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2.
Rheumatology (Oxford) ; 63(1): 198-208, 2024 Jan 04.
Article in English | MEDLINE | ID: mdl-37086441

ABSTRACT

OBJECTIVES: Granulomatosis with polyangiitis (GPA) is a chronic relapsing systemic autoimmune vasculitis. Current treatment of GPA is unsatisfactory, as it relies on strong immunosuppressive regimens, with either CYC or rituximab, which reduce the immunogenicity of several vaccines and are risk factors for a severe form of COVID-19. This emphasizes the need to identify new drug targets and to develop treatment strategies with less harmful side effects. Since CD4+ effector memory T cells (TEM) play a key role in the pathogenesis of GPA, we aimed in this study to modulate CD4+TEM cell activity via Kv1.3 blockade using the specific peptide inhibiter, ShK-186. METHODS: Peripheral blood samples from 27 patients with GPA in remission and 16 age- and sex-matched healthy controls (HCs) were pre-incubated in vitro in the presence or absence of ShK-186, followed by stimulation with phorbol myristate acetate, calcium ionophore and brefeldin-A. The effect of ShK-186 on the cytokine production (IFNγ, TNFα, IL-4, IL-17, IL-21) within total and subsets of CD4+ T helper (CD4+TH) cells were assessed using flow cytometry. RESULTS: ShK-186 reduced the expression level of IFNγ, TNFα, IL-4, IL-17 and IL-21 in CD4+TH cells from patients with GPA in vitro. Further analysis performed on sorted CD4+T cell subsets, revealed that ShK-186 predominantly inhibited the cytokine production of CD4+TEM cells. ShK-186 treatment reduced the production of the pro-inflammatory cytokines to the level seen in CD4+ TH cells from HCs. CONCLUSIONS: Modulation of cellular effector function by ShK-186 may constitute a novel treatment strategy for GPA with high specificity and less harmful side effects.


Subject(s)
Granulomatosis with Polyangiitis , Interleukin-17 , Humans , Memory T Cells , Granulomatosis with Polyangiitis/drug therapy , Interleukin-4 , Tumor Necrosis Factor-alpha , CD4-Positive T-Lymphocytes/metabolism , Cytokines/metabolism
3.
Front Immunol ; 14: 1311658, 2023.
Article in English | MEDLINE | ID: mdl-38152397

ABSTRACT

Background: Immune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response. Methods: Fully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and single B cell sequencing. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using in vitro and in vivo models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys. Results: Here, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated. Conclusion: These results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors.


Subject(s)
Antibodies, Monoclonal , Immune Checkpoint Inhibitors , Neoplasms , Animals , Humans , Mice , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Macaca fascicularis , Neoplasms/therapy , T-Lymphocytes , Tumor Microenvironment
4.
PLoS Pathog ; 14(12): e1007439, 2018 12.
Article in English | MEDLINE | ID: mdl-30576397

ABSTRACT

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.


Subject(s)
Antiviral Agents/pharmacology , Lassa Fever , Lassa virus/genetics , Virulence/drug effects , Virulence/genetics , Animals , Chlorocebus aethiops , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , HEK293 Cells , Humans , Lassa virus/drug effects , Mice , Mutation , Vero Cells , Viral Envelope Proteins/genetics
5.
Front Immunol ; 8: 1205, 2017.
Article in English | MEDLINE | ID: mdl-29018452

ABSTRACT

B cells are central to the pathogenesis of granulomatosis with polyangiitis (GPA), exhibiting both (auto)antibody-dependent and -independent properties. Class-switched memory B cells in particular are a major source of pathogenic autoantibodies. These cells are characterized by high expression levels of Kv1.3 potassium channels, which may offer therapeutic potential for Kv1.3 blockade. In this study, we investigated the effect of the highly potent Kv1.3 blocker ShK-186 on B cell properties in GPA in vitro. Circulating B cell subsets were determined from 33 GPA patients and 17 healthy controls (HCs). Peripheral blood mononuclear cells (PBMCs) from GPA patients, and HCs were stimulated in vitro in the presence and absence of ShK-186. The production of total and antineutrophil cytoplasmic antibodies targeting proteinase 3 (PR3-ANCA) IgG was analyzed by enzyme-linked immunosorbent assay and Phadia EliA, respectively. In addition, effects of ShK-186 on B cell proliferation and cytokine production were determined by flow cytometry. The frequency of circulating switched and unswitched memory B cells was decreased in GPA patients as compared to HC. ShK-186 suppressed the production of both total and PR3-ANCA IgG in stimulated PBMCs. A strong decrease in production of tumor necrosis factor alpha (TNFα), interleukin (IL)-2, and interferon gamma was observed upon ShK-186 treatment, while effects on IL-10 production were less pronounced. As such, ShK-186 modulated the TNFα/IL-10 ratio among B cells, resulting in a relative increase in the regulatory B cell pool. ShK-186 modulates the effector functions of B cells in vitro by decreasing autoantibody and pro-inflammatory cytokine production. Kv1.3 channel blockade may hold promise as a novel therapeutic strategy in GPA and other B cell-mediated autoimmune disorders.

6.
PLoS One ; 12(7): e0180762, 2017.
Article in English | MEDLINE | ID: mdl-28723914

ABSTRACT

BACKGROUND: Dalazatide is a specific inhibitor of the Kv1.3 potassium channel. The expression and function of Kv1.3 channels are required for the function of chronically activated memory T cells, which have been shown to be key mediators of autoimmune diseases, including psoriasis. OBJECTIVE: The primary objective was to evaluate the safety of repeat doses of dalazatide in adult patients with mild-to-moderate plaque psoriasis. Secondary objectives were to evaluate clinical proof of concept and the effects of dalazatide on mediators of inflammation in the blood and on chronically activated memory T cell populations. METHODS: Patients (n = 24) were randomized 5:5:2 to receive dalazatide at 30 mcg/dose, 60 mcg/dose, or placebo twice weekly by subcutaneous injection (9 doses total). Safety was assessed on the basis of physical and neurological examination and laboratory testing. Clinical assessments included body-surface area affected, Psoriasis Area and Severity Index (PASI), and investigator and patient questionnaires. RESULTS: The most common adverse events were temporary mild (Grade 1) hypoesthesia (n = 20; 75% placebo, 85% dalazatide) and paresthesia (n = 15; 25% placebo, 70% dalazatide) involving the hands, feet, or perioral area. Nine of 10 patients in the 60 mcg/dose group had a reduction in their PASI score between baseline and Day 32, and the mean reduction in PASI score was significant in this group (P < 0.01). Dalazatide treatment reduced the plasma levels of multiple inflammation markers and reduced the expression of T cell activation markers on peripheral blood memory T cells. LIMITATIONS: The study was small and drug treatment was for a short duration (4 weeks). CONCLUSION: This study indicates that dalazatide is generally well tolerated and can improve psoriatic skin lesions by modulating T cell surface and activation marker expression and inhibiting mediators of inflammation in the blood. Larger studies of longer duration are warranted.


Subject(s)
Kv1.3 Potassium Channel/antagonists & inhibitors , Potassium Channel Blockers/adverse effects , Proteins/adverse effects , Psoriasis/drug therapy , Adult , Double-Blind Method , Female , Humans , Hypesthesia/chemically induced , Male , Middle Aged , Paresthesia/chemically induced , Potassium Channel Blockers/pharmacology , Potassium Channel Blockers/therapeutic use , Proteins/pharmacology , Proteins/therapeutic use , Treatment Outcome
7.
Vaccine ; 35(15): 1964-1971, 2017 04 04.
Article in English | MEDLINE | ID: mdl-28279563

ABSTRACT

Vaccine adjuvants are essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Benzothiazoles/administration & dosage , DEAD Box Protein 58/metabolism , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Interferon Regulatory Factor-3/metabolism , Adjuvants, Immunologic/isolation & purification , Animals , Benzothiazoles/isolation & purification , Cell Line , Disease Models, Animal , Drug Evaluation, Preclinical , Female , High-Throughput Screening Assays , Humans , Mice, Inbred C57BL , Orthomyxoviridae Infections/prevention & control , Receptors, Immunologic , Survival Analysis
8.
Proc Natl Acad Sci U S A ; 114(10): E1825-E1832, 2017 03 07.
Article in English | MEDLINE | ID: mdl-28223528

ABSTRACT

Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.


Subject(s)
Cancer Pain/drug therapy , Hyperalgesia/drug therapy , Peptides/administration & dosage , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Analgesics, Opioid/adverse effects , Animals , Cancer Pain/chemically induced , Cancer Pain/genetics , Cancer Pain/pathology , Humans , Hyperalgesia/chemically induced , Hyperalgesia/genetics , Hyperalgesia/pathology , Ligands , Mice , Mice, Knockout , Neuralgia/chemically induced , Neuralgia/drug therapy , Neuralgia/genetics , Neuralgia/pathology , Nicotinic Antagonists/administration & dosage , Organoplatinum Compounds/adverse effects , Oxaliplatin , Receptors, GABA-B/genetics
9.
J Virol ; 90(5): 2372-87, 2015 Dec 16.
Article in English | MEDLINE | ID: mdl-26676770

ABSTRACT

UNLABELLED: The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family Flaviviridae, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus), and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera. IMPORTANCE: Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses, including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus, and Ebola virus. Our study thus identifies a class of small molecules with a novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant health care burden and/or that are known to cause infections with high case fatality rates.


Subject(s)
Antiviral Agents/pharmacology , Immunity, Innate/drug effects , Immunologic Factors/pharmacology , RNA Viruses/immunology , RNA Viruses/physiology , Virus Replication/drug effects , Animals , Antiviral Agents/isolation & purification , Cell Line , Gene Expression Profiling , Humans , Immunologic Factors/isolation & purification , Viral Load , Virus Cultivation
10.
FASEB J ; 28(9): 3952-64, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24891519

ABSTRACT

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.


Subject(s)
Autoimmune Diseases/prevention & control , Cnidarian Venoms/chemistry , Helminths/metabolism , Immunologic Memory/drug effects , Kv1.3 Potassium Channel/antagonists & inhibitors , Peptide Fragments/pharmacology , Potassium Channel Blockers/pharmacology , T-Lymphocytes/drug effects , Amino Acid Sequence , Animals , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Electrophysiology , Female , Fibroblasts/drug effects , Fibroblasts/immunology , Fibroblasts/metabolism , Humans , Hypersensitivity, Delayed/prevention & control , Magnetic Resonance Spectroscopy , Male , Mice , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Phylogeny , Protein Conformation , Rats , Rats, Inbred Lew , Receptors, CCR7/metabolism , Sequence Homology, Amino Acid , Structure-Activity Relationship , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
11.
Proc Natl Acad Sci U S A ; 110(24): E2239-48, 2013 Jun 11.
Article in English | MEDLINE | ID: mdl-23729813

ABSTRACT

Obesity is an epidemic, calling for innovative and reliable pharmacological strategies. Here, we show that ShK-186, a selective and potent blocker of the voltage-gated Kv1.3 channel, counteracts the negative effects of increased caloric intake in mice fed a diet rich in fat and fructose. ShK-186 reduced weight gain, adiposity, and fatty liver; decreased blood levels of cholesterol, sugar, HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity. These changes mimic the effects of Kv1.3 gene deletion. ShK-186 did not alter weight gain in mice on a chow diet, suggesting that the obesity-inducing diet enhances sensitivity to Kv1.3 blockade. Several mechanisms may contribute to the therapeutic benefits of ShK-186. ShK-186 therapy activated brown adipose tissue as evidenced by a doubling of glucose uptake, and increased ß-oxidation of fatty acids, glycolysis, fatty acid synthesis, and uncoupling protein 1 expression. Activation of brown adipose tissue manifested as augmented oxygen consumption and energy expenditure, with no change in caloric intake, locomotor activity, or thyroid hormone levels. The obesity diet induced Kv1.3 expression in the liver, and ShK-186 caused profound alterations in energy and lipid metabolism in the liver. This action on the liver may underlie the differential effectiveness of ShK-186 in mice fed a chow vs. an obesity diet. Our results highlight the potential use of Kv1.3 blockers for the treatment of obesity and insulin resistance.


Subject(s)
Insulin Resistance , Kv1.3 Potassium Channel/antagonists & inhibitors , Obesity/prevention & control , Proteins/pharmacology , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Adiposity/drug effects , Animals , Blood Glucose/metabolism , Diet , Energy Intake/drug effects , Energy Metabolism/drug effects , Fatty Liver/metabolism , Fatty Liver/physiopathology , Fatty Liver/prevention & control , Kv1.3 Potassium Channel/genetics , Kv1.3 Potassium Channel/physiology , Leptin/blood , Lipids/blood , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Motor Activity/drug effects , Obesity/genetics , Obesity/physiopathology , Oxygen Consumption/drug effects , Weight Gain/drug effects
12.
J Pharmacol Exp Ther ; 342(3): 642-53, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22637724

ABSTRACT

The Kv1.3 channel is a recognized target for pharmaceutical development to treat autoimmune diseases and organ rejection. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the pharmacokinetic-pharmacodynamic relationship for ShK-186 in rats and monkeys. The pharmacokinetic profile of ShK-186 was evaluated with a validated high-performance liquid chromatography-tandem mass spectrometry method to measure the peptide's concentration in plasma. These results were compared with single-photon emission computed tomography/computed tomography data collected with an ¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugate of ShK-186 to assess whole-blood pharmacokinetic parameters as well as the peptide's absorption, distribution, and excretion. Analysis of these data support a model wherein ShK-186 is absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC50 value for up to 7 days in monkeys. Pharmacodynamic studies on human peripheral blood mononuclear cells showed that brief exposure to ShK-186 resulted in sustained suppression of cytokine responses and may contribute to prolonged drug effects. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration. ShK-186's slow distribution from the injection site and its long residence time on the Kv1.3 channel contribute to the prolonged therapeutic effect of ShK-186 in animal models of autoimmune disease.


Subject(s)
Autoimmune Diseases/drug therapy , Kv1.3 Potassium Channel/antagonists & inhibitors , Proteins/pharmacology , T-Lymphocytes/drug effects , Absorption/drug effects , Absorption/immunology , Animals , Arthritis/drug therapy , Arthritis/immunology , Arthritis/metabolism , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Humans , Inhibitory Concentration 50 , Kv1.3 Potassium Channel/immunology , Kv1.3 Potassium Channel/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Macaca fascicularis , Potassium Channel Blockers/immunology , Potassium Channel Blockers/pharmacokinetics , Potassium Channel Blockers/pharmacology , Proteins/pharmacokinetics , Rats , Rats, Sprague-Dawley , Saimiri , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tissue Distribution/drug effects , Tissue Distribution/immunology
13.
J Virol ; 86(13): 7334-44, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22532686

ABSTRACT

There is a growing need for novel antiviral therapies that are broad spectrum, effective, and not subject to resistance due to viral mutations. Using high-throughput screening methods, including computational docking studies and an interferon-stimulated gene 54 (ISG54)-luciferase reporter assay, we identified a class of isoflavone compounds that act as specific agonists of innate immune signaling pathways and cause activation of the interferon regulatory factor (IRF-3) transcription factor. The isoflavone compounds activated the ISG54 promoter, mediated nuclear translocation of IRF-3, and displayed highly potent activity against hepatitis C virus (HCV) and influenza virus. Additionally, these agonists efficiently activated IRF-3 in the presence of the HCV protease NS3-4A, which is known to blunt the host immune response. Furthermore, genomic studies showed that discrete innate immune pathways centered on IRF signaling were regulated following agonist treatment without causing global changes in host gene expression. Following treatment, the expression of only 64 cellular genes was significantly induced. This report provides the first evidence that innate immune pathways dependent on IRF-3 can be successfully targeted by small-molecule drugs for the development of novel broad-spectrum antiviral compounds.


Subject(s)
Antiviral Agents/metabolism , Hepacivirus/immunology , Immunologic Factors/metabolism , Interferon Regulatory Factor-3/biosynthesis , Isoflavones/agonists , Orthomyxoviridae/immunology , Signal Transduction/drug effects , Hepacivirus/physiology , Humans , Immunity, Innate , Orthomyxoviridae/physiology , Protein Transport , Virus Replication
14.
Toxicon ; 59(4): 529-46, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-21867724

ABSTRACT

Electrophysiological and pharmacological studies coupled with molecular identification have revealed a unique network of ion channels--Kv1.3, KCa3.1, CRAC (Orai1 + Stim1), TRPM7, Cl(swell)--in lymphocytes that initiates and maintains the calcium signaling cascade required for activation. The expression pattern of these channels changes during lymphocyte activation and differentiation, allowing the functional network to adapt during an immune response. The Kv1.3 channel is of interest because it plays a critical role in subsets of T and B lymphocytes implicated in autoimmune disorders. The ShK toxin from the sea anemone Stichodactyla helianthus is a potent blocker of Kv1.3. ShK-186, a synthetic analog of ShK, is being developed as a therapeutic for autoimmune diseases, and is scheduled to begin first-in-man phase-1 trials in 2011. This review describes the journey that has led to the development of ShK-186.


Subject(s)
Autoimmune Diseases/drug therapy , Cnidarian Venoms/pharmacology , Immunologic Factors/pharmacology , Sea Anemones , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Cnidarian Venoms/pharmacokinetics , Drug-Related Side Effects and Adverse Reactions , Immunologic Factors/pharmacokinetics , Ion Channels/metabolism , Lymphocyte Activation/drug effects , Molecular Sequence Data , Protein Conformation
15.
J Virol ; 84(22): 11898-904, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20844035

ABSTRACT

The 2'-5' oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.


Subject(s)
2',5'-Oligoadenylate Synthetase/immunology , Antiviral Agents/immunology , Endoribonucleases/immunology , Extracellular Space/enzymology , Host-Pathogen Interactions , Virus Diseases/enzymology , Virus Diseases/immunology , Viruses/immunology , 2',5'-Oligoadenylate Synthetase/genetics , Animals , Cell Line , Endoribonucleases/genetics , Extracellular Space/immunology , Humans , Immunity, Innate , Mice , Mice, Inbred C57BL , Virus Diseases/virology , Virus Physiological Phenomena
17.
Obesity (Silver Spring) ; 16(4): 902-4, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18239580

ABSTRACT

Variants in the FTO gene have been strongly associated with obesity in a very large sample (38,759) of diabetic and control subjects. To replicate these findings, the previously reported SNP in the FTO gene (rs9939609, T/A) was genotyped in 5,607 subjects from five different Utah studies. The studies included a random sample of the Utah population, families selected for aggregation of extreme thinness, families selected for severe obesity, a series of unrelated severe obesity subjects, and families participating in a 25-year longitudinal study of cardiovascular disease and aging. Results show a strong significant increase in the rs9939609 A allele frequency with increasing BMI (P < 0.0001). In the longitudinal study, FTO genotypes were significantly associated with BMI at a baseline exam, a 2(1/2)-year follow-up exam and a 25-year follow-up exam using an additive genetic model. The mean genotype difference in BMI ranged from 1.3 to 2.1 kg/m(2) across exams. The genotype difference in BMI means was established in youth, and at-risk subjects under age 20 at baseline had a significantly larger 25-year BMI increase (10.0 for A/A; 9.7 for A/T, and 8.5 kg/m(2) for T/T, P = 0.05). We conclude that the BMI increases associated with FTO genotypes begin in youth and are maintained throughout adulthood.


Subject(s)
Aging/genetics , Body Mass Index , Obesity/genetics , Proteins/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Alpha-Ketoglutarate-Dependent Dioxygenase FTO , Cardiovascular Diseases/genetics , Child , Child, Preschool , Female , Follow-Up Studies , Gene Frequency , Genetic Predisposition to Disease , Genotype , Humans , Male , Middle Aged , Pedigree , Polymorphism, Single Nucleotide , Utah/epidemiology
18.
Vaccine ; 25(35): 6458-73, 2007 Aug 29.
Article in English | MEDLINE | ID: mdl-17651872

ABSTRACT

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents.


Subject(s)
Gene Expression Profiling , Monocytes/metabolism , Monocytes/virology , Respiratory Tract Infections/genetics , Vaccination , Vaccinia/genetics , Viral Vaccines/immunology , Yellow Fever/genetics , Adolescent , Adult , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Data Interpretation, Statistical , Female , Humans , Male , Oligonucleotide Array Sequence Analysis , RNA, Viral/biosynthesis , RNA, Viral/genetics , Regression Analysis , Respiratory Tract Infections/virology , Smallpox Vaccine/immunology , Vaccinia/virology , Vaccinia virus/immunology , Yellow Fever/virology , Yellow Fever Vaccine/immunology , Yellow fever virus/immunology
19.
BMC Genomics ; 8: 28, 2007 Jan 23.
Article in English | MEDLINE | ID: mdl-17244361

ABSTRACT

BACKGROUND: Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST)-derived array. RESULTS: We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR) of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS) sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from a closely related species. CONCLUSION: The number of different genes represented on microarrays for unfinished genomes can be greatly increased by matching known gene transcript annotations from a closely related species with sequence data from the unfinished genome. Signal intensity on both EST- and genome-derived arrays was highly correlated with probe distance from the 3' UTR, information often missing from ESTs yet present in early-stage genome projects.


Subject(s)
Genetic Techniques , Genome, Human , Genome , Oligonucleotide Array Sequence Analysis/instrumentation , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotides/chemistry , 3' Untranslated Regions , Animals , Brain/metabolism , Computational Biology/methods , DNA, Complementary/metabolism , Humans , Macaca mulatta , Nucleic Acid Hybridization , Signal Transduction , Spleen/metabolism
20.
Genome Biol ; 6(7): R60, 2005.
Article in English | MEDLINE | ID: mdl-15998449

ABSTRACT

We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.


Subject(s)
Genetic Variation , Macaca mulatta , Transcription, Genetic , Animals , Algorithms , Base Sequence , Cloning, Molecular , Databases, Nucleic Acid , DNA Primers , DNA, Complementary/genetics , Evolution, Molecular , Gene Library , Macaca fascicularis/genetics , Macaca nemestrina/genetics , Oligonucleotide Array Sequence Analysis , Sequence Homology, Nucleic Acid , Humans
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