Genotype and allele frequency of a 32-base pair deletion mutation in the CCR5 gene in various ethnic groups: absence of mutation among Asians and Pacific Islanders.

BACKGROUND: A 32-base pair (bp) deletion mutation in the beta-chemokine receptor CCR5 gene has been associated with resistance against human immunodeficiency virus type 1 (HIV-1) infection and disease. Large-scale studies conducted among Caucasians indicate that individuals who are homozygous for this deletion mutation (D32/D32) are protected against HIV-1 infection despite multiple high-risk exposures, whereas CCR5/ D32 heterozygotes have a slower progression to acquired immunodeficiency syndrome (AIDS). OBJECTIVE: To determine the genotype and allele frequencies of the CCR5 gene 32-bp deletion mutation among ethnically diverse non-Caucasian populations. METHODS: DNA, extracted from blood collected between 1980 and 1997 from 1912 individuals belonging to various ethnic groups, including 363 Caucasians, 303 Puerto Rican Hispanics, 150 Africans, 606 Asians, and 490 Pacific Islanders, were analyzed for the CCR5 gene 32-bp deletion mutation by a polymerase chain reaction (PCR)-based assay, using an oligonucleotide primer pair designed to discriminate CCR5 alleles without restriction endonuclease analysis. RESULTS: The comparative frequency of CCR5/D32 heterozygosity was 61 of 363 (16. 8%) in Caucasians, 17 of 303 (5.6%) in Puerto Rican Hispanics, 9 of 490 (1.8%) in Pacific Islanders, 0 of 606 (0%) in Asians, and 0 of 150 (0%) in Africans. CONCLUSIONS: The data confirm the high frequency of CCR5/D32 heterozygosity among Caucasians. Intermediate and low-level D32 allele frequencies among Puerto Rican Hispanics and Hawaiians could be attributed to recent European Caucasian gene flow. By contrast, the inability to detect the D32 allele among Asians and other Pacific Islander groups suggests that other mechanisms are responsible for resistance to HIV-1 infection in these populations.

Activation of T-cells by bryostatins: induction of the IL-2 receptor gene transcription and down-modulation of surface receptors.

Bryostatins are macrocyclic lactones isolated from the marine bryozoan Bugula neritina. They are currently evaluated for putative antineoplastic activity. Bryostatins bind and activate protein kinase C (PK-C), the cellular receptor for the phorbol ester, and elicit PK-D-dependent cellular functions. Such functions include the expression of the interleukin-2 receptor (IL-2R). Northern blot hybridization with a human IL-2R and an IL-2 cDNA showed that bryostatin 1 (bryo 1), like the phorbol ester, PMA, activates the IL-2R gene. Activation with bryo 1 or PMA in the presence of a calcium ionophore, A23187, increased IL-2 message. These findings indicate that calcium mobilization is necessary for bryo 1 or PMA induced IL-2 gene expression. Unlike PMA, bryo 1 did not cause a vigorous proliferative response of T-lymphocytes unless A23187 was added to the cultures. A bryostatin congener, bryo 13, was inactive in the above assays. Short-term treatment of T-cells with bryo 1 and PMA resulted in an equivalent down-regulation of surface CD3 and CD4 receptors without affecting the CD8 receptor. Bryo 1 or PMA mediated expression of surface IL-2R and T-cell proliferation induced by bryo 1 or PMA were sensitive to inhibition by the PK-C antagonists staurosporine (Sts) and H-7. In contrast, CD4 and CD3 down-regulation were resistant to H-7, but could be blocked by Sts, although the Sts concentration required to block bryo 1 or PMA-induced down-modulation was 2.5-fold higher than required to inhibit IL-2R expression and T-cell proliferation. These results indicate that bryostatins activate T-cell through PK-C.

Variation in the synthesis of membrane proteins of human T lymphocytes during mitogenic activation: recognition of a 70,000 dalton protein with anti-p23,30 heteroantiserum and a novel 42,000 dalton protein with anti-p44,12 heteroantiserum.

Membrane proteins of [35S]methionine-labeled, human T lymphocytes were analyzed by SDS polyacrylamide gradient slab gel electrophoresis and autoradiography each day during a 6-day period of activation with phytohemagglutinin or with concanavalin A. This process was characterized by the novel appearance and limited duration of synthesis of many proteins, in particular of 30, 35, 48, 50, and 55 kilodalton molecules in the early days of blast transformation and subsequently of 120, 125, 135, and 145 kilodalton proteins. The HLAA-A,-B antigens and beta 2-microglobulin, as recognized by anti-p44,12 serum, were synthesized by both resting and mitogen-activated T cells on each day of culture. But, an additional 42-kilodalton protein was recognized with this same antiserum on days 4 and 5 of activation. A 70-kilodalton protein, immunoprecipitated by anti-p23,30 (anti-HLA-protein, immunoprecipitated by anti-p23,30 (anti-HLA-DR) heteroantiserum, was synthesized principally on days 2 and 4 of mitogenic transformation. This molecule was absent from normal, resting T cells, the T cell line, CCRF-CEM, and the B cell line, Raji. In a parallel test, the same anti-p23,30 serum detected the conventional HLA-DR bimolecular glycoprotein complex of 29 and 34 kilodaltons in nonionic detergent solubilized Raji B cell membrane preparations. This study described in detail the molecular changes in the membrane proteins of activated T lymphocytes and included the definition of novel forms of HLA-A,B and HLA-DR associated molecules.

Membrane expression and synthesis of p23,30 (HLA-DR antigen) by human peripheral blood monocytes.

The synthesis and expression of protein complexes of 23,000 and 30,00 dalton (p23,30) HLA-DR antigen, and of 44,000 and 12,000 dalton (p44,12) HLA-A,B antigen and beta 2-microglobulin was demonstrated on human peripheral blood monocytes and in cultures of purified, adherent monocytes. In indirect immunofluorescence assays, a gradation in the intensity of staining with rabbit anti-p23,30 serum was present but clearly p23,30-negative, actively phagocytic monocytes were not found. In contrast the fluorescence intensity of staining with a serum to p44,12 (HLA-A,B antigens and beta 2-microglobulin) was constant on all macrophages. Macrophage HLA-DR antigen was shown in SDS gels of immunoprecipitates of 35S-methionine-labeled, detergent-solubilized membrane proteins to be composed of 29,000 and 34,000 dalton, noncovalently linked chains, which form was indistinguishable from that of B lymphoblastoid cell line HLA-DR antigens. The rate of synthesis of HLA-DR antigen was about 17% of that of synthesis of HLA-A,B antigens in adherent macrophage populations. The variable expression of p23,30 on peripheral blood monocytes was consistent with the view of the existence of subsets of such monocyte populations. These findings were compared to studies by others of the variable expression of Ia antigens on human, murine and guinea pig monocytes populations.