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Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-benzylthioinosines act as subversive substrates of T. gondii, but not human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Moreover, 7-deazaadenosine (tubercidin) was shown to be an excellent ligand of T. gondii adenosine kinase. Therefore, we synthesized 7-deaza-6-benzylthioinosine, and analogues with various substitutions at their phenyl ring, to increase the binding affinity of the 6-benzylthioinosines to T. gondii adenosine kinase. Indeed, the 7-deaza-6-benzylthioinosine analogues were better ligands of T. gondii adenosine kinase than the parent compounds, 6-benzylthioinosine and 7-deazainosine. Herein, we report the testing of the metabolism of these newly synthesized 7-deaza-6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the 7-deaza-6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the 7-deaza-6-benzylthioinosine analogues showed a selective antitoxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were 7-deaza-6-(p-methoxybenzylthio)inosine (IC(50)=4.6 microM), 7-deaza-6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=5.0 microM), and 7-deaza-6-(p-cyanobenzylthio)inosine (IC(50)=5.3 microM). These results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that 7-deaza-6-benzylthioinosines are potential antitoxoplasmic agents.

The powerful anti-inflammatory and immunosuppressive activities of IL-10 make it attractive for supplemental therapy in translational tolerance induction protocols. This is bolstered by reports of IL-10-mediated inhibition of innate immunity, association of human stem cell and nonhuman primate (NHP) islet allograft tolerance with elevated serum IL-10, and evidence that systemic IL-10 therapy enhanced pig islets survival in mice. IL-10 has not been examined as adjunctive immunosuppression in NHP. To enable such studies, we cloned and expressed rhesus macaque (RM) IL-10 fused to a mutated hinge region of human IgG1 Fc to generate IL-10/Fc(ala-ala). RM IL-10/Fc(ala-ala) was purified to approximately 98% homogeneity by affinity chromatography and shown to be endotoxin-free (0.008 EU/microg protein). The biological activity of IL-10/Fc(ala-ala) was demonstrated by (1) costimulation of the mouse mast cell line, MC/9 proliferation in a dose-dependent fashion, (2) suppression of LPS-induced septic shock in mice and (3) abrogation of LPS-induced secretion of proinflammatory cytokines/chemokines in vitro and in vivo in NHP. Notably, RM IL-10/Fc(ala-ala) had significantly greater potency than human IL-10/Fc(ala-ala) and exhibited a circulating half-life of approximately 14 days. The availability of this reagent will facilitate definitive studies to determine whether supplemental therapy with RM IL-10/Fc(ala-ala) can influence tolerance outcomes in NHP.

Optical coherence tomography (OCT) may revolutionize fundamental investigation and clinical management of age-related macular degeneration and other eye diseases. However, quantitative OCT interpretation is hampered due to uncertain sub-cellular correlates of reflectivity in the retinal pigment epithelium (RPE) and photoreceptor. The purpose of this study was twofold: 1) to test OCT correlates in the RPE, and 2) to demonstrate the feasibility of longitudinal OCT monitoring of sub-cellular RPE dynamics. A high resolution OCT was constructed to achieve dynamic imaging of frog eyes, in which light-driven translocation of RPE melanosomes occurred within the RPE cell body and apical processes. Comparative histological examination of dark- and light-adapted eyes indicated that the RPE melanin granule, i.e., melanosome, was a primary OCT correlate. In vivo OCT imaging of RPE melanosomes opens the opportunity for quantitative assessment of RPE abnormalities associated with disease, and enables longitudinal investigation of RPE kinetics correlated with visual function.

Summary CD4 + CD25 + regulatory T cells (Tregs) play an important role in allograft and self-tolerance and thus have potential therapeutic application in transplantation, autoimmunity, and allergy. Although nonhuman primate (NHP) provide the most accepted preclinical models for translational studies in allograft tolerance and infectious diseases, CD4 + CD25 + Tregs have been rarely studied in NHP. The low frequencies of Tregs in peripheral blood will likely necessitate ex vivo expansion to enable Tregs adaptive immune therapy in NHP and humans. Tregs were isolated by magnetic and flow sorting and then stimulated weekly with antirhesus CD3 clone FN18 and antihuman CD28-coated Dynal beads plus 100 U/ml rhIL-2. Under these conditions, the Tregs were expanded 300- to 2000-fold in 4 weeks. Expanded CD4 + CD25 + Tregs expressed high to moderate levels of FOXP3 as well as CD95, CD62L, CD69, and CCR7 surface antigens. Expanded rhesus Tregs were anergic and suppressed the proliferation of autologous peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion, and the suppression was partially reversed by anti-transforming growth factor (TGF)-β1 neutralizing antibody (Ab). These results demonstrate that rhesus macaque suppressive regulatory CD4 + CD25 + FOXP3 + Tregs can be efficiently expanded in vitro under rhesus-specific stimulation, which enables preclinical testing of Treg therapy in the NHP model.

The purine nucleoside analogue NBMPR {nitrobenzylthioinosine or 6-[(4-nitrobenzyl)thio]-9-β- d -ribofuranosylpurine} was selectively phosphorylated to its nucleoside 5′-monophosphate by Toxoplasma gondii but not mammalian adenosine kinase (EC 2.7.1.20 ). NBMPR was also cleaved in toxoplasma to its nucleobase, nitrobenzylmercaptopurine. However, nitrobenzylmercaptopurine was not a substrate for either adenosine kinase or hypoxanthine-guanine-xanthine phosphoribosyltransferase (EC 2.4.2.8 ). Because of this unique and previously unknown metabolism of NBMPR by the parasite, the effect of NBMPR as an antitoxoplasmic agent was tested. NBMPR killed T. gondii grown in human fibroblasts in a dose-dependent manner, with a 50% inhibitory concentration of approximately 10 μM and without apparent toxicity to host cells. Doses of up to 100 μM had no significant toxic effect on uninfected host cells. The promising antitoxoplasmic effect of NBMPR led to the testing of other 6-substituted 9-β- d -ribofuranosylpurines, which were shown to be good ligands of the parasite adenosine kinase (M. H. Iltzsch, S. S. Uber, K. O. Tankersley, and M. H. el Kouni, Biochem. Pharmacol. 49:1501–1512, 1995), as antitoxoplasmic agents. Among the analogues tested, 6-benzylthioinosine, p -nitrobenzyl-6-selenopurine riboside, N 6 -( p -azidobenzyl)adenosine, and N 6 -( p -nitrobenzyl)adenosine, like NBMPR, were selectively toxic to parasite-infected cells. Thus, it appears that the unique characteristics of purine metabolism in T. gondii render certain 6-substituted 9-β- d -ribofuranosylpurines promising antitoxoplasmic drugs.

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.