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OBJECTIVES: The human immunodeficiency virus protease inhibitor nelfinavir is substrate of polyspecific drug transporters encoded by ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCC2 (MRP2), and an inhibitor of BCRP, encoded by ABCG2. Genetic polymorphism in these genes may be associated with changes in transport function. METHODS: A comprehensive evaluation of single nucleotide polymorphisms (39 SNPs in ABCB1, 7 in ABCC1, 27 in ABCC2, and 16 in ABCG2), and inferred haplotypes was done to assess possible associations of genetic variants with cellular exposure of nelfinavir in vivo. Analysis used peripheral mononuclear cells from individuals receiving nelfinavir (n=28). Key results were re-examined in a larger sample size (n=129) contributing data on plasma drug levels. RESULTS AND CONCLUSIONS: There was no significant association between cellular nelfinavir area under the curve (AUC) and SNPs or haplotypes at ABCC1, ABCC2, ABCG2. There was an association with cellular exposure for two loci in strong linkage disequilibrium: ABCB1 3435C>T; AUCTT>AUCCT>AUCCC (ratio 2.1, 1.4, 1, Ptrend=0.01), and intron 26 +80T>C; AUCCC> AUCCT > AUCTT (ratio 2.4, 1.3, 1, Ptrend=0.006). Haplotypic analysis using tagging SNPs did not improve the single SNP association values.

Exposure to sunlight may cause sunburn, skin cancer or phototoxic reactions to certain drugs such as Hypericum extract. All these are ultraviolet B (UVB)-mediated reactions which may be modulated by individual genetic susceptibility. UVB exposure results in oxidative stress. Many products of oxidative stress are detoxified by glutathione-S-transferases mu 1 (GSTM1) and theta 1 (GSTT1). Deletion polymorphisms (genotype *0/*0) of GSTM1 and GSTT1 occur in 50% and 20% of Caucasians, respectively. By affecting the individual ability to detoxify oxidative stress-related products, they may influence the severity of the cutaneous photoreaction.Minimal erythema doses (MED) of UVB irradiation on the skin were determined in 110 subjects who were selected according to their GSTT1 genotype (28 GSTT1*0/*0, 54 GSTT1*A/*0, and 28 GSTT1*A/*A). Genotypes were detected with novel polymerase chain reaction (PCR) assays that allow the differentiation between homozygous and heterozygous GSTT1 and GSTM1 deletions.In the absence of GSTT1 enzyme, the susceptibility of individuals to UVB-induced inflammatory skin reactions increased significantly (p = 0.02, ANCOVA). 'Gene-equivalents' were calculated from the number of functional GSTM1 and GSTT1 alleles as a measure of the gene-dose. UVB sensitivity correlated with gene dose up to a threshold above which additional GSTT1 or GSTM1 alleles did not provide additional protection. Volunteers who were homozygously deficient in GSTT1 and GSTM1 were most sensitive to UVB. Interestingly, individuals with high GSTM1 gene-doses showed increased photosensitization after administration of Hypericum extract (St. John's wort).Individuals harboring the *0/*0 genotype of GSTT1 and/or GSTM1 showed enhanced UVB-induced cutaneous damage. Moreover, GST genotypes modulated Hypericum-induced photosensitization.

A PCR method is described for determining the expression of multiple heterogeneous mRNAs from single cells. The total mRNA pool of a single selected cell is subjected to reverse transcription and subsequent tailing with poly(dA). This cDNA is preamplified by a sequence non-specific PCR protocol using oligo(dT)-containing primers. The single cell cDNA library obtained permits the analysis of virtually unlimited numbers of mRNA species per cell using sequence-specific PCR. This procedure of multiple mRNA analysis enables phenotyping of any cell for its mRNA composition and could be used to study the cytokine mRNA expression of individual human T cells ex vivo. The method should greatly facilitate the analysis of combinatorial expression of known genes in any cell.

Lymphoid organs have been proposed to function as the major reservoir for the human immunodeficiency virus type 1 (HIV-1). Within lymphatic tissues germinal centers represent foci of rapidly proliferating B cells governed by the interaction between B and T cells and follicular dendritic cells (FDC). Accumulating evidence suggests an important role of FDC in the pathophysiology of the acquired immunodeficiency syndrome. Direct proof for the infectibility of FDC with HIV-1 has been lacking until recently when we were able to demonstrate a CD4-independent infection of FDC in vitro. Here we report that in vitro HIV-1-infected human FDC do not only contain proviral DNA, but also produce the virus, and transmit the infection to T cells. Furthermore, electron microscopical studies on ex vivo isolated FDC from simian immunodeficiency virus (SIV)-infected rhesus monkeys revealed typical virus budding. In addition, FDC from SIV-infected rhesus monkeys transmitted the infection to T cells in vitro. Due to this central role within the immune response FDC may serve as preferential targets for HIV both by trapping of virions on their surfaces and by productive infection. During disease FDC become productively infected and may, thus, be regarded as crucial elements in viral dissemination.

Cytokine gene expression was investigated in the germinal centre constituents of follicular dendritic cells and germinal centre T cells and compared to the mRNA expression of nongerminal centre tonsillar cells. Cells were isolated from human tonsils by preenrichment with MACS and subsequent FACS sorting. Cytokine gene expression was investigated by intronspanning RT-PCR for IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha. Frequency of cytokine-producing cells and the cytokine production pattern of single cells were determined by single cell PCR. Furthermore, cytokine protein expression was investigated by immunohistology. Using these methods, we found a strong production of IL-1 beta mRNA and protein in a small percentage of FDC. Germinal centre T cells showed production of IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha mRNA. The mRNAs of these cytokines could also be detected at the single cell level; as they were produced in a high percentage of germinal centre T cells, whereas immunohistological staining was negative, we conclude that a high percentage of germinal centre T lymphocytes produce IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha in low concentrations in contrast to T cells outside the germinal centre, in which strong cytokine production in few cells was shown earlier.

The structural variability of the external glycoproteins of primate immunodeficiency viruses, has, so far, been investigated exclusively by sequence comparison of the respective proviral genomes. We have examined the structural relationship amount the external glycoproteins from three specific human immunodeficiency viruses (HIF-1, HIV-2), three specific simian immunodeficiency viruses from macaques (SIVmac) and three specific SIV from African green monkeys (SIVagm) by peptide mapping. Differences among glycoproteins were most pronounced between HIV-1 and SIVmac, as well as HIV-2. Two specific glycoproteins from independent SIVagm isolates were closely related to HIV-1, whereas the glycoprotein from a third SIVagm isolate was more similar to those of SIVmac and HIV-2. Our analysis reflects the classification of primate immunodeficiency viruses into three groups, the HIV-2 and SIVmac viruses, the green monkey isolates and HIV-1.