Development of Potent Immune Modulators Targeting Stimulator of Interferon Genes Receptor.

Stimulator of interferon genes (STING) is an endoplasmic reticulum-membrane protein that plays important roles in cancer immunotherapy by activating innate immune responses. We designed and synthesized STING modulators and characterized compounds 4a and 4c that share a crucial amidobenzimidazole moiety. In vitro STING binding and cell-based activity assays demonstrated the potency and efficacy of the compounds that function as direct STING agonists by stimulating STING downstream signaling and promoting type I interferon immune responses. In vitro metabolic studies and the pharmacokinetic properties of the compounds led us to investigate their anticancer activity in an in vivo syngeneic model. Intravenous injection of compounds 4a and 4c significantly decreased tumor volume in a CT26 murine colorectal carcinoma model, and the immunological memory-derived cancer inhibition was observed in 4c-treated mouse models. The present results suggest the therapeutic potential of the compounds for cancer immunotherapy via STING-mediated immune activation.

Myxomavirus anti-inflammatory chemokine binding protein reduces the increased plaque growth induced by chronic Porphyromonas gingivalis oral infection after balloon angioplasty aortic injury in mice.

Thrombotic occlusion of inflammatory plaque in coronary arteries causes myocardial infarction. Treatment with emergent balloon angioplasty (BA) and stent implant improves survival, but restenosis (regrowth) can occur. Periodontal bacteremia is closely associated with inflammation and native arterial atherosclerosis, with potential to increase restenosis. Two virus-derived anti-inflammatory proteins, M-T7 and Serp-1, reduce inflammation and plaque growth after BA and transplant in animal models through separate pathways. M-T7 is a broad spectrum C, CC and CXC chemokine-binding protein. Serp-1 is a serine protease inhibitor (serpin) inhibiting thrombotic and thrombolytic pathways. Serp-1 also reduces arterial inflammation and improves survival in a mouse herpes virus (MHV68) model of lethal vasculitis. In addition, Serp-1 demonstrated safety and efficacy in patients with unstable coronary disease and stent implant, reducing markers of myocardial damage. We investigate here the effects of Porphyromonas gingivalis, a periodontal pathogen, on restenosis after BA and the effects of blocking chemokine and protease pathways with M-T7 and Serp-1. ApoE-/- mice had aortic BA and oral P. gingivalis infection. Arterial plaque growth was examined at 24 weeks with and without anti-inflammatory protein treatment. Dental plaques from mice infected with P. gingivalis tested positive for infection. Neither Serp-1 nor M-T7 treatment reduced infection, but IgG antibody levels in mice treated with Serp-1 and M-T7 were reduced. P. gingivalis significantly increased monocyte invasion and arterial plaque growth after BA (P<0.025). Monocyte invasion and plaque growth were blocked by M-T7 treatment (P<0.023), whereas Serp-1 produced only a trend toward reductions. Both proteins modified expression of TLR4 and MyD88. In conclusion, aortic plaque growth in ApoE-/- mice increased after angioplasty in mice with chronic oral P. gingivalis infection. Blockade of chemokines, but not serine proteases significantly reduced arterial plaque growth, suggesting a central role for chemokine-mediated inflammation after BA in P. gingivalis infected mice.

Formation of human IFN-beta complex with the soluble type I interferon receptor IFNAR-2 leads to enhanced IFN stability, pharmacokinetics, and antitumor activity in xenografted SCID mice.

Interferon-beta (IFN-beta) is biologically unstable under physiologic conditions in vitro and is cleared rapidly from the bloodstream on administration in vivo. In the present study, we demonstrate that a soluble recombinant form of the type I IFN receptor subunit, sIFNAR-2, can neutralize the bioactivity of type I IFNs at high concentrations and, at lower concentrations, causes an enhancement of IFN-beta-mediated antiviral activity. The in vitro enhancement is due to the specific interaction of IFN-beta with sIFNAR-2, followed by dissociation of IFN-beta from the complex over time in culture. In vivo, the serum half-life of IFN-beta is extended from minutes to hours when administered intravenously in mice as a sIFNAR-2-associated complex. Moreover, the antitumor effect of IFN-beta is increased by between 9-fold and 27-fold when injected as an sIFNAR-2-associated complex, as demonstrated by an increase in the mean survival time of immunodeficient mice challenged with human Burkitt lymphoma cell (Daudi) xenografts (sIFNAR-2-complexed vs. free IFN-beta treatment). These results show that on association with sIFNAR-2, IFN-beta is more stable in vitro and exhibits increased efficacy when administered in vivo. Administration as a complex with sIFNAR-2 may, therefore, provide a method of enhancing the delivery and effectiveness of type I IFNs.

Interferons: mechanisms of action and clinical applications.

PURPOSE OF REVIEW: Interferons are pleiotropic cytokines that exhibit important biologic activities, including antiviral, antitumor, and immunomodulatory effects. These cytokines have found important applications in clinical medicine, including the treatment of certain malignancies. The purpose of this review is to provide an update on basic and clinical research in the interferon field. RECENT FINDINGS: Significant advances have recently occurred in the field of type I interferon signal transduction. It is well known that the interferons transduce signals via activation of multiple signaling cascades, involving Jak kinases, signal transducer and activator of transcription proteins, Map kinases, and IRS and Crk proteins. Recent evidence indicates that the p38 Map kinase pathway plays an important role in type I interferon signaling in malignant cells and that its function is required for type I interferon-dependent gene transcription and generation of the antiproliferative of type I interferons. In clinical oncology, interferon-alpha remains an active and useful agent in the treatment of several malignant disorders, and efforts are underway to improve its efficacy by using different schedules and combinations with other agents. SUMMARY: This review summarizes the mechanisms of signal transduction of interferons and the emerging new concepts in this area. An update on the clinical applications of interferons in oncology is also provided, and potential translational applications, reflecting recent advances in the field, are discussed.

Chemokine-binding viral protein M-T7 prevents chronic rejection in rat renal allografts.

M-T7 is a myxoma virus-encoded protein that has been found to bind and disrupt human chemokine gradients. This study examined whether purified M-T7 could prevent chronic rejection in a rat renal allograft model. Fisher F344 renal allografts were transplanted into Lewis rats. Recipients were randomly grouped into two groups: control animals treated with cyclosporine alone and animals treated with cyclosporine combined with low-, medium- and high-dose M-T7 viral protein. The survival rate was not significantly different between allograft groups. Renal allografts treated with high-dose M-T7 demonstrated a significant reduction in tubular atrophy, glomerular atrophy, vascular hyalinization, cortical scarring, and lymphocyte infiltration. Morphometric analyses demonstrated that the high-dose M-T7 group also showed a significantly decreased amount of glomerulosclerosis and transplant arteriosclerosis. These data demonstrate for the first time that the immunoregulatory viral protein M-T7 can effectively attenuate chronic rejection in rat renal allografts.

Atividade parasiticida e/ou parasitostática de fibroblastos e macrofagos humanos ativados por rIFN-y e/ou rTNF-x

Estudamos o papel da degradação do triptofano pela indoleamina 2,3-dioxigenase (INDO) no controle da replicação do T. cruzzi ou T. gondii em fibroblastos e macrófagos humanos estimulados com rIFN-gama e/ou rTNF-alfa. O T. gondii foi utlizado como controle, por ser sensível à degradação do triptofano induzida pelo rIFN-gama. A estimulação de fibroblastos humanos com rIFN-gama e o rTNF-alfa inibiu consideravelmente o desenvolvimento do T. gondii, mas não influenciou o desenvolvimento do T. cruzi. O desenvolvimento do T. gondii foi triptofano dependente, enquanto que o do T. cruzi foi triptofano independente. Ao contrário dos fibroblastos parentais, os fibroblastos defectivos na via de transdução de sinal do IFN-gama (JAKI, JAK2 e STAT1alfa)não modularam o desenvolvimento intracelular de T. gondii e nem expressaram mRNA da INDO quando estimulados pelo rIFN-gama. Houve uma pequena indução de mRNA da INDO em fibroblastos parentais e mutantes (JAK2) estimulados com rTNF-alfa, que provavelmente foi estimulado através da indução da síntese de IFN-es em fibroblastos humanos. O rIFN-gama e/ou rTNF-alfa induziram expressiva atividade tripanosomicida em macrófagos humanos.

A estimulação de macrófagos humanos com rIFN-gama ou rIFN-gama + rTNF-alfa, produziu uma considerável expressão do mRNA da INDO, e pouca expressão foi detectada quando as células foram estimuladas apenas com rTNF-alfa. A adição de triptofano, ou do inibidor da INDO, norharmane, à cultura de macrófagos humanos ativados com rIFN-gama e/ou rTNF-alfa, não reverteu a inibição do crescimento do parasita , mostrando que a degradação do triptofano não é um mecanismo responsável pelo efeito tripanosomicida de macrófagos humanos ativados com rIFN-gama e/ou rTNF-alfa. Os catabólitos do triptofano (ácido quinolínico, ácido quinurênico e L-quinurenina)inibiram parcialmente o desenvolvimento do T. cruzi em macrófagos humanos. A amplificação por PCR do DNA T. cLsintese de Escherichia coli ou Neurospora crassa e Saccharomyces cerevisae e a hibridização cruzada via dot blot utilizando como sondas os produtos de PCR, sugerem a possibilidade da existência da enzima triptofano sintetase em T. cruzi, o que explicaria a insensibilidade do parasita à depleção do triptofano. Alternativamente reconhecemos que mais estudos devam ser feitos para a comprovação da existência da enzima em T. cruzi

Atividade parasiticida e/ou parasitostática de fibroblastos e macrofagos humanos ativados por rIFN-y e/ou rTNF-x / Parasiticides activity and/or parasitostática of fibroblasts and macrophages activated by human rIFN-y and / or rTNF-x

Estudamos o papel da degradação do triptofano pela indoleamina 2,3-dioxigenase (INDO) no controle da replicação do T. cruzzi ou T. gondii em fibroblastos e macrófagos humanos estimulados com rIFN-gama e/ou rTNF-alfa. O T. gondii foi utlizado como controle, por ser sensível à degradação do triptofano induzida pelo rIFN-gama. A estimulação de fibroblastos humanos com rIFN-gama e o rTNF-alfa inibiu consideravelmente o desenvolvimento do T. gondii, mas não influenciou o desenvolvimento do T. cruzi. O desenvolvimento do T. gondii foi triptofano dependente, enquanto que o do T. cruzi foi triptofano independente. Ao contrário dos fibroblastos parentais, os fibroblastos defectivos na via de transdução de sinal do IFN-gama (JAKI, JAK2 e STAT1alfa) não modularam o desenvolvimento intracelular de T. gondii e nem expressaram mRNA da INDO quando estimulados pelo rIFN-gama. Houve uma pequena indução de mRNA da INDO em fibroblastos parentais e mutantes (JAK2) estimulados com rTNF-alfa, que provavelmente foi estimulado através da indução da síntese de IFN-es em fibroblastos humanos. O rIFN-gama e/ou rTNF-alfa induziram expressiva atividade tripanosomicida em macrófagos humanos. A estimulação de macrófagos humanos com rIFN-gama ou rIFN-gama + rTNF-alfa, produziu uma considerável expressão do mRNA da INDO, e pouca expressão foi detectada quando as células foram estimuladas apenas com rTNF-alfa. A adição de triptofano, ou do inibidor da INDO, norharmane, à cultura de macrófagos humanos ativados com rIFN-gama e/ou rTNF-alfa, não reverteu a inibição do crescimento do parasita, mostrando que a degradação do triptofano não é um mecanismo responsável pelo efeito tripanosomicida de macrófagos humanos ativados com rIFN-gama e/ou rTNF-alfa. Os catabólitos do triptofano (ácido quinolínico, ácido quinurênico e L-quinurenina) inibiram parcialmente o desenvolvimento do T. cruzi em macrófagos humanos. A amplificação por PCR do DNA T. cLsintese de Escherichia coli ou Neurospora crassa e Saccharomyces cerevisae e a hibridização cruzada via dot blot utilizando como sondas os produtos de PCR, sugerem a possibilidade da existência da enzima triptofano sintetase em T. cruzi, o que explicaria a insensibilidade do parasita à depleção do triptofano. Alternativamente reconhecemos que mais estudos devam ser feitos para a comprovação da existência da enzima em T. cruzi.