ABSTRACT
Therapeutic vaccines to induce anti-tumor CD8 T cells have been used in clinical trials for advanced melanoma patients, but the clinical response rate and overall survival time have not improved much. We believe that these dismal outcomes are caused by inadequate number of antigen-specific CD8 T cells generated by most vaccines. In contrast, huge CD8 T cell responses readily occur during acute viral infections. High levels of type-I interferon (IFN-I) are produced during these infections, and this cytokine not only exhibits anti-viral activity but also promotes CD8 T cell responses. The studies described here were performed to determine whether promoting the production of IFN-I could enhance the potency of a peptide vaccine. We report that cyclic diguanylate monophosphate (c-di-GMP), which activates the stimulator of interferon genes, potentiated the immunogenicity and anti-tumor effects of a peptide vaccine against mouse B16 melanoma. The synergistic effects of c-di-GMP required co-administration of costimulatory anti-CD40 antibody, the adjuvant poly-IC, and were mediated in part by IFN-I. These findings demonstrate that peptides representing CD8 T cell epitopes can be effective inducers of large CD8 T cell responses in vaccination strategies that mimic acute viral infections.
Subject(s)
CD8-Positive T-Lymphocytes/drug effects , Cancer Vaccines/therapeutic use , Cyclic GMP/analogs & derivatives , Immunotherapy/methods , Melanoma, Experimental/therapy , Skin Neoplasms/therapy , Animals , Antibodies, Monoclonal/administration & dosage , CD40 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Cyclic GMP/administration & dosage , Cyclic GMP/adverse effects , Epitopes, T-Lymphocyte/administration & dosage , Humans , Interferon Type I/immunology , Lymphocyte Activation/drug effects , Melanoma, Experimental/immunology , Membrane Proteins/agonists , Mice , Mice, Inbred C57BL , Mice, Knockout , Poly I-C/administration & dosage , Receptors, Interferon/genetics , Skin Neoplasms/immunology , Vaccines, Subunit/therapeutic useABSTRACT
A large body of evidence indicates that nitric oxide (NO) and cGMP contribute to central sensitization of pain pathways during inflammatory pain. Here, we investigated the distribution of cyclic nucleotide-gated (CNG) channels in the spinal cord, and identified the CNG channel subunit CNGA3 as a putative cGMP target in nociceptive processing. In situ hybridization revealed that CNGA3 is localized to inhibitory neurons of the dorsal horn of the spinal cord, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. CNGA3 expression is upregulated in the superficial dorsal horn of the mouse spinal cord and in dorsal root ganglia following hindpaw inflammation evoked by zymosan. Mice lacking CNGA3 (CNGA3(-/-) mice) exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior in models of acute or neuropathic pain was normal. Moreover, CNGA3(-/-) mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Our results provide evidence that CNGA3 contributes in an inhibitory manner to the central sensitization of pain pathways during inflammatory pain as a target of NO/cGMP signaling.
Subject(s)
Cyclic GMP/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Inflammation/complications , Nitric Oxide/metabolism , Pain/etiology , Signal Transduction/physiology , Spinal Cord/metabolism , Analysis of Variance , Animals , Cyclic GMP/adverse effects , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Cyclic Nucleotide-Gated Cation Channels/deficiency , Cyclic Nucleotide-Gated Cation Channels/genetics , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Ganglia, Spinal/metabolism , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Inflammation/chemically induced , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microdissection , Naphthalenes/metabolism , Natriuretic Peptides/adverse effects , Pain/drug therapy , Pain/pathology , Pain Measurement/drug effects , Pain Measurement/methods , Pain Perception/drug effects , Peripheral Nervous System Diseases/drug therapy , Peripheral Nervous System Diseases/pathology , Physical Stimulation/adverse effects , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Stathmin/metabolism , Statistics, Nonparametric , Thionucleotides/pharmacology , Triazenes/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/metabolismABSTRACT
Several studies have reported the anxiolytic-like effects of various nitric oxide synthase inhibitors in distinct animal models. However, in the context of anxiety, the possible involvement of cyclic GMP, believed to be one of the main targets of NO, remains obscure. Cyclic GMP is degraded by the specific phosphodiesterases in the brain. Therefore, we studied the effect of the selective phosphodiesterase type 5 inhibitor sildenafil in the mouse elevated plus-maze test of anxiety and in the open field test of locomotion. We found that sildenafil (0.05-10 mg/kg i.p.) alone did not affect the behavior of animals in the plus-maze or open field tests, but the anxiogenic beta-carboline DMCM given in a subconvulsive dose (2 mg/kg i.p.) decreased the time spent on open arms in the elevated plus-maze. Treatment with the NO precursor L-arginine (200 mg/kg i.p.) did not modify the behavior of animals in the plus-maze, however, when sildenafil (1 mg/kg i.p.) was administered in combination with L-arginine (200 mg/kg i.p.), both the time spent on the open arms and the percentage of open arm visits were significantly decreased. We conclude that augmentation of the NO-cGMP cascade induces anxiogenic-like effect in mice.
Subject(s)
Anxiety/chemically induced , Cyclic GMP/adverse effects , Cyclic GMP/metabolism , Nitric Oxide/adverse effects , Nitric Oxide/metabolism , Animals , Arginine/administration & dosage , Arginine/adverse effects , Arginine/antagonists & inhibitors , Behavior, Animal/drug effects , Carbolines/administration & dosage , Carbolines/adverse effects , Carbolines/pharmacokinetics , Cyclic GMP/pharmacokinetics , Disease Models, Animal , Drug Therapy, Combination , Injections, Intraperitoneal , Male , Maze Learning/drug effects , Maze Learning/physiology , Mice , Motor Activity/drug effects , Nitric Oxide/pharmacokinetics , Piperazines/administration & dosage , Piperazines/adverse effects , Piperazines/antagonists & inhibitors , Purines , Sildenafil Citrate , Sulfones , Time FactorsABSTRACT
We evaluated the involvement of nitric oxide (NO) in the early hemodynamic response to uninephrectomy (UNX) in rats. Animals were uninephrectomized, and 48 h after removal of the kidney, the effect of infusing NG-nitro-L-arginine methyl ester (L-NAME) on renal function was studied. Glomeruli were isolated, and glomerular nitrite and cGMP productions were measured. In addition, endothelial constitutive NO synthase (NOS III) and inducible NO synthase (iNOS) were assessed by Western blot and by measuring the conversion of arginine to citrulline. UNX animals showed an increase in renal plasma flow that was inhibited by L-NAME in a higher proportion than in sham-operated (SO) animals. No differences were observed in systemic NO-dependent vascular tone, since mean arterial pressure showed similar increments in SO and UNX rats. Glomeruli from UNX animals showed an increase in glomerular nitrite production that was blunted by L-NAME addition. Also, cGMP levels were increased in glomeruli from UNX animals, and this increase was inhibited by L-NAME. Western blot analysis showed no differences in NOS III but a higher iNOS amount in glomeruli from UNX than in those from SO rats. No significant differences between UNX and SO rats were found in calcium-dependent NOS enzymatic activity in the renal cortex. However, calcium-independent enzymatic activity was markedly higher in the renal cortex of UNX than in those from SO animals. In conclusion, glomeruli from rats 48 h after UNX had a greater production of NO than those from SO animals. This increased glomerular NO production is based on an increase in the iNOS isoform. Increased glomerular NO synthesis seems to play a role in the decreased renal vascular resistance observed after unilateral nephrectomy in rats.
