Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-Dependent Variation in Quaternary Structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses

CXCL10 Can Inhibit Endothelial Cell Proliferation Independently of CXCR3

CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10) is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism

Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.</p> <p>Results</p> <p>The study presents association between TLR gene mutations and increased susceptibility to <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further <it>in silico </it>analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.</p> <p>Conclusion</p> <p>The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^thamino acid position in LRR motif (TLR1–LRR10) and 4^thresidue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.</p

HIV-1 Tat Co-Operates with IFN-γ and TNF-α to Increase CXCL10 in Human Astrocytes

HIV-associated neurological disorders (HAND) are estimated to affect 60% of the HIV infected population. HIV-encephalitis (HIVE), the pathological correlate of the most severe form of HAND is often characterized by glial activation, cytokine/chemokine dysregulation, and neuronal damage and loss. However, the severity of HIVE correlates better with glial activation rather than viral load. One of the characteristic features of HIVE is the increased amount of the neurotoxic chemokine, CXCL10. This chemokine can be released from astroglia activated with the pro-inflammatory cytokines IFN-γ and TNF-α, in conjunction with HIV-1 Tat, all of which are elevated in HIVE. In an effort to understand the pathogenesis of HAND, this study was aimed at exploring the regulation of CXCL10 by cellular and viral factors during astrocyte activation. Specifically, the data herein demonstrate that the combined actions of HIV-1 Tat and the pro-inflammatory cytokines, IFN-γ and TNF-α, result in the induction of CXCL10 at both the RNA and protein level. Furthermore, CXCL10 induction was found to be regulated transcriptionally by the activation of the p38, Jnk, and Akt signaling pathways and their downstream transcription factors, NF-κB and STAT-1α. Since CXCL10 levels are linked to disease severity, understanding its regulation could aid in the development of therapeutic intervention strategies for HAND

Interaction of Pattern Recognition Receptors with Mycobacterium Tuberculosis.

Tuberculosis (TB) is considered a major worldwide health problem with 10 million new cases diagnosed each year. Our understanding of TB immunology has become greater and more refined since the identification of Mycobacterium tuberculosis (MTB) as an etiologic agent and the recognition of new signaling pathways modulating infection. Understanding the mechanisms through which the cells of the immune system recognize MTB can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. A great challenge in chronic disease is to understand the complexities, mechanisms, and consequences of host interactions with pathogens. Innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the MTB. Several classes of pattern recognition receptors (PRRs) are involved in the recognition of MTB including Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs) and Nod-like receptors (NLRs) linked to inflammasome activation. Among the TLR family, TLR1, TLR2, TLR4, and TLR9 and their down-stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of TB. The inflammasome pathway is associated with the coordinated release of cytokines such as IL-1β and IL-18 which also play a role in the pathogenesis of TB. Understanding the cross-talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes. Abnormalities in PRR signaling pathways regulated by TB will affect disease pathogenesis and need to be elucidated. In this review we provide an update on PRR signaling during M. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities

Expression of the innate immune receptor LILRB5 on monocytes is associated with mycobacteria exposure.

Antigen presenting cells (APC) are critical components of innate immunity and consequently shape the adaptive response. Leukocyte Ig Like Receptors (LILR) are innate immune receptors predominantly expressed on myeloid cells. LILR can influence the antigen presenting phenotype of monocytic cells to determine the nature of T cell responses in infections including Mycobaterium leprae. We therefore investigated the relevance of LILR in the context of Mycobacterium tuberculosis. Real-time PCR studies indicated that the transcriptional profile of the orphan receptor LILRB5 was significantly up-regulated following exposure to mycobacteria. Furthermore, LILRA1 and LILRB5 were able to trigger signalling through direct engagement of mycobacteria using tranfectant cells incorporating a reporter system. We describe for the first time the expression of this receptor on T cells, and highlight the potential relevance to mycobacterial recognition. Furthermore, we demonstrate that crosslinking of this receptor on T cells increases proliferation of cytotoxic, but not helper, T cells

Syndromics: A Bioinformatics Approach for Neurotrauma Research

Substantial scientific progress has been made in the past 50 years in delineating many of the biological mechanisms involved in the primary and secondary injuries following trauma to the spinal cord and brain. These advances have highlighted numerous potential therapeutic approaches that may help restore function after injury. Despite these advances, bench-to-bedside translation has remained elusive. Translational testing of novel therapies requires standardized measures of function for comparison across different laboratories, paradigms, and species. Although numerous functional assessments have been developed in animal models, it remains unclear how to best integrate this information to describe the complete translational “syndrome” produced by neurotrauma. The present paper describes a multivariate statistical framework for integrating diverse neurotrauma data and reviews the few papers to date that have taken an information-intensive approach for basic neurotrauma research. We argue that these papers can be described as the seminal works of a new field that we call “syndromics”, which aim to apply informatics tools to disease models to characterize the full set of mechanistic inter-relationships from multi-scale data. In the future, centralized databases of raw neurotrauma data will enable better syndromic approaches and aid future translational research, leading to more efficient testing regimens and more clinically relevant findings

Therapeutic Potential of HDL in Cardioprotection and Tissue Repair

Epidemiological studies support a strong association between high-density lipoprotein (HDL) cholesterol levels and heart failure incidence. Experimental evidence from different angles supports the view that low HDL is unlikely an innocent bystander in the development of heart failure. HDL exerts direct cardioprotective effects, which are mediated via its interactions with the myocardium and more specifically with cardiomyocytes. HDL may improve cardiac function in several ways. Firstly, HDL may protect the heart against ischaemia/reperfusion injury resulting in a reduction of infarct size and thus in myocardial salvage. Secondly, HDL can improve cardiac function in the absence of ischaemic heart disease as illustrated by beneficial effects conferred by these lipoproteins in diabetic cardiomyopathy. Thirdly, HDL may improve cardiac function by reducing infarct expansion and by attenuating ventricular remodelling post-myocardial infarction. These different mechanisms are substantiated by in vitro, ex vivo, and in vivo intervention studies that applied treatment with native HDL, treatment with reconstituted HDL, or human apo A-I gene transfer. The effect of human apo A-I gene transfer on infarct expansion and ventricular remodelling post-myocardial infarction illustrates the beneficial effects of HDL on tissue repair. The role of HDL in tissue repair is further underpinned by the potent effects of these lipoproteins on endothelial progenitor cell number, function, and incorporation, which may in particular be relevant under conditions of high endothelial cell turnover. Furthermore, topical HDL therapy enhances cutaneous wound healing in different models. In conclusion, the development of HDL-targeted interventions in these strategically chosen therapeutic areas is supported by a strong clinical rationale and significant preclinical data.status: publishe

Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-γ in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-γ. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-γ upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-γ only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions