Search

We investigated the intracellular metabolism of S2242 (2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine), the only known antivirally active acyclic nucleoside analogue with the side chain substituted at the N7 position of the purine ring. Uptake of S2242 by CEM cells increased linearly with increasing extracellular concentrations of the compound and was blocked by inhibitors of nucleoside transport. S2242 was phosphorylated in a time- and concentration-dependent manner to its monophosphates, diphosphates, and triphosphates. Intracellular half-life of the diphosphates and triphosphates in CEM cells was approximately 3-6 hr. A strong correlation was found between the cytostatic action of the compound and its phosphorylation in different cell lines. In accord with the findings that (1) the cytostatic potential of S2242 is reversed by deoxycytidine (dCyd) and (2) the growth of deoxycytidine kinase-deficient (dCK-) cells is refractory to the inhibitory effect of S2242, the amount of metabolites formed from S2242 in the dCK- cell line was approximately one hundredth of that in the wild-type cells. The observation that purified dCK phosphorylates S2242 to its monophosphate further corroborates these results. The activity of S2242 against herpes simplex virus, varicella-zoster virus, and human herpesvirus type 6 was reversed by 50-100-fold on the addition of exogenous dCyd. Compound S2242 was not preferentially phosphorylated in herpes simplex virus 1-, varicella-zoster virus-, or human herpesvirus type 6-infected cells (Vero, human embryonic lung, and HSB-2 cells, respectively), and exogenously added dCyd reduced substantially the formation of S2242 metabolites in these cells. In human cytomegalovirus (HCMV)-infected human embryonic lung cells, a 5-25-fold increase in S2242 metabolite formation was observed compared with the noninfected cells, suggesting that an HCMV-encoded or -induced enzyme causes the specific phosphorylation of S2242. Exogenously added dCyd had little effect on the activity of S2242 against HCMV and on the phosphorylation of the compound in HCMV-infected cells. S2242 was not specifically phosphorylated by the HCMV-encoded UL-97 kinase in cells infected with a vaccinia/UL-97 recombinant. S2242 was found to be a substrate (K(m) = 90 microM) for purified human deoxyguanosine kinase; the latter enzyme was stimulated 3-4-fold in HCMV-infected cells.

We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.

Background Chronic inflammatory stimuli such as cytomegalovirus (CMV) infection and various genetic polymorphisms determining the inflammatory response are assumed to be important risk factors in atherosclerosis. We investigated whether patients with stable coronary artery disease (CAD) and homozygous for allele 2 of the interleukin 1 receptor antagonist (IL-1RA) gene and seropositive for CMV represent a group particular susceptible for recurrent cardiovascular events. Methods In a series of 300 consecutive patients with angiographically defined CAD a prospective follow-up was conducted (mean age 57.9 years, median follow-up time 38.2 months). Results No statistically significant relationship was found between CMV serostatus and IL-1RN*2 (alone or in combination) and risk for future cardiovascular events (CVE). The hazard ratio (HR) for a CVE given positive CMV-serology and IL-1RN*2 was 1.07 (95% confidence interval (CI) 0.32–3.72) in the fully adjusted model compared to seronegative CMV patients not carrying the IL-1RN*2 allele. In this prospective cohort study involving 300 patients with angiographically defined CAD at baseline, homozygousity for allele 2 of the IL-1 RA and seropositivity to CMV alone and in combination were not associated with an increased risk for cardiovascular events during follow-up; in addition, combination of the CMV-seropositivity and IL-1RN*2 allele were not associated with a proinflammatory response Conclusion Our study suggests that seropositivity to CMV and IL-1RA*2 genotype alone or in combination might not be a strong risk factor for recurrent cardiovascular events in patients with manifest CAD, and is not associated with levels of established inflammatory markers.