Publication: Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction
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Date
2006
Authors
Tanji, N. ; Ross, M.D. ; Tanji, K. ; Bruggeman, L.A. ; Markowitz, G.S. ; Klotman, P.E. ; D’Agati, V.D.
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
The localization of HIV-1 DNA in renal
tissues is critically important for understanding
pathogenesis of HIV-associated nephropathy (HIVAN),
but the clarification has been technically challenging.
We applied in situ polymerase chain reaction (IS-PCR)
to human renal tissues to demonstrate viral entry into the
renal epithelial cells in vivo.
To test the specificity of this method and to
determine the cell types infected, we used IS-PCR
followed by in situ hybridization (ISH) and IS-PCR
followed by immunohistochemistry and histochemical
counterstains.
Brief 2 hour fixation in 4% paraformaldehyde had
92.9% sensitivity and 100% specificity for detection of
viral DNA in renal biopsies of HIVAN patients,
compared to 70.8% sensitivity and 66.7% specificity in
renal biopsies fixed overnight in 10% formalin. Under
optimized conditions, the only signals detectable in HIV-
1 seronegative cases were false positives attributable to
renal tubular apoptosis. In HIVAN cases, positive signal
was observed in podocytes, parietal cells, renal tubular
cells, and interstitial leukocytes. Immunohistochemical
co-labeling for pan-T cell and macrophage markers
revealed that the interstitial leukocytes with positivity
for HIV-1 DNA included both T cells and macrophages Application of ISH after IS-PCR showed the same
distribution of signal as observed using IS-PCR alone,
confirming the specificity of the technique.
IS-PCR is a powerful technique to detect viral DNA
in human tissue sections, but requires proper use of
negative controls to set optimal fixation, protein
digestion, and amplification conditions.
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