This exploratory research proposal addresses the question of whether HHV6 infection of human glial precursor (hGRP) cells induces alterations in proliferation, cell-cycling, and differentiation that could plausibly contribute to the failure of myelin repair at sites of white matter injury in viral leukoencephalitides. By the use of novel, fluorescent-labeled preparations of purified, cell-free HHV6A and HHV6B (S.A.1) the """"""""direct"""""""" effects of virus infection upon hGRP cell cycling, differentiation, and maturation can be examined for the first time. For these experiments, standard methods for measuring cell proliferation (BrdU or Ki67), cell cycle profile (FACScan), and differentiation (immunological markers for cell surface antigens) will be examined. Surface markers for oligodendroglial differentiation include O1, 04, GalC, and MBP and for astrocytic differentiation, GFAP and S-100; these will be compared to expression of the early glial progenitor cell marker, A2B5. An alternative means by which viral infection may modulate precursor cell function is through """"""""indirect"""""""" effects of soluble factors, notably the induction of cytokines and other soluble factors produced by virus-infected lymphocytes and resident brain microglia (S.A.2). Using already-obtained microarray data of HHV6- infected lymphocytes and primary human microglia, obtained from surgical epilepsy resections, we have demonstrated the induction of soluble growth and differentiation factors, cyclins, chemokines, and their receptors, and a strong and charateristic Th-1 type pro-inflammatory response. Specific candidate molecules elaborated by HHV6A or B infected immune cells will therefore be examined for their effects on hGRP cell proliferation, cell-cycling, differentiation, and maturation by the addition of the recombinant molecules with or without specific blocking antisera to defined media, and measurement using the same markers and techniques as above. Potential confounding """"""""direct"""""""" effects of the virus will be eliminated through the use of 0.1 ? pore-size membranes in a transwell format which effectively excludes infectious virus.
| Mock, David J; Strathmann, Frederick; Blumberg, Benjamin M et al. (2006) Infection of murine oligodendroglial precursor cells with Human Herpesvirus 6 (HHV-6)--establishment of a murine in vitro model. J Clin Virol 37 Suppl 1:S17-23 |
