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Although the effect of recovery duration on caffeine-induced responses to multiple sprint work requires further investigation, the results of the present study show that caffeine has ergogenic properties with the potential to benefit performance in both single and multiple sprint sports.

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

In contrast to oncoviruses, lentiviruses do not require target cell division for integration into the host genome. Lentiviral vectors can therefore expand the spectrum of target cells susceptible to retroviral gene transfer. To analyze whether vectors based on simian immunodeficiency viruses (SIVs) could be used for gene transfer, a three-plasmid vector-packaging system was developed, in which Gag-Pol and the vector itself are of SIV origin, while Env is derived either from SIV, amphotropic murine leukemia virus (MuLV), or the G glycoprotein of vesicular stomatitis virus (VSV-G). To increase the safety of the SIV vector system, a self-inactivating SIV vector was constructed. After optimization of the SIV gag-pol expression plasmid, a minimal SIV vector, which contained only SIV sequences present on the multiply spliced nef transcript, could still be produced at titers of 2 x 10(5) infectious units/ml. Growth-arrested cells could be transduced with this vector even if vif, vpr, vpx, and nef had been deleted from the packaging construct and the vector.

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SUMMARYThe expression of CD4 antigen on the surface of LeuM3-positive human blood monocytes was found to be variable with 65 to 90~ of cells from 46 normal human volunteers being positive by dual staining flow cytometry. When monocytes adhered to plastic (but not when cultured on Teflon), a marked decrease in CD4 expression was observed between 1 and 24 h post-adherence. CD4 expression could not be detected in macrophages adhered to plastic for 5 days by using four anti-CD4 monoclonal antibodies in flow cytometry or direct immunofluorescence. Conversely an increasing proportion of adherent cells expressed LeuM3 and OKM5 surface antigens over the 5 days. CD4 mRNA levels were measured by slot-blot and Northern hybridization, and total cellular CD4 protein levels by immunoprecipitation. Both cellular mRNA and 'CD4 levels remained constant throughout the 5 day period but membrane CD4 protein levels were greatly reduced indicating that the down-regulation of CD4 was posttranslational. Infection with two of six fresh human immunodeficiency virus (HIV) isolates showed different kinetic patterns when tested on purified monocytes recently adhered to plastic and macrophages adherent for 5 days. HIV antigen and reverse transcriptase levels in infected monocyte cultures remained high for 3 to 4 weeks before detachment and necrosis of the cells occurred. Infection of macrophages generated much lower levels of antigen and reverse transcriptase which declined to very low or undetectable levels over 2 weeks, leaving persisting viable macrophages. One week after infection HIV nucleic acid was detected in 69 + 7~ of monocytes and 6 + 3~ of macrophages by in situ hybridization. Blocking experiments with anti-Leu3a monoclonal antibody suggested that HIV infection of 5 day adherent macrophages occurred mainly by a mechanism other than binding to CD4.