Highly active antiretroviral therapy (HAART) can suppress HIV replication to undetectable levels in plasma, but it is unlikely to eradicate cellular reservoirs of virus. Immunotherapies that are cytolytic may be useful adjuncts to drug therapies that target HIV replication. We have generated HIV-specific CD4(+) and CD8(+) T cells bearing a chimeric T-cell receptor (CD4zeta) composed of the extracellular and transmembrane domain of human CD4 (which binds HIVgp120) linked to the intracellular-zeta signaling chain of the CD3 T-cell receptor. CD4zeta-modified T cells can inhibit viral replication, kill HIV-infected cells in vitro, and survive for prolonged periods in vivo. We report the results of a phase II randomized trial of CD4zeta gene-modified versus unmodified T cells in 40 HIV-infected subjects on HAART with plasma viral loads <50 copies/ml. Serial analyses of residual blood and tissue HIV reservoirs were done for 6 months postinfusion. No significant between-group differences were noted in viral reservoirs following therapy. However, infusion of gene-modified, but not unmodified, T cells was associated with a decrease from baseline in HIV burden in two of four reservoir assays and a trend toward fewer patients with recurrent viremia. Both groups experienced a treatment-related increase in CD4(+) T-cell counts.

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An analytical isoelectric focussing technique has been developed to distinguish between different groups of R-plasmid-mediated trimethoprim-resistant dihydrofolate reductases. Those mediated by the R-plasmids R483 and R721, which are type I dihydrofolate reductases have isoelectric point (PI) values of 6.4 whereas those mediated by the R-plasmids R67bis and R27, which are type I1 enzymes, have PI values of 5.5. There has been some confusion as to the classification of the dihydrofolate reductases mediated by R-plasmids R388 and R751; however, our studies indicate that the one mediated by R388 is a type I1 enzyme, having a PI value of 5.5, whereas the one mediated by R751 is different from both the type I and type I1 enzymes having a PI value of 7.2. The differences in PI value of these three groups of trimethoprim-resistant enzymes were confirmed by DEAEcellulose chromatography at pH 8.5 where it was found that the type I enzymes were eluted with a different concentration of NaCl than were the type I1 enzymes including that mediated by R388, whereas the enzyme mediated by R751 did not bind to the DEAE-cellulose at this pH.A 'scale-down' technique was developed for analytical isoelectric focussing and was used to study the dihydrofolate reductases mediated by ten R-plasmids present in trimethoprim-resistant Escherichiu coli strains isolated from pigs. All these R-plasmids coded for type I enzymes and all also coded for resistance to streptomycin, indicating that the trimethoprim-resistance gene(s) were present on a transposon similar to Tn7.Bacteria resist trimethoprim when they posses R-plasmids that mediate the production of trimethoprim-resistant dihydrofolate reductase (DHFR) enzymes, thus allowing the cell to by-pass the action of the drug on the chromosomal trimethoprim-sensitive DHFR [I]. Pattishall et al. [2] differentiated between two groups of these resistant DHFRs (type I and 11) on the basis of the sensitivity of the enzymes to inhibitors, with type I1 being more resistant than type I enzymes. On this basis all the DHFRs studied by Amyes and Smith [3] appeared to be of type I ; however, using this criterion Tennhammar-Ekman and Skold [4] found one of the DHFRs studied by Amyes and Smith [3], that mediated by R-plasmid R388 which was the first R-plasmid-mediated trimethoprim-resistant DHFR to be discovered and characterized [1,5] Abbreviations. R-plasmids, resistance plasrnids; I D 5~, dose giving 50% inhibition; DHFR, dihydrofolate reductase; PI, isoelectric point; MTT, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyltetrazolium bromide.Enzyme. Dihydrofolate reductase or 5,6,7,8-tetrahydrofolate-NADP+ oxidoreductase (EC 1.5.1.3).Triviul Name. Trimethoprim, 2,4-diamino-5-(3', 4', 5'-trirnethoxybenzy1)-pyrimidine.Using these criteria it was concluded that the DHFRs mediated by both plasmids R388 and R751 were type I1 enzymes [6].Thus there appears to be some confusion as to the classification of these enzymes, with that mediated by R388 being classified as a type I [3] or as a type I1 [4,6] enzyme and that med...

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