Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.

Chronic immune activation and progression to AIDS are observed after SIV infection in macaques but not in natural host primate species. To better understand this dichotomy, we compared acute pathogenic SIV infection in pigtailed macaques (PTs) to non-pathogenic infection in African green monkeys (AGMs). SIVagm-infected PTs, but not SIVagm-infected AGMs, rapidly developed systemic immune activation, marked and selective depletion of IL-17-secreting (Th17) cells, and loss of the balance between Th17 and T regulatory (Treg) cells in blood, lymphoid organs, and mucosal tissue. The loss of Th17 cells was found to be predictive of systemic and sustained T cell activation. Collectively, these data indicate that loss of the Th17 to Treg balance is related to SIV disease progression.

In the current global AIDS pandemic, more than half of new human immunodeficiency virus type 1 (HIV-1) infections are acquired by women through intravaginal HIV exposure. For this study, we explored pathogenesis issues relevant to the development of effective vaccines to prevent infection by this route, using an animal model in which female rhesus macaques were exposed intravaginally to a high dose of simian immunodeficiency virus (SIV). We examined in detail the events that transpire from hours to a few days after intravaginal SIV exposure through week 4 to provide a framework for understanding the propagation, dissemination, and establishment of infection in lymphatic tissues (LTs) during the acute stage of infection. We show that the mucosal barrier greatly limits the infection of cervicovaginal tissues, and thus the initial founder populations of infected cells are small. While there was evidence of rapid dissemination to distal sites, we also show that continuous seeding from an expanding source of production at the portal of entry is likely critical for the later establishment of a productive infection throughout the systemic LTs. The initially small founder populations and dependence on continuous seeding to establish a productive infection in systemic LTs define a small window of maximum vulnerability for the virus in which there is an opportunity for the host, vaccines, or other interventions to prevent or control infection.The AIDS pandemic, already the most widespread pandemic in recorded human history, has claimed the lives of millions and continues relatively unabated for want of an effective vaccine or other means of prevention. Especially urgent is the need for effective vaccines and microbicides to prevent the vaginal transmission of human immunodeficiency virus type 1 (HIV-1), as women now account for close to 60 percent of newly acquired infections in Africa (27).The simian immunodeficiency virus (SIV)/rhesus monkey model of vaginal HIV transmission is clearly relevant to this objective. It has been used extensively to test vaccines (1,7,12,18,22) and microbicides (16,17,19,25,28) designed to prevent vaginal transmission. Moreover, pathogenesis studies relevant to the design and testing of vaccine and microbicide candidates that would be impossible in humans can be undertaken by use of this animal model. This model uses SIV, a primate lentivirus that is closely related to HIV (5) and that can be efficiently transmitted to macaques by vaginal inoculation of cell-free inocula (11,20,21). Further, the rhesus monkey is similar to humans with regard to the populations of target cells (10) and the physiology (4) and immunology (8, 9) of the female genital tract.Here we describe the use of this animal model to address the following two critical issues for the development of vaccines to prevent systemic infection following intravaginal transmission: the role of local propagation in establishing systemic infection and the dynamics of spread to the lymphatic tissues (LTs). The intravaginal inoculation mo...

In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8؉ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4 ؉ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4 ؉ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8 ؉ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4 ؉ T lymphocytes in the GALT. Thus, the virus-specific CD8 ؉ T-lymphocyte response is "too late and too little" to clear infection and prevent CD4 ؉ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8 ؉

The Aiptasia-Symbiodinium symbiosis is a promising model for experimental studies of cnidarian-dinoflagellate associations, yet relatively little is known regarding the genetic diversity of either symbiotic partner. To address this, we collected Aiptasia from 16 localities throughout the world and examined the genetic diversity of both anemones and their endosymbionts. Based on newly developed SCAR markers, Aiptasia consisted of two genetically distinct populations: one Aiptasia lineage from Florida and a second network of Aiptasia genotypes found at other localities. These populations did not conform to the distributions of described Aiptasia species, suggesting that taxonomic re-evaluation is needed in the light of molecular genetics. Associations with Symbiodinium further demonstrated the distinctions among Aiptasia populations. According to 18S RFLP, ITS2-DGGE and microsatellite flanker region sequencing, Florida anemones engaged in diverse symbioses predominantly with members of Symbiodinium Clades A and B, but also C, whereas anemones from elsewhere harboured only S. minutum within Clade B. Symbiodinium minutum apparently does not form a stable symbiosis with other hosts, which implies a highly specific symbiosis. Fine-scale differences among S. minutum populations were quantified using six microsatellite loci. Populations of S. minutum had low genotypic diversity and high clonality (R = 0.14). Furthermore, minimal population structure was observed among regions and ocean basins, due to allele and genotype sharing. The lack of genetic structure and low genotypic diversity suggest recent vectoring of Aiptasia and S. minutum across localities. This first ever molecular-genetic study of a globally distributed cnidarian and its Symbiodinium assemblages reveals host-symbiont specificity and widely distributed populations in an important model system.

microRNAs (miRNAs) play a crucial role in tissue development and the pathology of various diseases. However, the effects and roles of miRNAs in macrophage polarization have yet to be investigated. In this study, we analyzed and compared the miRNA expression profiles of bone marrow-derived macrophages (BMDMs) with two distinct polarizing conditions (classical macrophage activation 'M1' and alternative activation 'M2') using miRNA microarray. In total, 109 miRNAs were differentially expressed between M1 and M2. The differential expression of selected miRNAs was validated by real-time qRT-PCR: miR-181a, miR-155-5p, miR-204-5p and miR-451 were upregulated (fold change >2, P<0.05) and miR-125-5p, miR-146a-3p, miR-143-3p and miR-145-5p were downregulated (fold change <-2, P<0.05) in M1 compared with M2. In conclusion, our study may be useful for exploring the precise roles of miRNAs in macrophage differentiation and polarized activation processes in the future.

Recent studies have shown that stromal fibroblasts have a more profound influence on the initiation and progression of carcinoma than was previously appreciated. This study aimed at investigating the reciprocal relationship between cancer cells and their associated fibroblasts at both the molecular and cellular level in oral squamous cell carcinoma (OSCC). To identify key molecular regulators expressed by carcinoma-associated fibroblasts (CAF) that promote cancer cell invasion, microarrays were performed by comparing cocultured OSCC cells and CAF with monoculture controls. Microarray and real-time PCR analysis identified marked upregulation of the chemokine (C-C motif) ligand 7 (CCL7) in cocultured CAF. ELISA showed an elevated level of CCL7 secretion from CAF stimulated by coculture with OSCC cells. CCL7 promoted the invasion and migration of OSCC cells, and the invasiveness was inhibited by treatment with CCL7 neutralizing antibody. OSCC cells were shown to express CCR1, CCR2 and CCR3, receptors for CCL7, by RT-PCR. In addition, treatment with anti-CCR1 or anti-CCR3 antibody inhibited CCL7-induced OSCC cell migration, implicating that CCL7 promotes cancer cell migration through CCR1 and CCR3 on OSCC cells. Cytokine antibody array analysis of the supernatant from OSCC cell culture revealed that interleukin-1a was an inducer of CCL7 secretion by CAF. This study confirms the reciprocal relationship of the molecular crosstalk regulating the invasion of OSCC and describes new potential targets for future therapy.Carcinomas are malignant neoplasms derived from epithelial cells and are surrounded by specialized stroma, which orchestrate with cancer cells to regulate disease progression. 1-3 Carcinoma-associated fibroblasts (CAF) have been recognized as prominent modifiers of cancer initiation and progression. 4,5 For instance, it has been previously demonstrated that human prostatic CAF induce tumor formation from initiated but nontumorigenic human prostatic epithelial cells. 6 CAF also facilitate the invasiveness of otherwise noninvasive cancer cells when coinjected into mice. 7 The putative proinvasive effects of CAF may be mediated through either direct heterotypic cellÀcell contacts 8 or diffusible molecules, such as inflammatory mediators, cytokines and chemokines. 9,10 Chemokines have been shown to play an important role in tumor biology by influencing tumor growth, invasion and metastasis. 11 Chemokines are a family of small, structurally related cytokines with chemoattractant and activation properties that are involved in inflammatory reactions. 12 They are classified mainly into the CC and CXC subfamilies, according to the location of the first 2 cysteine residues, and are produced by a range of cell types, including fibroblasts. Various types of cancer cells also express chemokines and chemokine receptors, 11,[13][14][15] and their autocrine and paracrine roles in cancer progression are receiving increasing attention. For example, the CXC chemokine, CXCL12 (stromal cell-derived factor 1), secreted by CAF, r...

To define the ratio of simian immunodeficiency virus (SIV) RNA molecules to infectious virions in plasma, a ramp-up-stage plasma pool was made from the earliest viral RNA (vRNA)-positive plasma samples (collected approximately 7 days after inoculation) from seven macaques, and a set-point-stage plasma pool was made from plasma samples collected 10 to 16 weeks after peak viremia from seven macaques; vRNA levels in these plasma pools were determined, and serial 10-fold dilutions containing 1 to 1,500 vRNA copies/ml were made. Intravenous (i.v.) inoculation of a 1-ml aliquot of diluted ramp-up-stage plasma containing 20 vRNA copies infected 2 of 2 rhesus macaques, while for the set-point-stage plasma, i.v. inoculation with 1,500 vRNA copies was needed to transmit infection. Further, when the heat-inactivated set-point-stage plasma pool was mixed with ramp-up-stage virions, infection of inoculated macaques was blocked. Notably, 2 of 2 animals inoculated with 85 ml of a pre-ramp-up plasma pool containing <3 SIV RNA copies/ml developed SIV infections characterized by high levels of viral replication, demonstrating that "vRNA-negative" plasma collected from macaques in the pre-ramp-up stage is infectious. Furthermore, there is a high ratio of infectious virions to total virions in ramp-up-stage plasma (between 1:1 and 1:10) and a lower ratio in set-point-stage plasma (between 1:75 and 1:750). Heat-inactivated chronic-stage plasma can "neutralize" the highly infectious ramp-up-stage virions. These findings have implications for the understanding of the natural history of SIV and human immunodeficiency virus infection and transmission.