11th International AIDS Conference Vancouver, British Columbia — July 7-12, 1996

Int Conf AIDS 1996 Jul 7-12; 11:27 (abstract no. LB.B.6028)
Suzuki T, Tai H, Yoshie H, Jeannel D, Fournier S, Dupont B, de The G, Hara K; Niigata University School of Dentistry, Niigata, Japan. Fax: (+81) 25 223 3761. E-mail: takashi@dent.niigata-u.ac.jp.

OBJECTIVES: To determine whether the p24-positive monocyte/macrophages detected in gingival inflammatory tissue exudate (AIDS 1996, in press) are HIV-infected population within which viral replication occurs. Design: Twenty-three CDC stage IV AIDS patients participated in the study. Leukocyte infiltrates in gingival crevicular fluid (GCF) were recovered by crevicular washing. Leukocyte cellular RNA and genomic DNA were isolated and served as template for semi-nested PCR. Immunological detection, identification and characterization of pp24+ cells in GCF and blood were carried out by three-color flow cytometric analysis.

METHODS: RNA was extracted by the acetate-guanidine-phenol-chloroform method and genomic DNA by the Nal/chloroform method. For the RT-PCR amplification of viral RNA, 20-50 ng of the extracted RNA was reverse-transcribed with AMV reverse transcriptase. For the genomic DNA, 300-500 ng per sample was used. The gag sequence was amplified by a semi-nested PCR. First, a 611bp product corresponding to the sequence of 1551-2161 of the gag gene was amplified for 35 cycles using the SK38 upper primer and a lower primer (GM2:5'TGG CTC TGG TCT GCT CTG AA 3') designed for the purpose. The second amplification was performed for 25 cycles using 5 microliters of each endproduct from the first amplification as template and using the SK38 and SK39 pair to obtain the final 115 bp product corresponding to the oligonucleotides 1551-1665. For both DNA and RNA, human beta-actin served as internal control. Positive and negative controls were purchased from Perkin Elmer, USA. The specificity of the procedure was verified by direct sequencing of the endproducts.

RESULTS: PCR and RT-PCR of cellular DNA and RNA yielded positive signals from all samples, demonstrating the occurrence of viral integration and production in macrophage fraction in GCF leukocytes.

CONCLUSION: P24+ monocytes mobilized from the circulating pool to the HIV-P lesions differentiating into macrophages in situ may be considered as a within-mouth viral reservoir. Though saliva may neutralize free viruses and inhibit viral replication in MoPs, a direct contact with HIV-P-affected gingival lesion implies direct contact with a viral source, which gives rise to the necessity for a study combining the assessment of periodontal lesions and an analysis of human behaviour. Also, the presence of this potential viral reservoir gives rise to a novel viewpoint as to the oral transmission pathway of HIV.

960707
LBB6028

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