TIGIT Marks Exhausted T Cells,Correlates with Disease Progression,and Serves as a Target for Immune Restoration in HIV and SIV Infection

HIV infection induces phenotypic and functional changes to CD8⁺ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8⁺ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT⁺ and TIGIT⁺ PD-1⁺ CD8⁺ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8⁺ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8⁺ T cell effector responses. The frequency of TIGIT⁺ CD4⁺ T cells correlated with the CD4⁺ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.     

Historical demography and spatial genetic structure of the subterranean rodent Ctenomys magellanicus in Tierra del Fuego (Argentina)

Ctenomys (tuco‐tuco) is the most numerous genus of South American subterranean rodents and one of the most genetically diverse clades of mammals known. In particular, the genus constitutes a very interesting model for evolutionary studies of genetic divergence and conservation. Ctenomys magellanicus is the southernmost species of the group and the only one living in Isla Grande de Tierra del Fuego (Argentina). This species presents two chromosomal forms (Cm34 and Cm36) fragmented into demes distributed from the north region (steppe) to the south region (ecotone) of the island, respectively; no hybrids or overlapping areas were detected. To study the historical demography and the spatial genetic structure of the C. magellanicus population we used mitochondrial DNA (mtDNA) (D‐loop and cytochrome b) and microsatellite loci. Nine mtDNA haplotypes were identified, three of them belonging to the north and the other six to the south. Shared haplotypes between regions were not detected. mtDNA and microsatellite genotypes showed a marked pattern of population structure with low values of genetic flow between regions. The south is made up of small populations or isolated demes making up an endogamic metapopulation with unique alleles and haplotypes. Also, the results suggest a northward expansion process starting from an ancestral haplotype from the south. That population might have lived at a refuge through the adverse Pleistocene environmental conditions that took place at Tierra del Fuego. Results of this study are relevant to the conservation of C. magellanicus, suggesting that each region (north and south) might be considered as an Evolutionarily Significant Unit. © 2013 The Linnean Society of London     

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells

gp96 (GRP94) elicits antigen-presenting cell (APC) activation and can direct peptides into the cross- presentation pathways of APC. These responses arise through interactions of gp96 with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC. Previously, CD91, the alpha2-macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class-A (SR-A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re-presentation are inhibited by the SR-A inhibitory ligand fucoidin, although fucoidin was without effect on alpha2-macroglobulin binding or uptake. Ectopic expression of SR-A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR-A-/- mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR-A in the regulation of cellular responses to heat shock proteins.     

Calpain2 mediates Rab5-driven focal adhesion disassembly and cell migration

The early endosome protein Rab5 was recently shown to promote cell migration by enhancing focal adhesion disassembly through mechanisms that remain elusive. Focal adhesion disassembly is associated to proteolysis of talin, in a process that requires calpain2. Since calpain2 has been found at vesicles and endosomal compartments, we hypothesized that Rab5 stimulates calpain2 activity, leading to enhanced focal adhesion disassembly in migrating cells. We observed that calpain2 co-localizes with EEA1-positive early endosomes and co-immunoprecipitates with EEA1 and Rab5 in A549 lung carcinoma cells undergoing spreading, whereas Rab5 knock-down decreased the accumulation of calpain2 at early endosomal-enriched fractions. In addition, Rab5 silencing decreased calpain2 activity, as shown by cleavage of the fluorogenic substrate tBOC-LM-CMAC and the endogenous substrate talin. Accordingly, Rab5 promoted focal adhesion disassembly in a calpain2-dependent manner, as expression of GFP-Rab5 accelerated focal adhesion disassembly in nocodazole-synchronized cells, whereas pharmacological inhibition of calpain2 with N-acetyl-Leu-Leu-Met prevented both focal adhesion disassembly and cell migration induced by Rab5. In summary, these data uncover Rab5 as a novel regulator of calpain2 activity and focal adhesion proteolysis leading to cell migration.     

How competitive is the ‘pioneerclimax’ tree species Nothofagus alpina in pristine temperate forests#br#of Chile?

AimsNothofagusspecies are constitutive elements of the temperate forests along the Southern Andes Mountains. The deciduousNothofagus alpinais a fast-growing, but long-lived opportunistic pioneer species (‘pioneer-climax species’) and co-occurs with the evergreenN. dombeyi. We tested whetherN. alpinais competitively superior toN. dombeyiin mature stands and whether intra-specific competition inN. alpinais stronger than inter-specific competition withN. dombeyi.     

Pre-existing risk factor profiles in users and non-users of hormone replacement therapy: prospective cohort study in Gothenburg,Sweden

ObjectiveTo assess whether risk factor profiles for cardiovascular disease differed, before starting treatment, between women who would subsequently use hormone replacement therapy and those who would remain untreated.DesignProspective population study, initiated in 1968-9, with follow ups in 1974, 1980, and 1992.SettingGothenburg, Sweden.Participants1201 women born in 1918, 1922, and 1930, representative of women of the same age in the general population.Results179 of the 1202 women (14.9%) used hormone replacement therapy sometime during the 24 year follow up period. Multivariate models indicated that these women had significantly lower blood pressure, had less obesity, and belonged to a higher social group before the start of treatment than women who would remain untreated.ConclusionWomen who would subsequently use hormone replacement therapy were already at lower cardiovascular risk before the start of treatment than women who would remain untreated. Some of the claimed beneficial effects of treatment may thus be explained by women who would use hormone replacement therapy representing a healthier cohort than women who would remain untreated.

Key messages

• Many retrospective epidemiological studies have shown that hormone replacement therapy reduces the risk of cardiovascular disease
• Results from the prospective population study in Gothenburg show that there were already differences in risk factor profile of women before hormone replacement therapy was considered
• It is too early to recommend hormone replacement therapy for prevention of cardiovascular disease before controlled randomised studies have been performed

     

Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5'-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.     

Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones

Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.