Control of Parasitophorous Vacuole Expansion by LYST/Beige Restricts the Intracellular Growth of Leishmania amazonensis

The intracellular protozoan Leishmania replicates in parasitophorous vacuoles (PV) that share many features with late endosomes/lysosomes. L. amazonensis PVs expand markedly during infections, but the impact of PV size on parasite intracellular survival is still unknown. Here we show that host cells infected with L. amazonensis upregulate transcription of LYST/Beige, which was previously shown to regulate lysosome size. Mutations in LYST/Beige caused further PV expansion and enhanced L. amazonensis replication. In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth. Treatment of LYST/Beige over-expressing cells with vacuolin-1 reversed this phenotype, expanding PVs and promoting parasite growth. The opposite was seen with E-64d, which reduced PV size in LYST-Beige mutant cells and inhibited L. amazonensis replication. Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs. Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival

Nitric oxide short-circuits interleukin-12-mediated tumor regression

Interleukin-12 (IL-12) can promote tumor regression via activation of multiple lymphocytic and myelocytic eVectors. Whereas the cytotoxic mechanisms employed by T/NK/NKT cells in IL-12-mediated tumor kill are well deWned, the antitumor role of macrophage-produced cytotoxic metabolites has been more controversial. To this end, we investigated the speciWc role of nitric oxide (NO), a major macrophage eVector molecule, in post-IL-12 tumor regression. Analysis of tumors following a single intratumoral injection of slow-release IL-12 microspheres showed an IFN-dependent sevenfold increase in inducible nitric oxide synthase (iNOS) expression within 48 h. Flow cytometric analysis of tumor-resident leukocytes and in vivo depletion studies identiWed CD11b+ F4/80+ Gr1lo macrophages as the primary source of iNOS. Blocking of post-therapy iNOS activity with N-nitro-L-arginine methyl ester (L-NAME)dramatically enhanced tumor suppression revealing the inhibitory eVect of NO on IL-12-driven antitumor immunity. Superior tumor regression in mice receiving combination treatment was associated with enhanced survival and proliferation of activated tumor-resident CD8+ T-eVector/memory cells (Tem). These Wndings demonstrate that macrophageproduced NO negatively regulates the antitumor activity of IL-12 via its detrimental eVects on CD8+ T cells and identify L-NAME as a potent adjuvant in IL-12 therapy of cancer

Structure–Activity Relationships and Discovery of a G Protein Biased μ Opioid Receptor Ligand, [(3-Methoxythiophen-2-yl)methyl]({2-[(9<i>R</i>)‑9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan-9-yl]ethyl})amine (TRV130), for the Treatment of Acute Severe Pain

The concept of “ligand bias” at G protein coupled receptors has been introduced to describe ligands which preferentially stimulate one intracellular signaling pathway over another. There is growing interest in developing biased G protein coupled receptor ligands to yield safer, better tolerated, and more efficacious drugs. The classical μ opioid morphine elicited increased efficacy and duration of analgesic response with reduced side effects in β-arrestin-2 knockout mice compared to wild-type mice, suggesting that G protein biased μ opioid receptor agonists would be more efficacious with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased μ opioid receptor agonist, TRV130 ((<i><b>R</b></i>)-<b>30</b>). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for the treatment of acute severe pain

Effects of upregulated indoleamine 2, 3-dioxygenase 1 by interferon γ gene transfer on interferon γ-mediated antitumor activity

Interferon γ (IFN-γ), an anticancer agent, is a strong inducer of indoleamine 2, 3-dioxygenase 1 (IDO1), which is a tryptophan-metabolizing enzyme involved in the induction of tumor immune tolerance. In this study, we investigated the IDO1 expression in organs after IFN-γ gene transfer to mice. IFN-γ gene transfer greatly increased the mRNA expression of IDO1 in many tissues with the highest in the liver. This upregulation was associated with reduced L-tryptophan levels and increased L-kynurenine levels in serum, indicating that IFN-γ gene transfer increased the IDO activity. Then, Lewis lung carcinoma (LLC) tumor-bearing wild-type and IDO1-knockout (IDO1 KO) mice were used to investigate the effects of IDO1 on the antitumor activity of IFN-γ. IFN-γ gene transfer significantly retarded the tumor growth in both strains without any significant difference in tumor size between the two groups. By contrast, the IDO1 activity was increased only in the wild-type mice by IFN-γ gene transfer, suggesting that cells other than LLC cells, such as tumor stromal cells, are the major contributors of IDO1 expression in LLC tumor. Taken together, these results imply that IFN-γ gene transfer mediated IDO1 upregulation in cells other than LLC cells has hardly any effect on the antitumor activity of IFN-γ