Olig1 function is required for remyelination potential of transplanted neural progenitor cells in a model of viral-induced demyelination.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in cumulative neurologic deficits associated with progressive myelin loss. We have previously shown that transplantation of neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells (OPCs) that is associated with remyelination and axonal sparing. The current study examines the contributions of the transcription factor Olig1 on NPC differentiation and remyelination. Under defined conditions, NPCs preferentially differentiate into oligodendroglia whereas NPCs isolated from Olig1-deficient (Olig1-/-) mice exhibit enhanced differentiation into astrocytes. Transplantation of Olig1-/- and Olig1+/+ NPCs into JHMV-infected mice resulted in similar cell survival, proliferation, and selective migration to areas of demyelination. However, only recipients of wild type NPCs exhibited extensive remyelination compared to mice receiving Olig1-/- NPCs. In vivo characterization of NPCs revealed that Olig1+/+ NPCs preferentially differentiated into NG2-positive OPCs and formed processes expressing myelin basic protein that encircled axons. In contrast, the majority of transplanted Olig1-/- NPCs differentiated into GFAP-positive cells consistent with the astrocyte lineage. These results indicate that exogenous NPCs contribute to improved clinical and histological outcome and this is associated with remyelination by this donor population. Further, these findings reveal that Olig1function is required for the remyelination potential of NPCs after transplant, through specification and/or maintenance of oligodendroglial identity.

Surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

Inflammation induced by infection potentiates tau pathological features in transgenic mice.

Comorbidities that promote the progression of Alzheimer's disease (AD) remain to be uncovered and evaluated in animal models. Because elderly individuals are vulnerable to viral and bacterial infections, these microbial agents may be considered important comorbidities that could potentiate an already existing and tenuous inflammatory condition in the brain, accelerating cognitive decline, particularly if the cellular and molecular mechanisms can be defined. Researchers have recently demonstrated that triggering inflammation in the brain exacerbates tau pathological characteristics in animal models. Herein, we explore whether inflammation induced via viral infection, compared with inflammation induced via bacterial lipopolysaccharide, modulates AD-like pathological features in the 3xTg-AD mouse model and provide evidence to support the hypothesis that infectious agents may act as a comorbidity for AD. Our study shows that infection-induced acute or chronic inflammation significantly exacerbates tau pathological characteristics, with chronic inflammation leading to impairments in spatial memory. Tau phosphorylation was increased via a glycogen synthase kinase-3ß-dependent mechanism, and there was a prominent shift of tau from the detergent-soluble to the detergent-insoluble fraction. During chronic inflammation, we found that inhibiting glycogen synthase kinase-3ß activity with lithium reduced tau phosphorylation and the accumulation of insoluble tau and reversed memory impairments. Taken together, infectious agents that trigger central nervous system inflammation may serve as a comorbidity for AD, leading to cognitive impairments by a mechanism that involves exacerbation of tau pathological characteristics.

Cell replacement therapies to promote remyelination in a viral model of demyelination.

Persistent infection of the central nervous system (CNS) of mice with the neuroadapted JHM strain of mouse hepatitis (MHV) is characterized by ongoing demyelination mediated by inflammatory T cells and macrophages that is similar both clinically and histologically with the human demyelinating disease multiple sclerosis (MS). Although extensive demyelination occurs in mice persistently infected with MHV there is only limited remyelination. Therefore, the MHV model of demyelination is a relevant model for studying disease and evaluating therapeutic approaches to protect cells of the oligodendrocyte lineage and promote remyelination. This concept is further highlighted as the etiology of MS remains enigmatic, but viruses have long been considered as potential triggering agents in initiating and/or maintaining MS symptoms. As such, understanding mechanisms associated with promoting repair within the CNS in the context of a persistent viral infection is critical given the possible viral etiology of MS. This review focuses on recent studies using either mouse neural stem cells (NSCs) or human oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (hESC) to promote remyelination in mice persistently infected with MHV. In addition, the potential role for chemokines in positional migration of transplanted cells is addressed.

IFN-gamma-mediated suppression of coronavirus replication in glial-committed progenitor cells.

The neurotropic JHM strain of mouse hepatitis virus (JHMV) replicates primarily within glial cells following intracranial inoculation of susceptible mice, with relative sparing of neurons. This study demonstrates that glial cells derived from neural progenitor cells are susceptible to JHMV infection and that treatment of infected cells with IFN-gamma inhibits viral replication in a dose-dependent manner. Although type I IFN production is muted in JHMV-infected glial cultures, IFN-beta is produced following IFN-gamma-treatment of JHMV-infected cells. Also, direct treatment of infected glial cultures with recombinant mouse IFN-alpha or IFN-beta inhibits viral replication. IFN-gamma-mediated control of JHMV replication is dampened in glial cultures derived from the neural progenitor cells of type I receptor knock-out mice. These data indicate that JHMV is capable of infecting glial cells generated from neural progenitor cells and that IFN-gamma-mediated control of viral replication is dependent, in part, on type I IFN secretion.

The Anopheles gambiae vitellogenin gene (VGT2) promoter directs persistent accumulation of a reporter gene product in transgenic Anopheles stephensi following multiple bloodmeals.

Mosquitoes made resistant to pathogens through genetic engineering are proposed as a basis for developing a strategy to control disease transmission. Transgenic approaches that introduce exogenous antipathogen effector genes into mosquito genomes require cis-acting regulatory DNA to control tissue-, stage-, and sex-specific transgene expression. We show that control sequences derived from a vitellogenin-encoding gene of Anopheles gambiae, a major vector in sub-Saharan Africa, can direct expression of an exogenous gene in a tissue-, stage-, and sex-specific manner in Anopheles stephensi, a vector of urban malaria in southern Asia. Specific reporter gene expression was observed in fat-body tissues of transgenic blood-fed females, but not in transgenic males or non-blood-fed transgenic females. Multiple bloodmeals resulted in the continuous presence of reporter gene transcripts for at least 12 days. The persistent expression makes the heterologous promoter a good candidate for controlling transcription of engineered antipathogen effector genes in this important malaria vector.

HIV-1 prophylactic vaccine comprised of topical DermaVir prime and protein boost elicits cellular immune responses and controls pathogenic R5 SHIV162P3.

Topical DNA vaccination (DermaVir) facilitates antigen presentation to naive T cells. DermaVir immunization in mice, using HIV-1 Env and Gag, elicited cellular immune responses. Boosting with HIV-1 gp120 Env and p41 Gag augmented Th1 cytokine levels. Intramuscular DNA administration was less efficient in priming antigen-specific cytokine production and memory T cells. In rhesus macaques, DermaVir immunization induced Gag- and Env-specific Th1 and Th2 cytokines and generation of memory T cells. Boosting of DermaVir-primed serum antibody levels was noted following gp140(SHIV89.6P)/p27(SIV) immunization. Rectal challenge with pathogenic R5-tropic SHIV162P3 resulted in control of plasma viremia (4/5 animals) that was reflected in jejunum, colon and mesenteric lymph nodes. An inverse correlation was found between Gag- and Env-specific central memory T cell responses on the day of challenge and plasma viremia at set point. Overall, the topical DermaVir/protein vaccination yields central memory T cell responses and facilitates control of pathogenic SHIV infection.

T cell antiviral effector function is not dependent on CXCL10 following murine coronavirus infection.

The chemokine CXCL10 is expressed within the CNS in response to intracerebral infection with mouse hepatitis virus (MHV). Blocking CXCL10 signaling results in increased mortality accompanied by reduced T cell infiltration and increased viral titers within the brain suggesting that CXCL10 functions in host defense by attracting T cells into the CNS. The present study was undertaken to extend our understanding of the functional role of CXCL10 in response to MHV infection given that CXCL10 signaling has been implicated in coordinating both effector T cell generation and trafficking. We show that MHV infection of CXCL10(+/+) or CXCL10(-/-) mice results in comparable levels of T cell activation and similar numbers of virus-specific CD4+ and CD8+ T cells. Subsequent analysis revealed no differences in T cell proliferation, IFN-gamma secretion by virus-specific T cells, or CD8+ T cell cytolytic activity. Analysis of chemokine receptor expression on CD4+ and CD8+ T cells obtained from MHV-immunized CXCL10(+/+) and CXCL10(-/-) mice revealed comparable levels of CXCR3 and CCR5, which are capable of responding to ligands CXCL10 and CCL5, respectively. Adoptive transfer of splenocytes acquired from MHV-immunized CXCL10(-/-) mice into MHV-infected RAG1(-/-) mice resulted in T cell infiltration into the CNS, reduced viral burden, and demyelination comparable to RAG1(-/-) recipients of immune CXCL10(+/+) splenocytes. Collectively, these data imply that CXCL10 functions primarily as a T cell chemoattractant and does not significantly influence T cell effector response following MHV infection.

Optimal suppression of HIV replication by low-dose hydroxyurea through the combination of antiviral and cytostatic ('virostatic') mechanisms.

BACKGROUND: The hydroxyurea-didanosine combination has been shown to limit immune activation (a major pathogenic component of HIV/AIDS) and suppress viral load by both antiviral and cytostatic ('virostatic') activities. Virostatics action represent a novel approach to attack HIV/AIDS from multiple directions; however, the use of these drugs is limited by the lack of understanding of their dose-dependent mechanism of action and by fear of pancreatic toxicity, even though a large review of ACTG studies has shown that hydroxyurea does not increase the incidence of pancreatitis. METHODS: In vitro cytostatic and cytotoxic activity, inhibition of viral replication and immune activation by pharmacologically attainable plasma concentrations of hydroxyurea (10-100 micromol/l) and didanosine (1-5 micromol/l) were analyzed by cell proliferation, viability, apoptosis and infection assays using peripheral blood mononuclear cells. In vivo, 600, 900 and 1200 mg daily doses of hydroxyurea in combination with standard doses of didanosine and stavudine were studied in 115 randomized chronically infected patients. RESULTS: A cytostatic low (10 micromol/l) concentration of hydroxyurea inhibited cell proliferation and HIV replication in vitro. A gradual switch from cytostatic to cytotoxic effects was observed by increasing hydroxyurea concentration to 50-100 micromol/l, predicting that lower doses of hydroxyurea would be less toxic and more potent in vivo. The clinical results confirmed that 600 mg hydroxyurea was better tolerated, had fewer side effects and was more potent in suppressing HIV replication than the higher doses. CONCLUSIONS: A bimodal, dose-dependent, cytostatic-cytotoxic switch is an immune-based mechanism explaining the apparent paradox that lowering the dose of hydroxyurea to 600 mg daily induces maximal antiviral suppression in HIV-infected patients.

Hydroxyurea exerts a cytostatic but not immunosuppressive effect on T lymphocytes.

OBJECTIVE: To demonstrate that, despite a dose-dependent cytostatic effect, hydroxyurea (HU) does not have immunosuppressive effects. METHODS: The effects of HU on T lymphocyte proliferation parameters, activation phenotype and cytokine production were examined in vitro after exposure to clinically relevant concentrations of HU (10, 50, and 100 micromol/l). The effects of HU in vivo on CD4 T cell counts, viral load, activation phenotype and virus-specific response were examined in 17 Rhesus macaques infected with SIV(mac251) and randomized into three groups: untreated controls; treated with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) and didanosine (ddI) only; and treated with PMPA, didanosine, and HU. RESULTS: The in vitro inhibition of T lymphocyte proliferation confirmed the cytostatic effect of HU, with a linear dose-dependent effect; however, no relevant differences were found in the expression of activation markers between treated and untreated controls. Both T helper type 1 and type 2 cytokine production were enhanced by HU. Consistent with the in vitro results, a blunted increase of peripheral CD4 T cells was observed in vivo in the HU group, without relevant effects on the expression of activation markers, and SIV-specific T cell responses were not affected by HU. CONCLUSIONS: Hyper-proliferation of T-lymphocytes is a major factor contributing to HIV pathogenesis. HU exerts a cytostatic effect on T lymphocytes, without altering their activation and apparently without having an immunosuppressive effect. The increase in cytokine production at the single cell level might compensate for the decrease in the percentage of activated CD4 T lymphocytes, without overall impairment of HIV-specific immune responses.

Control of viral rebound through therapeutic immunization with DermaVir.

OBJECTIVE: To reconstitute immune responses capable of eliminating infected cells and suppressing viral load during chronic retroviral infection. DESIGN: : A topical, DNA-based therapeutic immunization (DermaVir) was designed to express most of the regulatory and structural viral genes in dendritic cells. METHODS: DermaVir alone and in combination with antiretroviral drugs was tested in chronically SIV-infected macaques. RESULTS: DermaVir provided virological, immunological and clinical benefit for SIV-infected macaques during chronic infection and AIDS. In combination with antiretroviral drugs, DermaVir augmented SIV-specific T-cell responses and enhanced control of viral load rebound during treatment interruptions. CONCLUSIONS: The results indicate the feasibility of therapeutic immunization even in immune compromised hosts, and suggest that DermaVir can complement antiretroviral drugs to sustain suppression of HIV-1 replication.

DermaVir: a novel topical vaccine for HIV/AIDS.

Human immunodeficiency virus (HIV) vaccines have the potential to improve antiretroviral drug treatment by inducing cytotoxic killing of HIV-infected cells. Prophylactic vaccines utilize new antigens to initiate immunity; however, in HIV-infected individuals the load of viral antigen is not the limiting factor for the restoration of immune responses. Here we describe a novel immunization strategy with DermaVir that improves viral antigen presentation using dendritic cells (DC). DermaVir contains a distinctive plasmid DNA expressing all HIV proteins except integrase to induce immune responses with broad specificity. The DNA is formulated to a mannosilated particle to target antigen-presenting cells and to protect the DNA from intracellular degradation. After topical application, DermaVir-transduced cells migrate from the skin to the draining lymph node and interdigitate as DermaVir-expressing, antigen-presenting DC. We compared the immunogenicity of topical and ex vivo DC-based DermaVir vaccinations in naive rhesus macaques. Both vaccinations induced simian immunodeficiency virus-specific CD4 helper and CD8 memory T cells detected by an in vivo skin test and an in vitro intracellular cytokine-based assay. Topical DermaVir vaccination represents an improvement upon existing ex vivo DC-based immunization technologies and may provide a new therapeutic option for HIV-infected patients.

Role of STAT6 and mast cells in IL-4- and IL-13-induced alterations in murine intestinal epithelial cell function.

Gastrointestinal nematode infections generally invoke a type 2 cytokine response, characterized by the production of IL-4, IL-5, IL-9, and IL-13. Among these cytokines, IL-4 and IL-13 exhibit a functional overlap that can be explained by the sharing of a common receptor or receptor component (IL-4Ralpha). Binding of IL-4 by either the type 1 or 2 IL-4R, or of IL-13 by the type 2 IL-4R, initiates Jak-dependent tyrosine phosphorylation of the IL-4Ralpha-chain and the transcription factor, STAT6. In the present study, we investigated: 1) whether IL-13 has effects on intestinal epithelial cells similar to those observed with IL-4, and 2) whether the effects of IL-4 and IL-13 depend on STAT6 signaling and/or mast cells. BALB/c, STAT6(-/-), and mast cell-deficient W/W(v) mice or their +/+ littermates were treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or with IL-13 for seven days. Segments of jejunum were mounted in Ussing chambers to measure mucosal permeability; chloride secretion in response to PGE(2), histamine, 5-hydroxytryptamine, or acetylcholine; and Na(+)-linked glucose absorption. IL-4C and IL-13 increased mucosal permeability, decreased glucose absorption, and decreased chloride secretion in response to 5-hydroxytryptamine. These effects were dependent on STAT6 signaling. Responses to PGE(2) and histamine, which were dependent on mast cells and STAT6, were enhanced by IL-4C, but not by IL-13. The effects of IL-4 and IL-13 on intestinal epithelial cell function may play a critical role in host protection against gastrointestinal nematodes.

Quantification of HIV-specific CD8 T cells by in vitro stimulation with inactivated viral particles.

OBJECTIVES: HIV-specific CD8 T cells play a central role in the immune control of virus replication. To further understand the role of CD8 T cells in clinical settings, there is a need for a diagnostic assay that quantifies HIV-specific CD8 T cells in all HIV-infected individuals. DESIGN: and methods: The CD8VIR (CD8 T cell-mediated virus-specific immune response) assay was designed to mimic viral load rebound by adding replication defective HIV particles to peripheral blood mononuclear cells. Antigen presenting cells processed the virus and presented most of the viral epitopes to T cells. Activated HIV-specific CD8 T cells were quantified by flow cytometry analysis as CD3CD8 IFNgamma producing T cells. RESULTS: The CD8VIR assay reproducibly detected a large proportion of functional HIV-specific CD8 T cells responding to viral load rebound. The whole HIV particle stimulation used in the CD8VIR assay was comparable to the sum of Gag, Pol, Env and Nef stimulations. The percentage of HIV-specific CD8 T cells also significantly correlated with the percentage of Gag-specific cytotoxicity measured by the traditional Cr release assay. HIV-specific CD8 T cells correlated with immune control of HIV in chronically infected patients. CONCLUSIONS: The CD8VIR assay quantifies the majority of HIV-specific CD8 T cells capable of killing HIV-infected cells during viral load rebound. This simple, versatile and reproducible assay can be performed from the specimen submitted for CD4 analysis. Upon clinical validation, the CD8VIR assay can be a new diagnostic tool to predict the control of viral load rebound after treatment interruption.

Control of HIV during a structured treatment interruption in chronically infected individuals with vigorous T cell responses.

PURPOSE: To study whether and under what circumstances HIV can be controlled in chronically infected patients. METHOD: Nine patients treated with hydroxyurea and didanosine (PANDAs) were compared with 7 patients on highly active antiretroviral therapy (HAART) during an 8-week treatment interruption. Both groups had similar baseline viral load, CD4 count, and length of treatment. Treatment was resumed if viral rebound >10,000 copies/mL (virological failure) or CD4 count decrease below 200 cells/mm(3) (immunological failure) occurred in two consecutive measurements. RESULTS: None of the PANDAs failed. Viral rebound was spontaneously contained, and CD4 count remained stable. Four out of 7 patients in the HAART group failed to control HIV by week 6 and had to restart therapy due to either viremia rebound or CD4 decrease. Before therapy interruption, the PANDAs had a vigorous HIV-specific T cell immune response (median CD4VIR 1.2%), while the HAART-treated patients did not (median CD4VIR 0.2%) (CD4VIR represents the percentage of HIV-specific CD4 subpopulation expressing IFN-gamma within the total CD4 population [CD3+, CD4+, IFN-gamma+]). This difference was statistically significant (p =.002). CONCLUSION: This study shows that HIV can be controlled during therapy interruption in patients with established infection, and that control of viral replication correlates with vigorous anti-HIV specific immune responses.

Methods of using interleukin 2 to enhance HIV-specific immune responses.

Interleukin 2 (IL-2) in combination with highly active antiretroviral therapy (HAART) can significantly increase CD4+ T cell counts but does not improve HIV-specific T cell-mediated immune responses that are associated with the control of viral replication. To characterize the immunomodulatory activity of IL-2 in HIV-infected individuals we studied the virus-specific immune response (VIR) by intracellular cytokine expression (interferon gamma, IFN-gamma) after mimicking HIV rebound in peripheral blood mononuclear cells (PBMCs). We found that the whole virus used as HIV antigen was able to activate HIV-specific T cells in the presence of a low concentration (50 IU/ml) of IL-2. Interestingly, increasing concentrations of IL-2 (400 or 1000 IU/ml) in combination with the same amount of HIV doubled the number of HIV-specific T cells. These cells were functionally intact because all of the IFN-gamma-producing CD8+ T cells contained perforin, a marker for cytotoxic T lymphocytes (CTLs). Induction of HIV-specific immune responses by IL-2 was not detected in the absence of HIV antigen both in vitro and in patients treated with HAART, indicating that IL-2 can amplify HIV-specific T cells in the presence of HIV antigen. Therefore, a combination of IL-2 with either structured treatment interruption, which results in a controlled viral load rebound, or with therapeutic vaccination is expected to improve HIV-specific T cell-mediated immune responses.