Progressive multifocal leukoencephalopathy (PML) is a debilitating demyelinating disease of the CNS caused by the infection and destruction Bumetanide of glial cells by JC virus (JCV) and is an AIDS-defining disease. reactivation of JCV by cytokine disturbances in the brain such as occur in HIV-1/AIDS. In this study we evaluated HIV-1/PML clinical samples and non-PML controls for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4. Consistent with our hypothesis HIV-1/PML tissue has high levels of Bumetanide TNF-α and TNFR1 expression and NF-κB and NFAT4 were preferentially localized to the nucleus. Keywords: Progressive multifocal leukoencephalopathy Human polyomavirus JC Tumor necrosis factor-α NF-κB NFAT4 proinflammatory cytokines viral reactivation INTRODUCTION The CNS demyelinating disease progressive multifocal leukoencephalopathy (PML) is characterized by a triad of histopathological features: demyelination bizarre astrocytes and enlarged oligodendrocytes with nuclear inclusion bodies [1 2 PML is manifested by motor deficits gait ataxia cognitive and behavioral changes language disturbances Bumetanide weakness or visual deficits with symptoms depending on the location and size of the lesions. It is caused Bumetanide by the ubiquitous polyomavirus JC (JCV) which infects most people in childhood as indicated by seroprevalence studies but thereafter is controlled by the immune system and becomes restricted to a persistent asymptomatic infection. However PML is rare and seen predominantly in individuals with underlying immune dysfunction most notably HIV-1/AIDS and in patients receiving immunomodulatory drugs such as natalizumab an α4β1 integrin inhibitor used to treat multiple sclerosis and Crohn’s disease [3]. Since the number of individuals that constitute the at-risk population is large PML has high public health significance. While seroprevalence studies show that most people are infected with JCV only very rarely and almost always under conditions of severe immune compromise does the virus reactivate from the persistent state and actively replicate causing cytolytic cell destruction. Replication of the virus occurs in the glia of the CNS PML i.e. astrocytes and oligodendrocytes thus leading to the generation of expanding demyelinated lesions and the associated pathologies of PML [4]. While the mechanism of reactivation remains unresolved our molecular and virological studies of JCV in primary human glial cultures have implicated transcription factors NF-κB [5] and NFAT4 [6]. The genome of JCV is a circular double-stranded DNA divided into three regions the early region encoding the viral early proteins (large and small T/t-antigens) late region encoding the late proteins (VP1 VP2 VP3 and agnoprotein) and the noncoding control region (NCCR) that controls transcription of both coding regions [7]. The NCCR binds multiple transcription factors that regulate JCV [8]. Cdx2 NF-κB [5] and NFAT4 [6] bind to a unique site in the NCCR and activate transcription of viral early and late genes. In turn these transcription factors are regulated by signal transduction pathways that lie downstream of pro-inflammatory cytokines which may be dysregulated in conditions that predispose to PML e.g. cytokine storms in HIV-1/AIDS. In experiments with cultured human glia we have found that TNF-α stimulates JCV transcription and that this effect is mediated through the same unique site in the JCV NCCR [9]. In addition epigenetic changes in the acetylation status of NF-κB can also activate JCV transcription [10 11 If the mechanisms that we have demonstrated in culture such as cytokine (TNF-α) stimulation of transcription factors (NF-κB and NFAT4) are at play during the pathogenesis of HIV-1/PML we would expect to detect these changes in cytokines and transcription factors in HIV-1/PML tissue compared to non-PML controls. In this context we evaluated brain tissues from HIV patients with and without PML for expression of TNF-α and its receptors and the subcellular localization of NF-κB p65 and NFAT4. If our hypothesis regarding the importance of TNF-α is correct we would expect Bumetanide to detect increased TNF-α in PML clinical samples and subcellular localization of NF-κB and NFAT4 to the nucleus. MATERIALS AND METHODS Clinical Samples Two sets of brain clinical samples were used.