Successful Application of Hyperbranched Multidisplacement Genomic Amplification to Detect HIV-1 Sequences in Single Neurons Removed from Autopsy Brain.

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Successful Application of Hyperbranched Multidisplacement Genomic Amplification to Detect HIV-1 Sequences in Single Neurons Removed from Autopsy Brain Sections by Laser Capture Microdissection Jorge E. Torres-Muñoz, Mariana Núñez, Carol K. Petito The Journal of Molecular Diagnostics Volume 10, Issue 4, Pages 317-324 (July 2008) DOI: 10.2353/jmoldx.2008.070074 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 CD8+ T lymphocyte immunohistochemistry of an AIDS patient with HIV-1 encephalitis in the CA4 subregion of the hippocampus. The CD8+ T lymphocytes localize to the microglial nodules of HIV-1 encephalitis (A) and diffusely infiltrate adjacent brain (B). Arrow indicates the cell-to-cell contacts between pyramidal neurons and perineuronal CD8+ T cells. Hematoxylin counterstain; original magnification ×400. The Journal of Molecular Diagnostics 2008 10, 317-324DOI: (10.2353/jmoldx.2008.070074) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: HIV encephalitis with infiltrating CD8+ T lymphocytes (brown) in the CA4 region of hippocampus, as seen with the laser capture microscope. B–D: Successful dissection and capture of a CD8+ pyramidal neuron (arrows). B shows the neuron and its perineuronal CD8+ T cell before microdissection. C shows the empty space on the tissue section that remains after successful microdissection; note that the perineuronal T cell has not been captured. D shows the Transfer Cap to which only the neuron adheres. Hematoxylin counterstain; original magnifications ×200 (A); ×400 (B–D). The Journal of Molecular Diagnostics 2008 10, 317-324DOI: (10.2353/jmoldx.2008.070074) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Real-time PCR SYBR Green Tm graph for β2-microglobulin (A) and HIV-1 nef (B). Vertical lines are used to point out PCR product Tm. The blue line with arrow shows the Tm of one positive control and several positive neuronal samples. The red line with arrow shows the Tm from negative control and several negative samples. Default green and yellow lines were not used. The Journal of Molecular Diagnostics 2008 10, 317-324DOI: (10.2353/jmoldx.2008.070074) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Housekeeping gene sequence amplification was related to whole-gene amplification before double-round PCR in single neurons (A) and to sample size in the HIVE and control groups (B) but was unaffected by postmortem intervals (C). The Journal of Molecular Diagnostics 2008 10, 317-324DOI: (10.2353/jmoldx.2008.070074) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 In housekeeping gene-positive neurons, HIV-1 viral sequences were more common in the HIVE cases than in the HIVnE cases; they were absent in uninfected controls (A). HIV-1 viral sequences were more common in housekeeping gene-positive neuronal samples that were in direct contact with perineuronal CD8+ T lymphocytes than in neuronal samples that had no contacts with adjacent cells (B). The Journal of Molecular Diagnostics 2008 10, 317-324DOI: (10.2353/jmoldx.2008.070074) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions