The major findings of our HTLV-I studies have been: 1) Increased HTLV-I specific CD8+ cells have been shown to be elevated in the peripheral blood and CSF of HAM/TSP patients and directly proportional to the amount of HTLV-I proviral DNA and RNA. These antigen-specific T cells are considered to be immunopathogenic and may be directly involved in virus-host interactions in the CNS 2) We demonstrated that spontaneous degranulation and IFN-gamma production (defined by CD107+ IFN-gamma+ cells) was correlated with proviral DNA load in CD14+ macrophages from HTLV-I-infected patients, and that enhanced IL-15 expression on CD14+ cells mediated the dysregulation of CD8+ T cells in HAM/TSP patients. The observation of HTLV-I infected macrophages is novel and suggests a wider tropism for this virus than previously reported. The effect of HTLV-I on CD14+ macrophages is also being investigated. 3) CD4+CD25+ regulatory T cells are important in the maintenance of immunological self-tolerance and in the prevention of autoimmune diseases. We have demonstrated that in HTLV-I infected CD4+CD25+ T cells of patients with HAM/TSP the expression of the forkhead transcription factor Foxp3, a specific marker of regulatory T cells, was lower than that of healthy individuals. In addition, we are investigating the demethylated Fox3 status of HAM/TSP patients and have shown that there is decreased demethylation in PBMC and CD4+CD25+ T cells compared to healthy donors. 4) Basic laboratory investigations have demonstrated the importance of the cytokine IL-15 in the life and death of lymphocytes and for its role in autoimmune disorders such as HAM/TSP. IL-15 is pivotally involved in the survival of CD8+ memory T-cells including self-directed cells and we have shown that in HAM/TSP, IL-15 is essential for the survival of HLA class I restricted virus antigen-specific effector and memory CD8+ T-cells. These CD8+ antigen-specific CTL are thought to play a major role in the immunopathogenesis of HAM/TSP since they have been localized in brain and spinal cord sections of patients. As IL-15 is a pro-inflammatory cytokine that stimulates the production of inflammatory cytokines, the release of IL-15 induced by HTLV-I tax in patients with HAM/TSP may underlie the pathogenesis of this autoimmune disease. The mode of action of IL-15 and its receptor subunits IL-15 and IL-15Ralpha are coordinately stimulated and expressed following HTLV-I tax stimulation. We have shown that this trans-stimulation can be virtually totally inhibited for NK and CD8+ T-cells by the addition of a humanized monoclonal antibody, Hu MiKbeta1, that blocks IL-15 binding to the IL-2/IL15Rbeta;receptor subunit expressed on these cells. Collaborative research by the Viral Immunology Section, NINDS and the Metabolism Branch, NCI have initiated a phase I clinical trial for the treatment of HAM/TSP using Hu MiK-beta-1 that blocks the action of IL-15. 5) The HTLV-I induced activation of nuclear factor kappa B (NFkappaB ) is considered a key event in the leukemogenesis leading to ATLL, but less has been demonstrated regarding the role of NFkappaB activation in the pathogenesis of HAM/TSP. We have shown evidence of both canonical and non-canonical NFKappaB activation in short-term cultures of PBMC from subjects with HAM/TSP, in the absence of exogenous stimulation. We show that the NFkappaB activation in HAM/TSP PBMC can be reversed by small molecule inhibitors. Inhibition of NFkappaB activation in HAM/TSP PBMC resulted in the inhibition of STAT5 activation and spontaneous lymphoproliferation (SP), two ex vivo correlates of the immune activation associated with HAM/TSP. These results indicate that NFkappaB activation plays a critical upstream role in the immune activation associated with HAM/TSP, and identify the NFkappaB pathway as a potential target for immune modulation in HAM/TSP. 6) This understanding of the roles of IL-2/IL-15 in the pathogenesis of HAM/TSP has led to targeted therapeutic approaches. Additionally, given the fact that IL-2/IL-15 signal through the common gamma chain (γC) receptor, a novel potentially more effective targeted therapeutic approach to HAM/TSP might consist of blocking the γC with an antagonizing peptide. This would inhibit the action of multiple cytokines implicated in the disease and might be better tolerated than monoclonal antibodies or small molecule inhibitors. BNZ-γis a novel anti-cytokine peptide that was designed based on the presence of a moderately conserved region among all the cytokines sharing the γC receptor, overlapping with the D-helix. It is a 19-mer peptide (BNZ-g-001) that, based on proliferation assays in cytokine-dependent human and murine cells lines, was found to selectively block signaling of IL-2, IL-15, and IL-9. We have examined the effects of BNZ- γon multiple in vitro markers of immune activation in HAM/TSP. The peptide significantly suppressed spontaneous proliferation (SP) of the PBMCs, reduced the frequency of Tax-specific CD8+ T-cells, and the degree of STAT5 phosphorylation after 20h of culture. It was as effective as a combination of monoclonal antibodies against CD25 and CD122 in reducing SP and STAT5 phosphorylation. 7) Using a genomic SNP analysis of a unique cohort of clinically and radiologically well-characterized MS patients enrolled in a BioMS biomarker study, we are identifying regions of the human genome that may be specific for MS versus a general, healthy population. In addition, we are identifying SNPs that may define subsets of MS patients with high burden of disease as measured by MRI. Once these SNPs are validated on additional MS patient cohorts, we will begin laboratory investigations to determine if gene products of these genomic regions will be informative biomarkers for this disease. 8) We continue to extend our work on the detection of the human herpesvirus (HHV-6) from brain resections of patients with mesial temporal lobe epilepsy and patients with neurologic complications following allogeneic bone marrow transplants. We have utilized quantitative real-time PCR for the detection of HHV-6 sequences and developed a novel electrochemiluminescent ELISA method for the quantitative detection of antibodies to HHV-6 IgG. We have screened large panels of sera from patients with MS, encephalitis and controls. Preliminary results suggest significantly more HHV-6 DNA in patients with encephalitis of unknown origin than previously reported. 9) Using a novel and highly sensitive digital droplet PCR (ddPCR) assay, we investigated the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and MS patients. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus. In our comparison of MS patients with healthy donors, we detected an elevated frequency of coinfection in MS saliva. Identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of HHV-6A and HHV-6B in health and disease. 10) A number of laboratory studies have demonstrated that virus infection can activate a variety of CNS-specific cells including microglia and astrocytes. The VIS has investigated the effects of both a human retrovirus (HTLV-I) and herpesvirus (HHV-6) in these cell populations, in vitro. We are attempting to translate these observations to the patient by imaging studies that can detect activated microglia/macrophage and reactive astrocytes. In collaboration with the Molecular Imaging Branch, NIMH, we have begun to investigate the expression of the peripheral benzodiazepine receptor (PBR) in the CNS that has been show be a marker of activated microglial activation.

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Anderson, Monique R; Enose-Akahata, Yoshimi; Massoud, Raya et al. (2014) Epigenetic modification of the FoxP3 TSDR in HAM/TSP decreases the functional suppression of Tregs. J Neuroimmune Pharmacol 9:522-32
Liu, Winston; Nair, Govind; Vuolo, Luisa et al. (2014) In vivo imaging of spinal cord atrophy in neuroinflammatory diseases. Ann Neurol 76:370-8
Sagar, Divya; Masih, Shet; Schell, Todd et al. (2014) In vivo immunogenicity of Tax(11-19) epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine. Vaccine 32:3274-84
Leibovitch, Emily C; Brunetto, Giovanna S; Caruso, Breanna et al. (2014) Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS One 9:e92328
Brunetto, Giovanna S; Massoud, Raya; Leibovitch, Emily C et al. (2014) Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations. J Neurovirol 20:341-51
Leibovitch, Emily C; Jacobson, Steven (2014) Evidence linking HHV-6 with multiple sclerosis: an update. Curr Opin Virol 9:127-33
Ablashi, Dharam; Agut, Henri; Alvarez-Lafuente, Roberto et al. (2014) Classification of HHV-6A and HHV-6B as distinct viruses. Arch Virol 159:863-70
Virtanen, J O; Wohler, J; Fenton, K et al. (2014) Oligoclonal bands in multiple sclerosis reactive against two herpesviruses and association with magnetic resonance imaging findings. Mult Scler 20:27-34
Leibovitch, Emily; Wohler, Jillian E; Cummings Macri, Sheila M et al. (2013) Novel marmoset (Callithrix jacchus) model of human Herpesvirus 6A and 6B infections: immunologic, virologic and radiologic characterization. PLoS Pathog 9:e1003138
Harberts, Erin; Datta, Dibyadeep; Chen, Selby et al. (2013) Translocator protein 18 kDa (TSPO) expression in multiple sclerosis patients. J Neuroimmune Pharmacol 8:51-7

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