title: Quantitation by competitive PCR of HIV-1 proviral DNA in epidermal Langerhans cells of HIV-infected patients.
abstract: 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.
authors.affiliation: Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Cimarelli
authors.forename: G
authors.initials: G
authors.lastname: Zambruno
authors.forename: A
authors.initials: A
authors.lastname: Marconi
authors.forename: G
authors.initials: G
authors.lastname: Girolomoni
authors.forename: U
authors.initials: U
authors.lastname: Bertazzoni
authors.forename: A
authors.initials: A
authors.lastname: Giannetti
mesh_terms: 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
chemical_list: D004279:DNA, Viral; D006684:HLA-DR Antigens; D016693:Receptors, Antigen, T-Cell, alpha-beta