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Substantial advancements have been made in our understanding of the complex replication cycle of, and immunopathology associated with HIV infection as well as the drugs used to treat the disease. The nucleoside reverse transcriptase inhibitors remain the cornerstones of current antiviral treatment modalities. Unfortunately, their longterm use often leads to adverse reactions and the emergence of virus mutants with decreased susceptibility to therapeutic agents. In addition to viral resistance, prolonged antiviral treatment may affect metabolic changes in the host cells that can diminish the efficacy of the treatment. Thus, both viral and cellular resistance mechanisms must be considered in the context of failing antiviral chemotherapy. This review article concerns the intracellular pharmacology of antiviral nucleoside analogues in human lymphoid cells and the possible impact of a newly identified nucleotide transporter on drug resistance.

Fixation/permeabilization methods used for the detection of intracellular antigens by flow cytometry often result in the destruction of cellular morphology and surface immunoreactivity, properties useful in flow cytometry for the characterization of cells in heterogeneous populations. In addition, a majority of these methods are incompatible with whole blood and require that peripheral blood leukocytes (PBLs) be purified prior to fixation. This article describes a new technique for the rapid detection of both intracellular and cell surface antigens, while preserving cell morphology, through the use of a single-step fixation/permeabilization reagent, ORTHO PermeaFix (OPF). OPF is compatible with whole blood, allowing for the direct preparation of PBLs without prior cell separation. An additional red blood cell lysing reagent was not required because RBC lysis occurred upon resuspension of OPF-treated whole blood samples in isotonic solution. Discrimination of leukocyte populations by light scatter after OPF treatment was comparable to matched unfixed live cells. In addition, absolute lymphocyte and white blood cell (WBC) counts were not significantly affected when OPF-treated cells were compared with unfixed cells. Treatment of whole blood from 7 normal donors showed no significant difference in percentage of cells positive for CD2, CD3, CD4, CD8, CD16, or CD19 between fixed and unfixed samples when cells were stained before fixation, and no difference in CD3, CD4, CD8, CD16, or CD19 percentages when cells were stained following fixation. Monoclonal antibodies specific for intracellular antigens located at various sites within the cell were tested on fixed samples. OPF-treated peripheral blood lymphocytes showed greater than 95% reactivity for the inner mitochondrial membrane protein bcl-2, and the cytoskeletal cytoplasmic protein vimentin. TIA-1, a cytolytic granule-associated protein, showed differential reactivity within lymphocyte subsets, from a low of 8 +/- 2% in CD4+ cells to 89 +/- 6% in CD16+ cells, when whole blood from five normal donors was fixed and stained. Reh cells treated with OPF showed greater than 95% reactivity for the internuclear protein TdT. A comparison of OPF with two other fixation/permeabilization procedures, 1% paraformaldehyde followed by 45% ethanol and 0.25% paraformaldehyde followed by 0.2% Tween 20, showed that only OPF could be used both prior to or following cell surface staining with no effect on antigen detection while allowing optimal detection of all of the intracellular antigens tested.

The acyclic nucleoside phosphonates (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) inhibited herpes simplex virus-1 replication in Vero cells, and the IC50 values ranged from 4 microM (for HPMPC and HPMPA) to 40 microM (for PMEA). Pretreatment of cells with HPMPC for 12-24 hr induced an effective antiviral state, and the cells maintained this antiviral state for7 days. In contrast, much larger amounts (approximately 2.5-5 x IC50 doses) of PMEA or HPMPA were required to establish an antiviral state, which lasted for only approximately 24 or 72 hr, respectively. A 12-hr treatment of the cells with the phosphonates was required for the establishment of optimal antiviral activity; surprisingly, longer durations of exposure to PMEA (but not HPMPA or HPMPC) resulted in diminished antiviral effect. We investigated the metabolism of PMEA and HPMPC to determine the cellular basis for these differences. The cellular uptake of HPMPC was approximately 8-fold greater than that of PMEA. The levels of the PMEA metabolites PMEA monophosphate and PMEA diphosphate increased for approximately 12 hr and plateaued thereafter. PMEA and its metabolites were cleared from the cells with a half-life of 4.9 hr. In contrast, the HPMPC metabolites HPMPC monophosphate (HPMPCp) and HPMPC diphosphate (HPMPCpp) accumulated throughout the 24-hr study period and, at equimolar drug concentrations (25 microM), reached intracellular levels approximately 2-3-fold greater than those of the PMEA metabolites. HPMPC also differed from PMEA in its capacity to generate a phosphodiester metabolite (HMPCp-choline), which was a predominant metabolite in HPMPC-treated cells. In addition, the rates of disappearance of intracellular metabolites of the two drugs were significantly different. Thus, the decay of HPMPCpp was quite slow and biphasic (t1/2 = 24 and 65 hr) and that of HMPCp-choline was monophasic (t1/2 = 87 hr). Together, these factors can explain the differing antiviral potencies seen with PMEA and HPMPC. The possible role of the choline adduct in the expression of antiviral activity of the drug remains to be elucidated, but the adduct may serve as an intracellular store for the long term maintenance of active HPMPCpp in cells. The results also highlight the extent of diversity in the cellular pharmacology and antiviral activities of the acyclic nucleoside phosphonates.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent with activity against herpes viruses and retroviruses, including human immunodeficiency virus, but its metabolism and mechanism of action remain unclear. We have isolated a human T lymphoid cell line (CEMr-1) that is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEMr-1 cells, compared with the parental cells. This mutant showed cross-resistance to the related acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-phosphonylmethoxyethyl)guanine and the lipophilic prodrug bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine-( bispome-PMEA), as well as partial resistance to the purine nucleosides 2-chlorodeoxyadenosine, 2-fluro-9-beta-D-arabinosylfuranosyladenine, and adenosine, but did not show resistance to 2'-deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [3H]PMEA and [3H]-bispom-PMEA in the mutant and parental cells. The analysis of radioactive products by high pressure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compared with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bispom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with the parental cells, the variant cells showed a approximately 7-fold increase in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabolism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribosyl-1-pyrophosphate synthetase, showed no significant change in the two cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in the cellular efflux of PMEA as the major basis for the resistant phenotype.

In the present study, we explored the suitability of a new cell fixative (ORTHO PermeaFix, OPF) for the detection by flow cytometry of intracellular molecules while preserving the cell surface immunoreactivity, scatter features and morphology. The effect of OPF was investigated on whole blood of ten normal donors, and on separated blasts of 17 leukemic patients. OPF fixation for 45 min to 24 h maintained the morphology of lymphoid cells with minimal cellular distortion and scatter changes, and only slightly modified cell surface immunoreactivity. For at least 1 week following fixation, the cells were still suitable for immunostaining with monoclonal antibodies that recognize the main lymphoid populations. These included CD3, CD4 and CD8 for T-cell subsets, CD19 and CD16 for B lymphocytes and NK cells, and CD45 for leukocyte common antigen (LCA). The OPF fixation of leukemic cells allowed the simultaneous detection of nuclear TdT in conjunction with membrane CD19, and with membrane and/or cytoplasmic CD22 in common-ALL, as well as with cytoplasmic CD3 in T-ALL cases. Our findings suggest that with the introduction of this new fixative into the routine laboratory service, a number of convenient and practical arrangements can be made which increase the efficiency of immunodiagnosis. Small laboratories with no inhouse flow-cytometric facilities can now accumulate OPF-treated whole blood samples for at least 3-4 days and send these to reference laboratories. In addition, the immunodiagnosis of acute leukemia is greatly facilitated by combination staining for membrane and intracellular antigens both at diagnosis and when the analysis of minority populations is warranted for detecting minimal disease.

The metabolism and cytostatic effects of 3'-azido-3'-deoxythymidine (AZT), one of the most effective agents being used in the treatment of acquired immunodeficiency syndrome, were investigated in the CCRF-CEM line of human T lymphoid cells. The concentration of drug required to inhibit cell growth by 50% (CD50) was significantly lower when the cells were exposed to AZT for 24 hr (CD50 = 50 microM), as compared with 48 or 96 hr (CD50 = 225 and greater than 300 microM, respectively). AZT at 25 microM blocked the progression of cells in S phase for about 12 hr, but this effect was reversed by 24 hr, despite the continued presence of drug in the medium. At this drug concentration, the level of dTTP decreased to about 75% of the control level by 4 hr but rebounded to 30% above normal by 8 hr of drug exposure. dGTP and dATP pool sizes were unchanged, whereas the dCTP pool increased 5-fold. The time course of these biochemical changes indicated that the onset of S phase arrest was not directly related to the decrease in deoxynucleoside triphosphate pools. CCRF-CEM cells incubated with 25 microM AZT accumulated about 0.9 mM 5'-monophosphate (AZTMP) after 4 hr whereas levels of the 5'-di- and 5'-triphosphates (AZTDP and AZTTP) plateaued at about 2 and 5 microM, respectively. After this period, there was a rapid decrease in AZTMP accumulation, to one third its initial level by 24 hr, whereas AZTDP and AZTTP pools decreased to only about 70%. The loss in AZT nucleotide formation with time of drug exposure was associated with a concomitant accumulation of AZTMP in the medium. Cellular excretion of AZTMP was not associated with any detectable cell lysis or leakage of other cellular metabolites. The ability of CCRF-CEM cells to excrete AZTMP may be an important factor limiting the biochemical and biological effects of the drug.

The use of chromium dioxide particles as a solid support for very sensitive and rapid immunoassays, is the result of the combination of large surface area (40 m2) and high protein uptake capacity (40 mg/g) allowing rapid capture kinetics and high binding capacity. Magnetic and physical properties of these particles give a rapid separation, a complete resuspension and a rapid high-efficiency washing, highly desirable characteristics for efficient automation of immunoassays. Good precision and accuracy, exemplified by excellent recovery, parallelism and correlation were demonstrated. Test results prove that the technology is highly flexible and applicable to a variety of assay formats.

Strains of Neisseria gonorrhoeae were treated with pyocin 611 131 (pyocin 103) from Pseudomonas aeruginosa PA103, and isogenic resistant variants were isolated. The interaction of pyocin-sensitive and isogenic pyocin-resistant strains with wheat germ agglutinin (WGA) agglutinated all pyocin-sensitive, but not pyocin-resistant, strains. Binding of WGA to three pyocin-sensitive strains and their isogenic pyocin-resistant variants was examined quantitatively by using fluorescein-conjugated lectin. Pyocin-resistant strains maximally bound one-third to one-eighth the quantity of WGA bound by isogenic-sensitive strains. Linear Scatchard plots revealed homogeneous WGA-binding sites on three pyocin-sensitive and one pyocin-resistant strains. Biphasic Scatchard plots, obtained with two pyocin-resistant strains, show that WGA-binding sites in these strains are heterogeneous. The number of WGA-binding sites for pyocin-sensitive organisms ranged from 8 x 10(5) to 1 x 10(6) sites per coccus and from 1 x 10(5) to 3 x 10(5) sites per coccus for pyocin-resistant strains. The apparent association constant for WGA binding to pyocin-sensitive strains ranged from 3 x 10(6) to 6 x 10(6) liters/mol and from 6 x 10(6) to 1 x 10(7) liters/mol for pyocin-resistant strains. Gonococcal lipopolysaccharide was shown to serve as the pyocin 103 receptor by inhibition of pyocin activity. Lipopolysaccharide from a pyocin 103-resistant strain was not able to inhibit pyocin 103 activity. Pyocin 103 resistance was correlated with a structural alteration involving N-acetylglucosamine residues in gonococcal lipopolysaccharide. Based on interactions with wheat germ, soybean, and ricin lectins, a model of lipopolysaccharide structure in N. gonorrhoeae is presented.

Homologous antisera were raised against lipopolysaccharides (LPSs) isolated from pyocin 103-sensitive JW31 strain Neisseria gonorrhoeae and its isogenic, pyocin-resistant variant, JW31R. Changes in immunochemical reactivity of LPS antigen associated with pyocin-resistance were examined by enzyme-linked immunosorbent assay, employing homologous and heterologous anti-LPS immune sera. The acquisition of pyocin 103 resistance is accompanied by a loss in LPS antigen reactivity with homologous anti-LPS. The variant LPS of pyocin 103-resistant mutants is immunogenic and displays a new, distinct antigenic specificity shared with other pyocin 103-resistant variant gonococcal strains. The acquisition of pyocin 103 resistance by JW31 strain gonococci is also accompanied by a striking loss of LPS cross-reactivity with antistreptococcal polysaccharide reagents having an antibody combining site specificity directed against the chemically defined lactose polymer from Streptococcus faecalis cell wall and pneumococcal type 14 capsular polysaccharide. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, JW31 and JW31R LPSs show banding patterns characteristic of microheterogeneous, rough-type LPS devoid of O-side chains. Immunoblot transfer analysis of gel-separated gonococcal LPS antigens shows a difference in the pattern of antibody binding by homologous versus cross-reactive anti-LPS, which suggests a heterogeneity in the distribution of cross-reactive determinants among LPS molecules.

The exact route of metabolism of tiazofurin, a novel nucleoside with antitumor activity, is controversial. Using human cell lines severely deficient in salvage nucleotide enzymes, we were able to identify the route of activation in tiazofurin metabolism. With loss of adenosine kinase activity by mutation in two lymphoblastoid cell lines, CCRF-CEM and WI-L2, the growth sensitivity to tiazofurin decreased by 6- and 3-fold, respectively. In contrast, the mutant lines were about 3000- to 1500- and 16- to 4-fold more resistant to the structurally similar tiazofurin analogues pyrazofurin and ribavirin, respectively. Other mutants with defective deoxycytidine or uridine kinase activity showed normal sensitivity to all three analogues. Both cell lines with defective adenosine kinase activity accumulated about 50% wild-type levels of tiazofurin-5'-monophosphate and thiazole-4-carboxamide adenine dinucleotide analogue of tiazofurin at cytotoxic concentrations of the drug. Extracts of wild-type lymphoblasts catalyzed the phosphorylation of tiazofurin in the presence of adenosine 5'-triphosphate and Mg2+. Loss of adenosine kinase activity in the mutant extract eliminated this phosphorylating activity for tiazofurin consistent with the notion that adenosine kinase catalyzes phosphorylation of tiazofurin. However, an enzyme activity that catalyzed the phosphorylation of tiazofurin in the presence of inosine-5'-monophosphate as donor and Mg2+ was detected in the extracts of both wild-type cells and adenosine kinase-deficient mutants. The monophosphate donor specificity, divalent metal, high salt requirement, and nucleoside acceptor specificity of this enzyme activity paralleled that of a 5'-nucleotidase (EC 3.1.3.5) which catalyzes inosine phosphorylation. In addition, tiazofurin phosphorylation was competitively inhibited by inosine and the apparent Ki value was similar to the apparent Km value for inosine phosphorylation. These results indicate that two enzymes, adenosine kinase and a cytoplasmic 5'-nucleotidase, are functionally important anabolizing enzymes for tiazofurin in human cells.