Quantitative Viral Outgrowth Assay to Measure the Functional SIV Reservoir in Myeloid Cells.

The role of CD4+ T cells in HIV infection and the latent reservoir, that is, latently infected cells that harbor replication competent virus, has been rigorously assessed. We have previously reported a quantitative viral outgrowth assay (QVOA) for SIV that demonstrated the frequency of latently infected CD4+ T cells is approximately 1 in a million cells, similar to that of HIV infected individuals on ART. However, the frequency of productively infected monocytes in blood and macrophages in tissues has not been similarly studied. Myeloid cells are infected during acute HIV and SIV infection; however, unlike lymphocytes, they are resistant to the cytopathic effects of the virus. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a stable viral reservoir. A better understanding of the number of productively infected macrophages is critical to understanding the role of infected myeloid cells as a viral reservoir. In order to assess the functional latent reservoir. we have developed specific QVOAs for monocytes in blood, and macrophages in spleen, BAL and brain, which are described in detail in this chapter.

Cerebrospinal fluid markers that predict SIV CNS disease.

Predictive cerebrospinal fluid markers would provide valuable tools for tracking the development and progression of HIV CNS disease. In this study, expression of IL-6, MCP-1, and viral RNA in cerebrospinal fluid collected from SIV-inoculated macaques during acute, asymptomatic, and terminal stages of infection was quantitated to determine whether one or several of these parameters paralleled the severity of SIV encephalitis. Animals that developed moderate to severe SIV encephalitis had significantly elevated levels of CSF IL-6, MCP-1, and SIV RNA during asymptomatic infection and persisting through terminal disease as compared to animals developing mild or no CNS disease.

Accumulation of beta-amyloid precursor protein in axons correlates with CNS expression of SIV gp41.

Axonal damage represented by accumulation of beta-amyloid precursor protein (beta-APP) develops in numerous central nervous system (CNS) diseases including human immunodeficiency virus (HIV) infection. To study the underlying mechanisms of axonal damage associated with HIV CNS infection, the amount of axonal beta-APP immunostaining in the corpus callosum of 24 simian immunodeficiency virus (SIV)-infected macaques and 3 control macaques was measured by computerized image analysis. The amounts of beta-APP accumulation were then compared with time post-inoculation, extent and character of CNS inflammation, and viral load in the CNS measured by the amount of immunohistochemical staining for the viral transmembrane protein gp41. Significant increases over control values were present in 10 of 24 SIV-infected animals. SIV encephalitis was present in 9 of the 10 animals with elevated beta-APP Increases in beta-APP correlated most strongly with levels of SIV gp41 in the brain (p = 0.005), but significant associations with macrophage infiltration and microglial activation (p = 0.04) and infiltration by cytotoxic lymphocytes (p = 0.05) also were identified. These data demonstrate that beta-APP accumulation in the white matter of SIV-infected macaques develops during SIV infection in close correlation with levels of viral replication and may serve as a sensitive marker of neuronal/axonal damage mediated by viral proteins.

Alterations in blood-brain barrier glucose transport in SIV-infected macaques.

The neurological manifestations of HIV infection may be in part due to alterations in the blood-brain barrier. These may be caused by structural changes in the barrier or may consist of subtle metabolic or biochemical disturbances in barrier function. In the CNS, the family of glucose transporter proteins plays a key role in controlling movement of glucose across cell membranes. The 55 kDa isoform of glucose transporter 1 (GLUT1) regulates import of glucose from blood to brain across the endothelial cells of the blood-brain barrier (BBB), whereas the 45 kDa form of GLUT1 predominantly regulates nonvascular glial glucose uptake. In this study, expression of 55 and 45 kDa forms of GLUT1 in different regions of the brain from 18 SIV-infected macaques was measured by quantitative immunoblot and then compared with the severity of SIV encephalitis to determine whether neurologic disease is related to altered glucose metabolism at the BBB and in brain parenchyma. An inverse relationship was found between severity of SIV encephalitis and expression of the endothelial 55 kDa isoform of GLUT1 at the BBB in cortical grey matter, caudate nucleus, and cerebellum. A similar relationship also was found for the glial 45 kDa GLUT1 isoform in cortical grey matter. In addition, a significant increase in 55 kDa GLUT1 expression was found in caudate nucleus during the early stages of infection. In the brains of macaques with moderate to severe encephalitis, 55 kDa GLUT1 expression had declined to pre-infection levels. These GLUT1 alterations at the BBB and in glial cells may reflect severe disturbances in the CNS microenvironment that contribute to CNS dysfunction.

Sleep reduction in night-shift workers: is it sleep deprivation or a sleep disturbance disorder?

Two explanations of the reported reduction in night-shift workers' sleep length have emerged. One argues that these reductions in sleep are related to sleep disturbances which may require medical treatment as disorders. The other argues that sleep reduction is mainly due to a sleep deficit caused by the voluntary choice to delay sleep onset and reduce the sleep duration which requires a systems approach aimed at increasing sleep length and reducing the effect of a sleep deficit at the workplace. Using survey data from 1078 industrial shift workers, this study demonstrates that permanent night workers reporting sleep difficulties, as well as those who do not, report sleep reductions. This finding is interpreted as supporting, in part, the position that night-shift workers suffer primarily from a self-induced sleep deficit.