MTS interferon assay: a simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt.

MTS, a tetrazolium dye, is reduced by hydrogenases in living cells to a water-soluble formazan. When it is added to the medium at the end of a cytopathic effects (CPE) inhibition interferon assay, the formazan formed diffuses into the medium; the resultant optical density directly and quantitatively measures how much cellular damage has been produced by the challenge virus in the presence of different amounts of interferon. The use of MTS has considerable advantages in that after it is added, no further steps, such as washing of the cells, extraction of dye, or other manipulations, are needed.

Expression of a channel-like pathway for adenosine transport in Leishmania donovani promastigotes.

L. donovani promastigotes (MHOM/ET/67/HA3) transport adenosine by a route that is sensitive to inhibition by a nonspecific channel blocker, propranolol. At the logarithmic and stationary phases of growth, the transport of 1 microM-3H-adenosine was significantly inhibited (40-50%) by 100 microM-propranolol. In contrast, a strain of Leishmania donovani promastigotes clonally selected to grow in defined medium was only slightly (approximately 10%) inhibited at the logarithmic but not at the stationary phase. These results suggest that the differences in expression of adenosine in the parasites previously reported, may be related to uptake by a channel-like pathway in the promastigotes.

Nucleoside transporters in Leishmania major: diversity in adenosine transporter expression or function in different strains.

Cytotoxic nucleoside derivatives may become useful in the treatment of parasitic infections. As part of our drug development studies, the effect of a number of nucleosides (100 microM) on the cellular transport of 3H-adenosine and 3H-inosine (each at 1 microM) in promastigotes from four Leishmania major strains was investigated. When 3H-inosine was used as permeant, all strains exhibited essentially the same inhibition profile, with unlabeled inosine, guanosine, formycin B, and 3'-deoxyinosine being strongly inhibitory, and adenosine-related compounds such as 2'-deoxyadenosine and tubercidin being inactive. However, when 3H-adenosine was used as permeant, considerable differences in the inhibition profiles were noted among strains. Thus, both inosine transporter-selective nucleosides such as inosine and guanosine and adenosine transporter-selective nucleosides such as 2'-deoxyadenosine and tubercidin showed variable activity as inhibitors of 3H-adenosine transport in different strains. These observations indicated that an adenosine transporter was variably expressed in different strains, and that inhibition profiles for adenosine transport indicated cellular entry via both the inosine and adenosine transporters. The existence of different types of adenosine transporters as an alternative explanation could not be ruled out. The apparent uniform expression of an inosine transporter among different species and strains of Leishmania suggests that inosine derivatives may be useful as anti-leishmanial drugs.

A carbocyclic nucleoside analogue is a TNF-alpha inhibitor with immunosuppressive action: role of prostaglandin E2 and protein kinase C and comparison with pentoxifylline.

Tumor necrosis factor-alpha (TNF-alpha) is associated with several acute and chronic inflammatory conditions. New therapies directed at inhibiting TNF-alpha will be important in treating pathological processes mediated by TNF-alpha. In this study, we studied and compared the effect of the carbocyclic nucleoside analogue (9-[(1R, 3R)-trans-cyclopentan-3-ol] adenine) with pentoxifylline on modulating TNF-alpha production. The carbocyclic nucleoside analogue inhibited TNF-alpha production in a dose-dependent manner (1 microM-1 mM) by stimulated peripheral blood mononuclear cells and cell lines of both monocyte (THP-1) and T-lymphocyte phenotypes (CEM x 174). The drug potently inhibited TNF production in cells stimulated by endotoxin, the superantigen (staphylococci enterotoxin A), the mitogen (phytohemagglutinin), and the protein kinase C activator (phorbol myristate acetate) with ED50 ranging from 5 to 30 microM. At moderate concentrations, the carbocyclic nucleoside analogue inhibited superantigen (ED50 = 300 microM) and alloantigen (mixed lymphocyte reaction) T cell proliferative responses (ED50 = 150 microM). The involvement of protein kinase C and prostaglandin E2 (PGE2), mediators that regulate TNF-alpha production, was also investigated. Unlike PTX, the nucleoside analogue did not upregulate PGE2 production. The inhibition of TNF-alpha production appeared to be mediated at least partly by PKC, since the nucleoside analogue caused suppression of PKC activity in stimulated cells. The results show that the carbocyclic nucleoside analogue is a TNF-alpha inhibitor that may be appropriate in the therapy of TNF-alpha-associated complications. The suppressive properties of the carbocyclic nucleoside analogue on antigen and alloantigen (mixed lymphocyte reaction) responses may be appropriate in disease conditions in which inhibiting both TNF-alpha and T-cell reactivity is desirable.