Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC].

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.

Selection and characterization of human immunodeficiency virus type 1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine.

Human immunodeficiency virus (HIV) type 1 (HIV-1) variants were selected for resistance to the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine (dOTFC), by use of the infectious molecular clone HIV HXB2D and the human T-cell line MT-4. The dOTFC-resistant variants that were selected were 10-fold less sensitive than wild-type virus, and cloning and sequencing of the complete reverse transcriptase (RT)-coding region identified the mutation M184V. Studies with mutated recombinant HXB2D virus confirmed the importance of the M184V mutation in conferring resistance to (-)dOTFC in MT-4 cells, although no difference in sensitivity was observed in primary cells. The M184V substitution also displayed decreased susceptibility to (+)dOTFC. Selection with (+)dOTFC also produced variants which were 10-fold more resistant than the wild type, and a novel mutation, D67G, was identified following cloning and sequencing of the RT genes. The D67G mutation was introduced into HXB2D by site-directed mutagenesis, and the data obtained confirmed the importance of this mutation in conferring resistance to both (+)dOTFC and (-)dOTFC. Mutated recombinant molecular clone HXB2D-D67G was further selected with (+)dOTFC, and three of six clones sequenced contained both the D67G and M184V mutations, while the other three of the six clones contained only the D67G mutation. Clinical isolates of HIV-1 which are (-) 2'-deoxy-3'-thiacytidine-resistant also displayed resistance to both (+)dOTFC and (-)dOTFC.

Mechanism of action and in vitro activity of 1',3'-dioxolanylpurine nucleoside analogues against sensitive and drug-resistant human immunodeficiency virus type 1 variants.

(-)-Beta-D-1',3'-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 microM when evaluated against HIV-1(IIIB) in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2', 3'-dideoxy-3'-thiacytidine (3TC) but 5- to 10-fold less potent than 3'-azido-2',3'-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2',3'-dideoxyinosine, and 2',3'-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 microM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

A high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors.

A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 has been developed for human cytomegalovirus (HCMV) antiviral susceptibility testing and adapted to a microtiter plate format. Optimal conditions were determined and the standard routine assay was calibrated with a viral input of 0.05-0.10 plaque forming unit (p.f.u.)/cell with a density of 2000 cells/well in a 96-well microtiter plate for an incubation period of 7 days. Ganciclovir (9-(2-hydroxy-1(hydroxymethyl) ethyoxymethyl) guanine; DHPG), and cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; HPMPC) were used as positive control test compounds to validate the assay. The effective EC50 concentration values obtained with the two antiviral compounds in the present assay were in good agreement with plaque reduction assay results performed in parallel experiments. This method presents the advantage of being easy and rapid to perform, reliable, reproducible, and convenient for use in a high throughput screening capacity.

Selection and characterization of HIV-1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC).

Human immunodeficiency virus type 1 (HIV-1) variants were selected for resistance against the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC), using the infectious molecular clone HXB2D grown in the MT-4 line of human T cells. The variants selected with (+) dOTC were approximately 6-7-fold less sensitive than wild-type virus to this drug. Cloning and sequencing of the complete reverse transcriptase (RT) coding region of these variants identified the M1841 mutation and further selection with virus containing the M1841 substitution led to the appearance of an M184V mutation. In contrast, selection experiments performed with (-) dOTC yielded variants capable of growing in drug concentrations as high as 100 microM, but phenotypic analysis of these viruses revealed near wild-type 50% inhibitory concentration (IC50) values for this compound. Site-directed mutagenesis experiments in which the M1841 and M184V mutations were introduced into HXB2D confirmed the importance of these mutations when viruses were grown in MT4 cells. However, wild-type IC50 values in regard to both (-) and (+) dOTC were obtained when these recombinant viruses were grown in cord blood mononuclear cells (CBMC). Clinical isolates of HIV-1 resistant to lamivudine and containing the M184V substitution also displayed low-level resistance to both (-) and (+) dOTC when grown in CBMC. Finally, cell-free RT assays were performed in the presence of either (-) dOTC triphosphate, (+) dOTC triphosphate, or the triphosphate of a racemic mixture of (+) and (-) dOTC with wild-type and mutated M184V-containing recombinant RT. The data demonstrate chain termination effects of these compounds with regard to both wild-type and mutated enzyme and that the latter was approximately twofold less sensitive than the former to these drugs.

Potent antitumor activity of a novel nucleoside analogue, BCH-4556 (beta-L-dioxolane-cytidine), in human renal cell carcinoma xenograft tumor models.

Beta-L-Dioxolane-cytidine (BCH-4556) is a novel anticancer nucleoside analogue with a stereochemically unnatural beta-L configuration. This compound was previously shown to have a potent antitumor activity in human prostate and hepatocellular xenograft tumor models (K. L. Grove et al., Cancer Res., 55: 3008-3011, 1995). Herein, we extended the efficacy validation of BCH-4556 to renal cell carcinoma (RCC) cell lines both in vitro and in vivo. In vitro cytotoxicity and proliferation inhibition determinations in human RCC cell lines CAKI-1, CAKI-2, 786-0, and A498 produced IC50 concentrations ranging from 15-35 nM. In vivo antitumor activity was consistent with the in vitro sensitivity. BCH-4556 was very effective in human RCC tumor xenograft models, including CAKI-1, A498, RXF-393, and SN12C carcinomas. Very good responses were observed in animals bearing CAKI-1, A498, and RXF-393 RCC tumors given i.p. doses of 10, 25, and 50 mg/kg twice a day for 5 days, with complete regression recorded in most of the animals tested. Curative activity was also observed, with 40-60% of animals remaining tumor free in all three RCC models at the day of study termination. Significant tumor shrinkage was also evident in the SN12C model. BCH-4556 efficacy evaluation in the orthotopic subrenal capsule tumor models demonstrated a potent tumor growth inhibition against human CAKI-1 xenografts and tumor stasis against mouse Renca tumors. BCH-4556 was also effective in inhibiting the growth of rebound CAKI-1 tumors after the administration of a second treatment cycle. The observed antitumor activity of BCH-4556 in several RCC human solid tumor xenografts, including the lethal RXF-393 model, warrants further investigation of this novel nucleoside analogue in clinical trials of RCC.

Adenosine A3 receptor agonists inhibit murine macrophage tumor necrosis factor-alpha production in vitro and in vivo.

Adenosine and related analogs have been shown to regulate a variety of cell functions through different classes of adenosine receptors. Murine J774.1 macrophage cells were found to predominantly express adenosine A3 receptor RNA relative to adenosine A1 receptor or adenosine A2 receptor RNA. Adenosine receptor agonists, in a dose-dependent manner characteristic of the adenosine A3 receptor, blocked endotoxin-induction of the TNF-alpha gene and TNF-alpha protein expression in the J774.1 macrophage cell line. The adenosine A3 receptor antagonist BW-1433 dose-dependently reversed this adenosine receptor agonist inhibitory effect on TNF-alpha gene expression. Thus, the binding of adenosine receptor agonists to the adenosine A3 receptor interrupts the endotoxin CD14 receptor signal transduction pathway and blocks induction of cytokine TNF-alpha, revealing a novel cross-talk between the murine adenosine A3 receptor and the endotoxin CD14 receptor in J774.1 macrophages.

AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1.

Interleukin-1 (IL-1) -alpha and -beta are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo.

Inhibition of superantigen-induced proinflammatory cytokine production and inflammatory arthritis in MRL-lpr/lpr mice by a transcriptional inhibitor of TNF-alpha.

We have used fas-defective MRL-lpr/lpr mice to study the effects of the staphylococcal enterotoxin superantigens on the development of autoimmune, inflammatory joint disease in animals that are susceptible to the development of rheumatoid arthritis-like disease. We show that systematic administration by a single i.p. injection of staphylococcal enterotoxin B (SEB; 10 micrograms/mouse) caused a mild, inflammatory arthritis +30 days postchallenge in the knee joints of young (< 2-mo-old) MRL-lpr/lpr mice, but not aged-matched MRL +/+ mice. In aged (> 8-mo-old) MRL-lpr/lpr mice, but not in aged MRL +/+ mice, SEB caused a severe, inflammatory arthritis, as assessed histologically, and systemic autoimmune disease, including glomerulonephritis and autoantibody production. Furthermore, in aged MRL-lpr/lpr mice, SEB but not heat-denatured SEB caused acute weight loss and elevated levels of serum proinflammatory cytokines. Compared with highly purified peritoneal macrophages obtained from either aged MRL +/+, young MRL-lpr/lpr, or young MRL +/+, peritoneal macrophages obtained from aged MRL-lpr/lpr mice constitutively expressed 2- to 10-fold greater levels of TNF-alpha, IL-1 beta, IL-6, and IL-10, and produced elevated amounts of these cytokines when treated in vitro with SEB. SEB-challenged aged MRL-lpr/lpr mice treated with anti-TNF mAb (100 micrograms/mouse; every other day), anti-V beta 8 TCR mAb (250 micrograms/mouse; every other day), or orally with the novel TNF-alpha inhibitor MDL 201,449A (9-[(1R, 3R)-trans-cyclopentan-3-ol] adenine; 25 mg/kg/day) exhibited reduced inflammatory arthritis, autoantibody formation, and serum TNF-alpha levels, but not IL-10 levels, after +30 days of treatment. These data suggest that SEB is an extremely potent macrophage-activating factor in vitro and in vivo, enhancing several aspects of autoimmune disease in MRL-lpr/lpr mice, and that anti-TNF therapies may have potential use in inflammatory arthritis.

Activation of adenosine A3 receptors on macrophages inhibits tumor necrosis factor-alpha.

Murine macrophage-derived tumor necrosis factor alpha (TNF-alpha) gene expression has been shown to be dramatically induced by bacterial lipopolysaccharide, and to be dependent upon nuclear factor-kappa B (NF-kappa B) binding sites in its promoter for the lipopolysaccharide induction. Murine J774.1 macrophage cells were found to predominantly express the adenosine A3 receptor RNA relative to adenosine A1 receptor or adenosine A2 receptor RNA. Adenosine receptor agonists, in a dose-dependent manner characteristic of the adenosine A3 receptor, blocked the endotoxin induction of the TNF-alpha gene and TNF-alpha protein expression in the J774.1 macrophage cell line. The adenosine A3 receptor antagonist BW-1433 dose-dependently reversed this adenosine inhibitory effect on TNF-alpha gene expression. Thus, the binding of adenosine receptor agonists to the adenosine A3 receptor interrupts the endotoxin CD14 receptor signal transduction pathway and blocks induction of cytokine TNF-alpha, revealing a novel cross-talk between the murine adenosine A3 receptor and the endotoxin CD14 receptor in J774.1 macrophages.

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

Carbocyclic nucleosides as inhibitors of human tumor necrosis factor-alpha production: effects of the stereoisomers of (3-hydroxycyclopentyl)adenines.

A series of four structurally related carbocyclic nucleosides (6a, 6b, 10a, and 10b) were synthesized and evaluated for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) production from human primary macrophages. These compounds had little effect on the production of IL-1 beta and IL-6. It was determined that compound 10a was the most potent inhibitor of TNF-alpha production (IC50 = 10 microM), having 2-5-fold more activity compared to its enantiomer 10b or its diastereomers 6a and 6b. In addition, these compounds were also tested for their ability to protect mice against lethal challenges of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Compound 10a showed superior protective effects (100% protection) compared to its enantiomer 10b or its diastereomers 6a and 6b when it was administered to mice which were challenged with 3 times the LD100 dose of LPS.

Refined crystal structure of the interleukin-1 receptor antagonist. Presence of a disulfide link and a cis-proline.

Interleukin-1 (IL-1) molecules are cytokines involved in the acute-phase response against infection and injury. Three naturally occurring IL-1 molecules are known, two agonists: IL-1 alpha and IL-1 beta, and one antagonist, the IL-1 receptor antagonist (IL-1ra). Although IL-1 action protects the organism by enhancing the response to pathogens, its overproduction can lead to pathology and has been implicated in disease states that include septic shock, rheumatoid arthritis, graft versus host disease and certain leukemias. The crystal structure of IL-1ra has been solved at 0.21-nm resolution by molecular replacement using the IL-1 beta structure as a search model. The crystals contain two independent IL-1ra molecules which are very similar. IL-1ra has the same fold as IL-1 alpha and IL-1 beta. The fold consists of twelve beta-strands which form a six-stranded beta-barrel, closed on one side by three beta-hairpin loops. Cys69 and Cys116 are linked via a disulfide bond and Pro53 has been built in the cis-conformation. Comparison of the IL-1ra structure with the IL-1 alpha and IL-1 beta structures present in the Protein Data Bank shows that a putative receptor interaction region, involving the N-terminus up to the beginning of strand beta 1 and the loops D and G, is very different in the three IL-1 molecules. Other putative interaction regions, as identified with mutagenesis studies, are structurally conserved and rigid, allowing precise and specific interactions with the IL-1 receptor.

Specific interaction of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid with human cellular CD4.

We recently demonstrated that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) prevented the infection of T cells by human immunodeficiency virus type-1 (Cardin et al., J. Biol. Chem. 266, 13355, 1991). In the present study we have used a panel of monoclonal antibodies (mabs) against a variety of human leukocyte antigens to characterize the interaction of DIDS by flow cytometry with various T cell surface molecules. DIDS blocked the specific immunoreactivity of mabs OKT4A and Leu 3A with CD4 on human leukemic T cells (JM) and human mononuclear lymphocytes with an IC50 approximately 30 microM. The membrane distal (D1) and proximal (D3 and D4) domains of CD4 remained blocked for up to 5 hr of culture and returned to control levels of expression after 24 hr, reflecting the rate of membrane turnover of the CD4-DIDS complex. The binding frequencies (% positive) for anti-CD2, -CD3, -CD5, -CD6, -CD7, -CD8, -CD11a, -CD14, -CD18, -CD19, -CD45, -T cell receptor, and -HLA-DR were not significantly affected. However, there was a partial reduction in the antigen density of CD2, CD5, CD8, and CD11b. The selective interaction of DIDS with CD4 suggests that antagonism of the virus receptor may account in part for the antiviral properties of the stilbene disulfonate.

Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing.

Swainsonine, a known inhibitor of the alpha-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at suboptimal doses of interleukin-2; the amount needed to increase LAK activity is 100-1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 microM) concentrations of swainsonine showed a 3-4 fold increase in D-[3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by D-[2-3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 microM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and N-glycanase, but was susceptible to cleavage by Jack bean alpha-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with D-[2-3H]mannose from mouse B16F10 melanoma and baby hamster kidney cells subsequent to swainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed the presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.

Immunomodulation by an inhibitor of S-adenosyl-L-homocysteine hydrolase: inhibition of in vitro and in vivo allogeneic responses.

The response of murine T cells to MHC class II determinants on allogeneic cells induces helper T cell activation and the development of cytotoxic T cells. We have recently established that an S-adenosyl-L-homocysteine hydrolase inhibitor, (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (MDL 28,842), is a potent immunosuppressive agent which selectively inhibits T cell activation. In this report we characterize the effect of MDL 28,842 on in vitro and in vivo models of transplant rejection. In vitro, MDL 28,842 inhibited the generation of cytotoxic T cells in the murine mixed lymphocyte reaction with an IC50 of less than 0.1 microM. MDL 28,842 (1.0 microM) totally inhibited the generation of cytotoxic T cells when added up to 3 days after the initiation of culture with no apparent cell toxicity. In vivo, MDL 28,842 given by gavage at 5.0, 2.5, or 1.0 mg/kg/day inhibited the increase in popliteal lymph node weight induced by injection of allogeneic spleen cells into the footpad. MDL 28,842 was also evaluated in a model of graft rejection. Skin allografts on animals given MDL 28,842 at 5 mg/kg/day (ip) for the first 6 days following transplantation survived for 12.2 days, compared to 8.7 days for control animals. Cyclosporin A (CSA) given at 5.0 mg/kg/day did not prolong graft survival. The combination of MDL 28,842 and CSA was not any more effective than MDL 28,842 alone. Based on these findings, we suggest that MDL 28,842 is useful in the prevention of allograft rejection.

Selective inhibition of T cell activation by an inhibitor of S-adenosyl-L-homocysteine hydrolase.

Defects in the enzymes involved in the pathway of S-adenosylmethionine (AdoMet) metabolism, or inhibition of those enzymes, results in profound immunodeficiency. We have examined MDL 28,842, a novel irreversible inhibitor of S-adenosyl-L-homocysteine hydrolase (AdoHcyase), an enzyme involved in AdoMet metabolism, to determine its effect on the immune system and to investigate its potential as an immunosuppressive agent. The stimulation of human mononuclear cell proliferation in vitro with Con A, a T cell mitogen, and PWM, a T-dependent B cell mitogen, were inhibited by MDL 28,842. The 50% inhibitory concentration for both were 0.33 microM. In murine spleen cells, MDL 28,842 was a potent, nontoxic, inhibitor of Con A-stimulated T cell proliferation (IC50 = 0.19 microM) but did not affect LPS-induced B cell proliferation. This selective suppression was also observed when enriched murine T and B cells were stimulated with mitogens, although S-adenosyl-L-homocysteine (AdoHcy), the substrate of AdoHcyase, was similarly elevated in both populations. In addition to proliferation in response to a number of stimuli, IL-2 production and the expression of IL-2R by mitogen-stimulated T cells were inhibited by MDL 28,842. These results suggest a direct effect of MDL 28,842 on T cells. In vivo, the antibody response to a T cell-dependent Ag, OVA, was inhibited by MDL 28,842. The response of splenic T cells from these animals to OVA in vitro were similarly depressed compared with controls. The results demonstrate that MDL 28,842 is a potent nontoxic immunosuppressive agent, which has selectivity for T cells and therefore may be useful in the treatment of T cell-mediated disorders, such as autoimmune disease and tissue transplantation.

Identification of a specific receptor for interleukin-1 in vascular smooth muscle cells: regulation by interleukin-1 and interleukin-6.

A fluorescently labeled ligand was utilized to establish the existence of an interleukin-1 (IL-1) receptor in vascular smooth muscle. The binding of the phycoerythrin-labeled IL-1 beta to the murine T cell line, EL-4, was examined as a positive control. The phycoerythrin-labeled IL-1 beta identified a specific IL-1 receptor in the EL-4 cells. Vascular smooth muscle cells were also positively stained by the fluorescent ligand. The binding of phycoerythrin-labeled IL-1 beta to these cells was saturable and reversed by 100-fold excess unlabeled IL-1 beta. Incubation of the vascular smooth muscle cells with IL-1 beta (25 ng/ml) or IL-6 (250 ng/ml) for 18 h increased and decreased, respectively, the percentage of cells positively stained by phycoerythrin-labeled IL-1 beta which suggests these cytokines regulate IL-1 receptor expression in these cells. These data indicate a specific receptor for IL-1 exists in vascular smooth muscle cells.

Human aortic endothelial cells express the type I but not the type II receptor for interleukin-1 (IL-1).

Endothelial cells (EC) are very responsive to the proinflammatory cytokine interleukin-1 (IL-1). EC are induced by IL-1 to secrete chemotactic factors and to increase expression of cell surface adhesion molecules leading to increased leukocyte adhesion. Activated EC further contribute to the inflammatory response by secreting additional cytokines. IL-1 interacts with EC through high-affinity cell-surface receptors. However, the low number of receptors present on EC has made characterization difficult. Further, recent evidence has suggested diversity in the responses of EC from different regions of the vascular system. Interested in the effect of IL-1 on early atherosclerotic lesion formation, we have characterized the IL-1 receptors on human aortic endothelial cells (HAEC). Using a direct binding assay, we found that HAEC have 1,000-3,000 IL-1 receptors per cell and bind IL-1 alpha with a Kd of 3.5 x 10(-10) M. We found that a monoclonal antibody specific for the type I receptor completely blocks IL-1 alpha binding. The blocking antibody also completely inhibits the IL-1 induced increase in intracellular adhesion molecule 1 (ICAM-1) expression by HAEC. Using solution hybridization and ribonuclease protection with an antisense probe, a sensitive method for detection of low abundance mRNA species we found that HAEC as well as human umbilical vein EC (HUVEC) have significant levels of mRNA for the type I IL-1 receptor. To test whether HAEC might also contain transcripts for the type II IL-1 receptor, we compared levels of mRNAs by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from total RNA. We found only transcripts for the type I receptor and not the type II receptor in HAEC. Based on this data, we conclude that aortic endothelial cells respond to IL-1 through the type I receptor.

Regulation of IL-2 production by mononuclear cells from rheumatoid arthritis synovial fluids.

Products of polyamine oxidation down-regulate IL-2 production by peripheral blood T cells. We show here that the production of IL-2 by rheumatoid arthritis synovial fluid mononuclear cells is inversely correlated with the concentrations of polyamines in these cells. In addition, the inhibition of polyamine biosynthesis or oxidation in cultures of these cells enhances their ability to produce IL-2. Our findings suggest that polyamine oxidation plays an important role in the suppression of T cell function characteristic of rheumatoid arthritis synovial fluids.