Quantitation by competitive PCR of HIV-1 proviral DNA in epidermal Langerhans cells of HIV-infected patients.
Langerhans cells (LC) belong to the dendritic cell family and represent the principal antigen presenting cells populating squamous epithelia. We have reported the presence of human immunodeficiency virus Type 1 (HIV-1) proviral DNA and RNA in purified LC from the epidermis of seropositive patients. The aim of this study was to quantify HIV-1 proviral DNA in LC of infected patients using a competitive polymerase chain reaction (PCR) assay. Bulk epidermal cell (EC) suspensions were obtained from the skin of nine AIDS patients and six seronegative subjects. Purified LC and LC-depleted EC were prepared by immunomagnetic separation using an anti-CD1a monoclonal antibody. LC preparations did not contain T cells, as assessed by reverse transcription PCR analysis of the T cell receptor beta-chain gene (C region). In addition, no CD14+ cells could be detected in LC fractions by immunostaining of cytospin preparations. To quantify HIV-1 DNA, a new competitive PCR system was devised using SK145/150 as primers (gag) and a competitor plasmid DNA with a modified sequence (209 instead of 142 bp). The number of HIV-1 DNA copies found in the LC of AIDS patients ranged from 107 to 3,645/10(5) LC. In contrast, LC-depleted EC from the same subjects were all negative. The results indicate that in AIDS patients the frequency of infected LC is comparable to that reported for peripheral blood CD4+ T cells.
Cimarelli
A
A
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Zambruno
G
G
Marconi
A
A
Girolomoni
G
G
Bertazzoni
U
U
Giannetti
A
A
D000163:Acquired Immunodeficiency Syndrome / Q000382:microbiology* / Q000473:pathology; D000328:Adult; D000368:Aged; D001667:Binding, Competitive; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D018189:Immunomagnetic Separation; D007801:Langerhans Cells / Q000382:microbiology*; D008297:Male; D008875:Middle Aged; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D012397:Rosette Formation; D014158:Transcription, Genetic
D004279:DNA, Viral; D006684:HLA-DR Antigens; D016693:Receptors, Antigen, T-Cell, alpha-beta