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1.  Dynamic interaction between astrocytes and infiltrating PBMCs in context of neuroAIDS 
Glia  2014;63(3):441-451.
HIV-mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells (peripheral blood mononuclear cells (PBMCs)). We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary-derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either β-catenin-dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10-fold increase in HLA-DR and a 5- fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2-fold, which was mediated by IFNγ in a Stat-3-dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte-secreted Wnts exert an anti-HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation.
PMCID: PMC4293283  PMID: 25331637
2.  HIV Infection Accelerates Gastrointestinal Tumor Outgrowth in NSG-HuPBL Mice 
HIV infection is a risk factor for the tumorigenesis including non-AIDS-defining cancers such as those of the gastrointestinal tract. However, the mechanisms underlying such cancer outgrowth are still unknown. Furthermore, combined HIV/cancer studies are difficult to evaluate using primate models or in the clinical patient setting. To understand the mechanisms of tumor outgrowth in the context of HIV infection, we adopted a humanized mouse model permissive to infection and cancer as well as an in vivo humanized mouse challenge with colon cancer in the context of HIV infection. Immunodeficient NOD SCID IL-2R–/– mice were immunologically reconstituted by adoptive transfer of 107 HIV-negative donor peripheral blood leukocytes and challenged with 106 HCT116 human colon cancer cells. A group of mice was treated with antiretroviral therapy. Tumor microenvironment and epithelial tissues in the context of HIV infection were analyzed using immunohistochemistry. We demonstrate that HIV-infected humanized mice develop significantly larger tumors than uninfected mice (p<0.05). Epithelial cell proliferation in HIV-infected mice is significantly enhanced in comparison to proliferation in uninfected mice (p<0.01). Moreover, the activation of β-catenin, an important step in intestinal epithelial cell proliferation and tumorigenesis, is elevated in the tumors of HIV-infected mice (p<0.0001). Importantly, antiretroviral therapy reverses these pathological processes independently of CD4+ T cell return. These findings model the ability of HIV infection to result in tumor outgrowth that is evident in HIV-positive patients and lend insight into previously unrecognized mechanisms that may underlie this pathology.
PMCID: PMC4076996  PMID: 24593860
3.  β-catenin/TCF-4 signaling regulates susceptibility of macrophages and resistance of monocytes to HIV-1 productive infection 
Current HIV research  2014;12(3):164-173.
Cells of the monocyte/macrophage lineage are an important target for HIV-1 infection. They are often at anatomical sites linked to HIV-1 transmission and are an important vehicle for disseminating HIV-1 throughout the body, including the central nervous system. Monocytes do not support extensive productive HIV-1 replication, but they become more susceptible to HIV-1 infection as they differentiate into macrophages. The mechanisms guiding susceptibility of HIV-1 replication in monocytes versus macrophages are not entirely clear. We determined whether endogenous activity of β-catenin signaling impacts differential susceptibility of monocytes and monocyte-derived macrophages (MDMs) to productive HIV-1 replication. We show that monocytes have an approximately 4-fold higher activity of β-catenin signaling than MDMs. Inducing β-catenin in MDMs suppressed HIV-1 replication by 5-fold while inhibiting endogenous β-catenin signaling in monocytes by transfecting with a dominant negative mutant for the downstream effector of β-catenin (TCF-4) promoted productive HIV-1 replication by 6-fold. These findings indicate that β-catenin/TCF-4 is an important pathway for restricted HIV-1 replication in monocytes and plays a significant role in potentiating HIV-1 replication as monocytes differentiate into macrophages. Targeting this pathway may provide a novel strategy to purge the latent reservoir from monocytes/macrophages, especially in sanctuary sites for HIV-1 such as the central nervous system.
PMCID: PMC4331035  PMID: 24862328
4.  Porcupine Is Not Required for the Production of the Majority of Wnts from Primary Human Astrocytes and CD8+ T Cells 
PLoS ONE  2014;9(3):e92159.
Wnts are small secreted glycoproteins that are highly conserved among species. To date, 19 Wnts have been described, which initiate a signal transduction cascade that is either β-catenin dependent or independent, culminating in the regulation of hundreds of target genes. Extracellular release of Wnts is dependent on lipidation of Wnts by porcupine, a membrane-bound-O-acyltransferase protein in the endoplasmic reticulum. Studies demonstrating the requirement of porcupine for Wnts production are based on cell line and non-human primary cells. We evaluated the requirement for porcupine for Wnts production in human primary astrocytes and CD8+ T cells. Using IWP-2, an inhibitor of porcupine, or siRNA targeting porcupine, we demonstrate that porcupine is not required for the release of Wnt 1, 3, 5b, 6,7a, 10b, and 16a. While IWP had no effect on Wnt 2b release, knockdown of porcupine by siRNA reduced Wnt 2b release by 60%. These data indicate that porcupine-mediated production of Wnts is context dependent and is not required for all Wnts production, suggesting that alternative mechanisms exist for Wnts production.
PMCID: PMC3960167  PMID: 24647048
5.  Mouse Models of Multiple Sclerosis: Experimental Autoimmune Encephalomyelitis and Theiler’s Virus-Induced Demyelinating Disease 
Experimental autoimmune encephalomyelitis (EAE) and Theiler’s Murine Encephalitis Virus-Induced Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question being asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed. In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro proliferation, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assessment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.
PMCID: PMC3583382  PMID: 22933080
Multiple sclerosis; Experimental autoimmune encephalomyelitis; EAE; Emulsion; Active induction; Adoptive transfer; T cell blasts; Encephalitogenic; Neurodegeneration; Theiler’s murine encephalomyelitis virus-induced demyelinating disease; PLP; MOG; Myelin; MBP; VP2; VP3; Relapsing–remitting; Epitope spreading
6.  Virus Expanded Regulatory T Cells Control Disease Severity in the Theiler’s Virus Mouse Model of MS 
Journal of autoimmunity  2011;36(2):142-154.
Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) serves as virus-induced model of chronic progressive multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and a progressive autoimmune demyelinating disease mediated by myelin-specific T cells activated via epitope spreading. In contrast, virus is rapidly cleared by a robust CTL response in TMEV-IDD-resistant C57BL/6 mice. We investigated whether differential induction of regulatory T cells (Tregs) controls susceptibility to TMEV-IDD. Infection of disease-susceptible SJL/J, but not B6 mice, leads to rapid activation and expansion of Tregs resulting in an unfavorable CNS ratio of Treg:Teffector cells. In addition, anti-CD25-induced inactivation of Tregs in susceptible SJL/J, but not resistant B6, mice results in significantly decreased clinical disease concomitant with enhanced anti-viral CD4+, CD8+ and antibody responses resulting in decreased CNS viral titers. This is the first demonstration that virus-induced Treg activation regulates susceptibility to autoimmune disease differentially in susceptible and resistant strains of mice and provides a new mechanistic explanation for the etiology of infection-induced autoimmunity.
PMCID: PMC3046315  PMID: 21273044
T regulatory cells; CD8+ T cells; Theiler’s virus; demyelination; Multiple sclerosis; chronic viral infection
7.  Human FasL Gene Is a Target of β-Catenin/T-Cell Factor Pathway and Complex FasL Haplotypes Alter Promoter Functions 
PLoS ONE  2011;6(10):e26143.
FasL expression on human immune cells and cancer cells plays important roles in immune homeostasis and in cancer development. Our previous study suggests that polymorphisms in the FasL promoter can significantly affect the gene expression in human cells. In addition to the functional FasL SNP -844C>T (rs763110), three other SNPs (SNP -756A>G or rs2021837, SNP -478A>T or rs41309790, and SNP -205 C>G or rs74124371) exist in the proximal FasL promoter. In the current study, we established three major FasL hyplotypes in humans. Interestingly, a transcription motif search revealed that the FasL promoter possessed two consensus T-cell factor (TCF/LEF1) binding elements (TBEs), which is either polymorphic (SNP -205C>G) or close to the functional SNP -844C>T. Subsequently, we demonstrate that both FasL TBEs formed complexes with the TCF-4 and β-catenin transcription factors in vitro and in vivo. Co-transfection of LEF-1 and β-catenin transcription factors significantly increased FasL promoter activities, suggesting that FasL is a target gene of the β-catenin/T-cell factor pathway. More importantly, we found that the rare allele (-205G) of the polymorphic FasL TBE (SNP -205C>G) failed to bind the TCF-4 transcription factor and that SNP -205 C>G significantly affected the promoter activity. Furthermore, promoter reporter assays revealed that FasL SNP haplotypes influenced promoter activities in human colon cancer cells and in human T cells. Finally, β-catenin knockdown significantly decreased the FasL expression in human SW480 colon cancer cells. Collectively, our data suggest that β-catenin may be involved in FasL gene regulation and that FasL expression is influenced by FasL SNP haplotypes, which may have significant implications in immune response and tumorigenesis.
PMCID: PMC3191176  PMID: 22022540
8.  A critical role for virus-specific CD8+ CTLs in protection from Theiler’s virus-induced demyelination in disease-susceptible SJL mice 
Virology  2010;402(1):102-111.
Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is a relevant mouse model of multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and development of a chronic T cell-mediated autoimmune demyelinating disease triggered via epitope spreading to endogenous myelin epitopes. Potent CNS-infiltrating CD8+ T cell responses to TMEV epitopes have previously been shown to be induced in both disease-susceptible SJL/J and resistant C57BL/6 mice, in which the virus is rapidly cleared. Specific tolerization of SJL CD8+ T cells specific for the immunodominant TMEV VP3159–166 epitope has no effect on viral load or development of clinical TMEV-IDD, but adoptive transfer of activated CD8+ VP3159–166-specific T cell blasts shortly after TMEV infection to boost the early anti-viral response leads to clearance of CNS virus and protection from subsequent TMEV-IDD. These studies have important implications for vaccine strategies and treatment of chronic infections in humans.
PMCID: PMC2872027  PMID: 20381109

Results 1-8 (8)