Kamel Khalili

Cellular reservoirs of Hiv-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry

Objectives The majority of HIV-1-infected individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including AIDS dementia complex, encephalitis, and various other disorders of the CNS. The present study sought to evaluate the cellular reservoirs and expression patterns of HIV-1 in brain tissue to gain further understanding of HIV-1 neuropathogenesis. Design CNS tissue, obtained post-mortem from 22 patients with AIDS and four HIV-1-seronegative controls, was analyzed. Methods CNS samples were evaluated using a combination of in situ DNA polymerase chain reaction (PCR), reverse transcriptase (RT)-initiated in situ PCR, and immunohistochemistry. By utilizing this triple-staining methodology, HIV-1 proviral DNA and HIV-1-specific mRNA can be identified at the single cell level. Results HIV-1 was detected in all 22 AIDS brain specimens and in none of the four brains from HIV-1-seronegative individuals. The most commonly infected cells in AIDS brains were microglia cells and macrophages, but variable levels of HIV-1 infection were demonstrated in many of the major histological cell types within the CNS, including neurons, microvascular endothelial cells (MVEC) and astrocytes. The presence of HIV-1-infected cells was not uniform with infected cells unevenly distributed throughout the brain parenchyma. The degree of HIV-1 mRNA expression varied from 39–65% of the cells in the CNS harboring HIV-1 provirus. Choroid plexus and MVEC exhibited relatively high levels of productive infection. Conclusions These findings demonstrate that several cell types in the CNS, in addition to microglia or macrophages, may become infected with HIV-1 in vivo with variable levels of HIV-1 mRNA expression. The diverse cellular reservoirs for HIV-1 in the CNS may be critically linked to the molecular mechanisms involved in HIV-1 neuropathogenesis. In addition, in vivo infection of MVEC, and possibly cells in the choroid plexus, may directly contribute to penetration of the blood-brain barrier by HIV-1.

CNS invasion by CD14+/CD16+peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection

Increases in circulating CD14+/CD16+ monocytes have been associated with HIV dementia; trafficking of these cells into the CNS has been proposed to play an important role in the pathogenesis of HIV-induced neurological disorders. This model suggests that events outside the CNS leading to monocyte activation initiate the process leading to HIV dementia. To investigate the role of this activated monocyte subset in the pathogenesis of HIV dementia, we examined brain specimens from patients with HIV encephalopathy (HIVE), HIV without encephalopathy, and seronegative controls. An accumulation of perivascular macrophages was observed in HIVE. The majority of these cells identified in microglial nodules and in the perivascular infiltrate were CD14+/CD16+. P24 antigen colocalized with both CD14 and CD16 suggesting that the CD14+/CD16+ macrophage is a major reservoir of HFV-1 infection in CNS. Using CD45/LCA staining, the perivascular macrophage was distinguished from resident microglia. In addition to perivascular and nodular localizations, CD16 also stained ramified cells throughout the white matter. These cells were more ramified and abundant than cells positive for CD14 in white matter. Double staining for p24 and CD16 suggests that these cells were often infected with HIV-1. The prominent distribution of CD14+ cells in HIVE prompted our analysis of soluble CD14 levels in cerebrospinal fluid. Higher levels of soluble CD14 (sCD14) were observed in patients with moderate-to-severe HIV dementia, suggesting the utility of sCD14 as a surrogate marker. CD14+/CD16+ monocytes may play a role in other neurological disorders and sCDl4 may be useful for evaluating these conditions.

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

Significance For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly. AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

Molecular Biology, Epidemiology, and Pathogenesis of Progressive Multifocal Leukoencephalopathy, the JC Virus-Induced Demyelinating Disease of the Human Brain

SUMMARY Progressive multifocal leukoencephalopathy (PML) is a debilitating and frequently fatal central nervous system (CNS) demyelinating disease caused by JC virus (JCV), for which there is currently no effective treatment. Lytic infection of oligodendrocytes in the brain leads to their eventual destruction and progressive demyelination, resulting in multiple foci of lesions in the white matter of the brain. Before the mid-1980s, PML was a relatively rare disease, reported to occur primarily in those with underlying neoplastic conditions affecting immune function and, more rarely, in allograft recipients receiving immunosuppressive drugs. However, with the onset of the AIDS pandemic, the incidence of PML has increased dramatically. Approximately 3 to 5% of HIV-infected individuals will develop PML, which is classified as an AIDS-defining illness. In addition, the recent advent of humanized monoclonal antibody therapy for the treatment of autoimmune inflammatory diseases such as multiple sclerosis (MS) and Crohns disease has also led to an increased risk of PML as a side effect of immunotherapy. Thus, the study of JCV and the elucidation of the underlying causes of PML are important and active areas of research that may lead to new insights into immune function and host antiviral defense, as well as to potential new therapies.

Cerebral Hypoperfusion Generates Cortical Watershed Microinfarcts in Alzheimer Disease

Background and Purpose— The watershed cortical areas are the first to be deprived of sufficient blood flow in the event of cerebral hypoperfusion and will be the sites of watershed microinfarcts. Cerebral hypoperfusion is associated with Alzheimer disease (AD), but information regarding the occurrence of watershed cortical infarcts in AD is lacking. Methods— Brains of 184 autopsy cases (105 definite AD cases and 79 age-matched controls) were selected and analyzed by histochemical and immunohistochemical techniques. The 3-dimensional reconstruction of the whole cerebrum, with 3-mm spaced serial sections, was performed in 6 AD cases to study the intrahemispheric and interhemispheric distribution of the cortical microinfarcts. Results— A significant association (P =0.001) was found between the occurrence of watershed cortical infarcts and AD (32.4% versus 2.5% in controls). The microinfarcts were restricted to the watershed cortical zones. Congophilic angiopathy was revealed to be an important risk factor. Perturbed hemodynamic factors (eg, decreased blood pressure) may play a role in the genesis of cortical watershed microinfarcts. Conclusions— In AD, cerebral hypoperfusion induces not only white matter changes but cortical watershed microinfarcts as well, further aggravating the degenerative process and worsening dementia. To prevent the formation of watershed cortical microinfarcts in AD, monitoring blood pressure and treating arterial hypotension are essential.

HIV-1 associated dementia: symptoms and causes

Despite the use of highly active antiretroviral therapy (HAART), neuronal cell death remains a problem that is frequently found in the brains of HIV-1-infected patients. HAART has successfully prevented many of the former end-stage complications of AIDS, however, with increased survival times, the prevalence of minor HIV-1 associated cognitive impairment appears to be rising among AIDS patients. Further, HIV-1 associated dementia (HAD) is still prevalent in treated patients as well as attenuated forms of HAD and CNS opportunistic disorders. HIV-associated cognitive impairment correlates with the increased presence in the CNS of activated, though not necessarily HIV-1-infected, microglia and CNS macrophages. This suggests that indirect mechanisms of neuronal injury and loss/death occur in HIV/AIDS as a basis for dementia since neurons are not themselves productively infected by HIV-1. In this review, we discussed the symptoms and causes leading to HAD. Outcome from this review will provide new information regarding mechanisms of neuronal loss in AIDS patients.

Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat.

The broad range of histological lesions associated with HIV‐1 are somewhat subtle relative to the clinical manifestations that occur as a result of HIV infection. Although it is clear that HIV has a causative role in CNS disease, dementia appears to be a consequence of the infiltration of inflammatory cells and cytokine dysregulation rather than the amount of virus in CNS. The HIV transregulatory protein Tat plays an important intracellular as well as extracellular role in the dysregulation of cytokines. The cytokines and possibly chemokines that are induced by Tat modify the action of astrocytes such that the survival of neurons is compromised. Pathogenetic alteration induced by Tat involves a series of interactions between circulating monocyte/macrophages, endothelial cells, and astrocytes. Cytokine dysregulation induced by viral infection and extracellular Tat leads to alterations in expression of adhesion molecules and promotes migration of non‐infected inflammatory cells into the CNS compartment. We demonstrate here that recombinant HIV‐1 Tat protein introduced by stereotaxic injection into mouse brain can induce pathologically relevant alterations including macrophage invasion as well as astrocytosis. The mechanism of destruction of the CNS by Tat appears to involve autocrine and paracrine pathways that depend not only on Tat, but cytokine and chemokine signaling pathways that are altered by viral infection. In this review, we discuss various pathogenic effects of Tat in brain cells and provide experimental evidence for an increased TNF‐α level in CSF in mice injected intracerebrally with Tat protein. J. Leukoc. Biol. 65: 458–465; 1999.

Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy

HIV-1 infection can lead to severe central nervous system (CNS) clinical syndromes in more than 50% of HIV-1 positive individuals. Progressive multifocal leukoencephalopathy (PML) is the frequent opportunistic infection of the CNS which is seen in as high as 5% of AIDS patients. Results from previous cell culture studies showed that the HIV-1 regulatory protein, Tat can potentiate transcription of the human neurotropic virus, JCV, the causative agent for PML in cells derived from the human CNS. In this communication we examine the presence of the HIV-1 regulatory protein, Tat, as well as the HIV-1 and JCV structural proteins, p24 and VP1, respectively in AIDS/PML clinical samples. We demonstrate high level expression of the JCV capsid protein, VP1, in oligodendrocytes and to some degree in astrocytes of AIDS with PML. In HIV-1+ samples expression of HIV-1 core protein, p24 was detected in perivascular monocytic cells and to a lesser extent in astrocytes and endothelial cells. A lack of p24 expression in oligodendrocytes suggested no infection of these cells with HIV-1. Interestingly, HIV-1 Tat was detected in various infected cells as well as in uninfected oligodendrocytes from HIV-1+ tissue, supporting the earlier in vitro findings that secreted Tat from the infected cells can be localized in the neighboring uninfected cells. The presence of Tat in oligodendrocytes was particularly interesting as this protein can up-modulate JCV gene transcription and several key cell cycle regulatory proteins including cyclin E, Cdk2, and pRb. The data presented here provide in vivo evidence for a role of HIV-1 Tat in the pathogenesis of AIDS/PML by acting as a positive regulatory protein that affects the expression of JCV and other cell regulatory proteins in the CNS.

Human neurotropic polyomavirus, JCV, and its role in carcinogenesis.

A number of recent studies have reported the detection of the ubiquitous human polyomavirus, JC virus (JCV), in samples derived from several types of neural as well as non-neural human tumors. The human neurotropic JCV was first identified as the etiologic agent of the fatal demyelinating disease, progressive multifocal leukoencephalopathy, which usually occurs in individuals with defects in cell-mediated immunity, including AIDS. However, upon mounting evidence of the oncogenic potential of the viral regulatory protein, T-antigen, and JCVs oncogenecity in a broad range of animal models, studies were initiated to determine its potential involvement in human carcinogenesis. Initially, the most frequently observed tumors in rodent models, including medulloblastoma, astrocytoma, glioblastoma, and other neural-origin tumors were analysed. These studies were followed by analysis of non-neural tumors such as colorectal carcinomas. In a subset of each tumor type examined, JC viral genomic DNA sequences could be detected by PCR and confirmed by Southern blot hybridization or direct sequencing. In a smaller subset of the tumors, the expression of T-antigen was observed by immunohistochemical analysis. Owing to the established functions of T-antigen including its ability to interact with tumor suppressor proteins such as Rb and p53, and its ability to influence chromosomal stability, potential mechanisms of JCV T-antigen-mediated cellular dysregulation are discussed. Further, as increasing evidence suggests that T-antigen is not required for maintenance of a transformed phenotype, a hit-and-run model for T-antigen-induced transformation is proposed.

Purα Is Essential for Postnatal Brain Development and Developmentally Coupled Cellular Proliferation As Revealed by Genetic Inactivation in the Mouse

ABSTRACT The single-stranded DNA- and RNA-binding protein, Purα, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA−/− mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA−/− mice. Levels of proliferation were also absent or low in several other tissues of the PURA−/− mice, including those of myeloid lineage, whereas those of PURA+/− mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Purα and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA+/+ mice, but both were absent in dendrites of PURA−/− mice. Purα and Cdk5 can be coimmunoprecipitated from brain lysates of PURA+/+ mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Purα in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.