome patients: does age play a role?

QUESTION UNDER STUDY: To assess which high-risk acute coronary syndrome (ACS) patient characteristics played a role in prioritising access to intensive care unit (ICU), and whether introducing clinical practice guidelines (CPG) explicitly stating ICU admission criteria altered this practice. PATIENTS AND METHODS: All consecutive patients with ACS admitted to our medical emergency centre over 3 months before and after CPG implementation were prospectively assessed. The impact of demographic and clinical characteristics (age, gender, cardiovascular risk factors, and clinical parameters upon admission) on ICU hospitalisation of high-risk patients (defined as retrosternal pain of prolonged duration with ECG changes and/or positive troponin blood level) was studied by logistic regression. RESULTS: Before and after CPG implementation, 328 and 364 patients, respectively, were assessed for suspicion of ACS. Before CPG implementation, 36 of the 81 high-risk patients (44.4%) were admitted to ICU. After CPG implementation, 35 of the 90 high-risk patients (38.9%) were admitted to ICU. Male patients were more frequently admitted to ICU before CPG implementation (OR=7.45, 95% CI 2.10-26.44), but not after (OR=0.73, 95% CI 0.20-2.66). Age played a significant role in both periods (OR=1.57, 95% CI 1.24-1.99), both young and advanced ages significantly reducing ICU admission, but to a lesser extent after CPG implementation. CONCLUSION: Prioritisation of access to ICU for high-risk ACS patients was age-dependent, but focused on the cardiovascular risk factor profile. CPG implementation explicitly stating ICU admission criteria decreased discrimination against women, but other factors are likely to play a role in bed allocation.

Variation in novel exons (RACEfrags) of the MECP2 gene in Rett syndrome patients and controls.

The study of transcription using genomic tiling arrays has lead to the identification of numerous additional exons. One example is the MECP2 gene on the X chromosome; using 5'RACE and RT-PCR in human tissues and cell lines, we have found more than 70 novel exons (RACEfrags) connecting to at least one annotated exon.. We sequenced all MECP2-connected exons and flanking sequences in 3 groups: 46 patients with the Rett syndrome and without mutations in the currently annotated exons of the MECP2 and CDKL5 genes; 32 patients with the Rett syndrome and identified mutations in the MECP2 gene; 100 control individuals from the same geoethnic group. Approximately 13 kb were sequenced per sample, (2.4 Mb of DNA resequencing). A total of 75 individuals had novel rare variants (mostly private variants) but no statistically significant difference was found among the 3 groups. These results suggest that variants in the newly discovered exons may not contribute to Rett syndrome. Interestingly however, there are about twice more variants in the novel exons than in the flanking sequences (44 vs. 21 for approximately 1.3 Mb sequenced for each class of sequences, p=0.0025). Thus the evolutionary forces that shape these novel exons may be different than those of neighboring sequences.

Sub-cortical and brainstem sites associated with chemo-stimulated increases in ventilation in humans.

We investigated the neural basis for spontaneous chemo-stimulated increases in ventilation in awake, healthy humans. Blood oxygen level dependent (BOLD) functional MRI was performed in nine healthy subjects using T2 weighted echo planar imaging. Brain volumes (52 transverse slices, cortex to high spinal cord) were acquired every 3.9 s. The 30 min paradigm consisted of six, 5-min cycles, each cycle comprising 45 s of hypoxic-isocapnia, 45 s of isooxic-hypercapnia and 45 s of hypoxic-hypercapnia, with 55 s of non-stimulatory hyperoxic-isocapnia (control) separating each stimulus period. Ventilation was significantly (p<0.001) increased during hypoxic-isocapnia, isooxic-hypercapnia and hypoxic-hypercapnia (17.0, 13.8, 24.9 L/min respectively) vs. control (8.4 L/min) and was associated with significant (p<0.05, corrected for multiple comparisons) signal increases within a bilateral network that included the basal ganglia, thalamus, red nucleus, cerebellum, parietal cortex, cingulate and superior mid pons. The neuroanatomical structures identified provide evidence for the spontaneous control of breathing to be mediated by higher brain centres, as well as respiratory nuclei in the brainstem.

Basal and antigen-induced exposure of the proline-rich sequence in CD3ε.

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.

The evolutionary history of the CD209 (DC-SIGN) family in humans and non-human primates

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K(A)/K(S) values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigens

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells.

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

Immunology taught by lung dendritic cells.

Dendritic cells (DCs) are leukocytes specialised in the uptake, processing, and presentation of antigen and fundamental in regulating both innate and adaptive immune functions. They are mainly localised at the interface between body surfaces and the environment, continuously scrutinising incoming antigen for the potential threat it may represent to the organism. In the respiratory tract, DCs constitute a tightly enmeshed network, with the most prominent populations localised in the epithelium of the conducting airways and lung parenchyma. Their unique localisation enables them to continuously assess inhaled antigen, either inducing tolerance to inoffensive substances, or initiating immunity against a potentially harmful pathogen. This immunological homeostasis requires stringent control mechanisms to protect the vital and fragile gaseous exchange barrier from unrestrained and damaging inflammation, or an exaggerated immune response to an innocuous allergen, such as in allergic asthma. During DC activation, there is upregulation of co-stimulatory molecules and maturation markers, enabling DC to activate naïve T cells. This activation is accompanied by chemokine and cytokine release that not only serves to amplify innate immune response, but also determines the type of effector T cell population generated. An increasing body of recent literature provides evidence that different DC subpopulations, such as myeloid DC (mDC) and plasmacytoid DC (pDC) in the lungs occupy a key position at the crossroads between tolerance and immunity. This review aims to provide the clinician and researcher with a summary of the latest insights into DC-mediated pulmonary immune regulation and its relevance for developing novel therapeutic strategies for various disease conditions such as infection, asthma, COPD, and fibrotic lung disease.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Williams-Beuren syndrome TRIM50 encodes an E3 ubiquitin ligase.

Williams-Beuren syndrome (WBS) is a neurodevelopmental and multisystemic disease that results from hemizygosity of approximately 25 genes mapping to chromosomal region 7q11.23. We report here the preliminary description of eight novel genes mapping within the WBS critical region and/or its syntenic mouse region. Three of these genes, TRIM50, TRIM73 and TRIM74, belong to the TRIpartite motif gene family, members of which were shown to be associated to several human genetic diseases. We describe the preliminary functional characterization of these genes and show that Trim50 encodes an E3 ubiquitin ligase, opening the interesting hypothesis that the ubiquitin-mediated proteasome pathway might be involved in the WBS phenotype.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.