RESUMEN
Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Disbiosis/inmunología , Inflamación/inmunología , Interferón gamma/metabolismo , Macrófagos/inmunología , Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato/genética , Animales , Autofagia , Enfermedad Crónica , Femenino , Microbioma Gastrointestinal/inmunología , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/metabolismo , Sexismo , Transducción de SeñalRESUMEN
RATIONALE: Stratification of asthma at the molecular level, especially using accessible biospecimens, could greatly enable patient selection for targeted therapy. OBJECTIVES: To determine the value of blood analysis to identify transcriptional differences between clinically defined asthma and nonasthma groups, identify potential patient subgroups based on gene expression, and explore biological pathways associated with identified differences. METHODS: Transcriptomic profiles were generated by microarray analysis of blood from 610 patients with asthma and control participants in the U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) study. Differentially expressed genes (DEGs) were identified by analysis of variance, including covariates for RNA quality, sex, and clinical site, and Ingenuity Pathway Analysis was applied. Patient subgroups based on DEGs were created by hierarchical clustering and topological data analysis. MEASUREMENTS AND MAIN RESULTS: A total of 1,693 genes were differentially expressed between patients with severe asthma and participants without asthma. The differences from participants without asthma in the nonsmoking severe asthma and mild/moderate asthma subgroups were significantly related (r = 0.76), with a larger effect size in the severe asthma group. The majority of, but not all, differences were explained by differences in circulating immune cell populations. Pathway analysis showed an increase in chemotaxis, migration, and myeloid cell trafficking in patients with severe asthma, decreased B-lymphocyte development and hematopoietic progenitor cells, and lymphoid organ hypoplasia. Cluster analysis of DEGs led to the creation of subgroups among the patients with severe asthma who differed in molecular responses to oral corticosteroids. CONCLUSIONS: Blood gene expression differences between clinically defined subgroups of patients with asthma and individuals without asthma, as well as subgroups of patients with severe asthma defined by transcript profiles, show the value of blood analysis in stratifying patients with asthma and identifying molecular pathways for further study. Clinical trial registered with www.clinicaltrials.gov (NCT01982162).
Asunto(s)
Corticoesteroides/uso terapéutico , Asma/sangre , Asma/tratamiento farmacológico , Perfilación de la Expresión Génica/métodos , Corticoesteroides/sangre , Adulto , Análisis por Conglomerados , Estudios de Cohortes , Europa (Continente) , Femenino , Humanos , Masculino , Análisis por Micromatrices/estadística & datos numéricos , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Transcriptoma/efectos de los fármacosRESUMEN
The IL-17 pathway is an established driver of psoriasis pathogenesis. We examined the detailed molecular and cellular effects of blockade of IL-17 signaling in human psoriatic skin before and following treatment with brodalumab, a competitive inhibitor of the IL-17 Receptor A subunit. Thousands of aberrantly expressed genes in lesional skin normalized within 2 weeks following brodalumab treatment, with conversion of the lesional psoriasis transcriptome to resemble that seen in nonlesional skin. Keratinocyte-expressed genes appeared to normalize rapidly, whereas T cell-specific normalization occurred over six weeks. The three IL-17 ligand genes that are upregulated in lesional skin, IL17A, IL17C, and IL17F, were all downregulated in a dose-dependent manner following brodalumab treatment. Cellular measures also showed a similar pattern with dramatic decreases in keratinocyte hyperplasia within one week, and decreases in infiltrating leukocytes occurred over a longer timescale. Individuals with the highest brodalumab exposure showed normalization of both IL-17-responsive genes and the psoriasis transcriptome, whereas subjects with lower exposures showed transient or incomplete molecular responses. Clinical and molecular response appeared dependent on the extent of brodalumab exposure relative to the expression of IL-17 ligand genes, and reduction of IL-17 signaling into the nonlesional range was strongly correlated with normalization of the psoriasis transcriptome. These data indicate that blockade of IL-17 signaling in psoriatic skin leads to rapid transcriptomal changes initially in keratinocyte-expressed genes, followed by normalization in the leukocyte abnormalities, and demonstrates the essential role of the IL-17R on keratinocytes in driving disease pathogenesis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Psoriasis/genética , Receptores de Interleucina-17/antagonistas & inhibidores , Piel/efectos de los fármacos , Piel/metabolismo , Transcriptoma , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Psoriasis/tratamiento farmacológico , Piel/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
PURPOSE: To investigate the relationships between AMG 811 exposure, concentration changes in serum IFN-γ, and IFN-γ-induced protein 10 (CXCL10), and to identify important contributions of baseline covariates to these relationships. METHODS: A mechanism based pharmacokinetic (PK)-pharmacodynamic (PD) model was developed. A target mediated disposition model was used to describe AMG 811 and target IFN-γ interaction. CXCL10 was predicted to be driven by estimated free IFN-γ levels. RESULTS: For an average systemic lupus erythematosus (SLE) subject, the linear clearance (CL) of AMG 811 was 0.176 L/day, and the central (Vc) and peripheral (Vp) volumes of distribution were 1.48 and 2.12 L, respectively. Body weight was found to correlate with CL, Vc, Vp, and inter compartment clearance (Q); and age was found to correlate with Vc. The relationship between estimated free serum IFN-γ concentration levels and serum CXCL10 in logarithmic scales was best described by a linear model with slope and intercept estimated to be 0.197 and -0.3, respectively. CONCLUSIONS: The largest observed reduction of serum CXCL10 concentration was achieved at the highest AMG 811 dose tested (180 mg SC). This model enables simulations of AMG 811 PK-PD profiles under various dosing regimens to support future clinical studies.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Inmunoglobulina G/metabolismo , Interferón gamma/antagonistas & inhibidores , Modelos Lineales , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Método Doble Ciego , Femenino , Humanos , Inmunoglobulina G/uso terapéutico , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Despite recent advances in qualitative proteomics, the automatic identification of peptides with optimal sensitivity and accuracy remains a difficult goal. To address this deficiency, a novel algorithm, Multiple Search Engines, Normalization and Consensus is described. The method employs six search engines and a re-scoring engine to search MS/MS spectra against protein and decoy sequences. After the peptide hits from each engine are normalized to error rates estimated from the decoy hits, peptide assignments are then deduced using a minimum consensus model. These assignments are produced in a series of progressively relaxed false-discovery rates, thus enabling a comprehensive interpretation of the data set. Additionally, the estimated false-discovery rate was found to have good concordance with the observed false-positive rate calculated from known identities. Benchmarking against standard proteins data sets (ISBv1, sPRG2006) and their published analysis, demonstrated that the Multiple Search Engines, Normalization and Consensus algorithm consistently achieved significantly higher sensitivity in peptide identifications, which led to increased or more robust protein identifications in all data sets compared with prior methods. The sensitivity and the false-positive rate of peptide identification exhibit an inverse-proportional and linear relationship with the number of participating search engines.
Asunto(s)
Péptidos/análisis , Proteómica/métodos , Motor de Búsqueda , Algoritmos , Secuencia de Consenso , Bases de Datos de Proteínas , Reacciones Falso Positivas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Biomarker assays are often conducted on whole blood samples in the course of drug development studies. Because bacterial lipopolysaccharide (LPS) (endotoxin) contamination is known to cause spontaneous cytokine production by monocytes, contamination of blood collection tubes may interfere with biomarker assay results. METHODS: Whole blood from healthy donors was collected into plastic or glass sodium (Na(+))-heparin Vacutainer() blood collection tubes and heparinized syringes. Samples were analyzed for phosphoprotein response, cytokine production, and RNA expression. Tubes were tested for endotoxin contamination by use of the limulus amoebocyte lysate assay. RESULTS: Results of phospho-flow cytometry, branched DNA (bDNA), and ELISA assays indicated that a specific lot (#5339582) of plastic Na(+)-heparin Vacutainer tubes was highly contaminated with an endotoxinlike substance, and contamination was confirmed by the limulus amoebocyte lysate assay. Analysis of multiple-analyte panels revealed that analytes whose changed expression was predictive of LPS stimulation were increased when whole blood was incubated in contaminated tubes for 6 or 18 h. Two additional lots of plastic tubes tested had detectable amounts of endotoxin sufficient to strongly alter phospho-flow cytometry analyses, as determined by the fold change in phosphorylation of p38 mitogen-activated protein kinase in response to tumor necrosis factor alpha and LPS. In contrast, 3 lots of glass tubes had substantially lower levels of spontaneous blood activation. CONCLUSIONS: Endotoxin contamination associated with tubes from 3 lots of a particular type of plastic Na(+)-heparin Vacutainer tube dramatically affected biomarker assay measurements. Prescreening these tubes is suggested before their use in clinical sample analysis.
Asunto(s)
Anticoagulantes , Recolección de Muestras de Sangre/instrumentación , Endotoxinas/análisis , Contaminación de Equipos , Heparina , Biomarcadores/sangre , Proteína C-Reactiva/biosíntesis , Proteína C-Reactiva/genética , Quimiocina CCL2/sangre , Quimiocina CCL2/genética , Quimiocina CCL7/sangre , Quimiocina CCL7/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Interleucina-1beta/sangre , Interleucina-6/sangre , Interleucina-6/genética , Lipopolisacáridos/farmacología , Fosforilación , Plásticos , ARN Mensajero/sangre , Componente Amiloide P Sérico/biosíntesis , Componente Amiloide P Sérico/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/sangreRESUMEN
The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS(-/-) mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Antígeno B7-1/inmunología , Citocinas/metabolismo , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Schistosoma mansoni/inmunología , Células Th2/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/inmunología , Antígeno B7-1/metabolismo , Movimiento Celular , Citocinas/inmunología , Femenino , Ligando Coestimulador de Linfocitos T Inducibles , Proteína Coestimuladora de Linfocitos T Inducibles , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T/inmunologíaRESUMEN
Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28(-/-) mice, Th2-mediated airway inflammation was not induced in CD28(-/-) mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS(-/-) mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD28/inmunología , Enfermedades Pulmonares/inmunología , Células Th2/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Histocitoquímica , Inmunidad Celular/inmunología , Inmunoglobulina E/sangre , Proteína Coestimuladora de Linfocitos T Inducibles , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organismos Libres de Patógenos EspecíficosRESUMEN
Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided design, synthesis, structure-activity relationships, and pharmacological characterization of 2-amino-6-phenylpyrimido[5',4':5,6]pyrimido[1,2- a]benzimidazol-5(6 H)-ones, a new class of compounds that are potent inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)amino)pyrimido[5',4':5,6]pyrimido-[1,2- a]benzimidazol-5(6 H)-one ( 25), exhibits potent inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in vivo models of T-cell activation and arthritis, respectively.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Artritis/tratamiento farmacológico , Bencimidazoles/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinonas/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Femenino , Inyecciones Intradérmicas , Interleucina-2/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Estereoisomerismo , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).
Asunto(s)
Amidas/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Linfocitos T/efectos de los fármacos , Administración Oral , Amidas/síntesis química , Amidas/química , Animales , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Interleucina-2/antagonistas & inhibidores , Interleucina-2/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: Interferon-γ (IFNγ) is implicated in the pathogenesis of discoid lupus erythematosus (DLE). This study sought to evaluate a single dose of AMG 811, an anti-IFNγ antibody, in patients with DLE. METHODS: The study was designed as a phase I randomized, double-blind, placebo-controlled crossover study of the pharmacodynamics, safety, and clinical efficacy of AMG 811 in patients with DLE. Patients received a single subcutaneous dose of AMG 811 (180 mg) or placebo. The patients in sequence 1 received AMG 811 followed by placebo, while those in sequence 2 received placebo followed by AMG 811. Pharmacodynamic end points included global transcriptional analyses of lesional and nonlesional skin, IFNγ blockade signature (IGBS) transcriptional scores in the skin and blood, keratinocyte IFNγ RNA scores, and serum levels of CXCL10 protein. Additional end points were efficacy outcome measures, including the Cutaneous Lupus Erythematosus Disease Area and Severity Index, and safety outcome measures. RESULTS: Sixteen patients with DLE were enrolled in the study (9 in sequence 1 and 7 in sequence 2). AMG 811 treatment reduced the IGBS score (which was elevated in DLE patients at baseline) in both the blood and lesional skin. The keratinocyte IFNγ RNA score was not affected by administration of AMG 811. Serum CXCL10 protein levels (which were elevated in the blood of DLE patients) were reduced with AMG 811 treatment. The AMG 811 treatment was well tolerated but did not lead to statistically significant improvements in any of the efficacy outcome measures. CONCLUSION: AMG 811 treatment led to changes in IFNγ-associated biomarkers and was well tolerated, but no significant clinical benefit was observed in patients with DLE.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunosupresores/uso terapéutico , Interferón gamma/inmunología , Lupus Eritematoso Discoide/tratamiento farmacológico , Adulto , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Quimiocina CXCL10/inmunología , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Inmunosupresores/farmacocinética , Interferón gamma/genética , Lupus Eritematoso Discoide/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: To evaluate safety, pharmacokinetics and pharmacodynamics of anti-interferon (IFN)-γ monoclonal antibody AMG 811 in subjects with SLE without or with lupus nephritis (LN). METHODS: In this phase Ib, randomised, multiple-dose escalation study (NCT00818948), subjects without LN were randomised to subcutaneous AMG 811 (6, 20 or 60 mg) or placebo and subjects with LN were randomised to subcutaneous AMG 811 (20, 60 or 120 mg) or placebo every four weeks for three total doses. Outcomes included incidence of adverse events (AEs); pharmacokinetics; levels of serum proteins (CXCL-10, interleukin 18, monocyte chemotactic protein-1); changes in gene transcript profiles and clinical parameters (Safety of Estrogen in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) scores, proteinuria, anti-double-stranded DNA (anti-dsDNA) antibodies, C3 complement, C4 complement). RESULTS: Fifty-six subjects enrolled (28 SLE without LN; 28 with LN). Baseline mean SELENA-SLEDAI scores were 2.2 and 12.0 for SLE subjects without and with LN, respectively. Most subjects reported an AE; no meaningful imbalances were observed between AMG 811 and placebo. Pharmacokinetic profiles were similar and mostly dose-proportional in subjects without or with LN. AMG 811 treatment reduced CXCL-10 protein levels and blood-based RNA IFN-γ Blockade Signature compared with placebo. Reductions were less pronounced and not sustained in subjects with LN, even at the highest dose tested, compared with subjects without LN. No effect on SELENA-SLEDAI scores, proteinuria, C3 or C4 complement levels, or anti-dsDNA antibodies was observed. CONCLUSION: AMG 811 demonstrated favourable pharmacokinetics and acceptable safety profile but no evidence of clinical impact. IFN-γ-associated biomarkers decreased with AMG 811; effects were less pronounced and not sustained in LN subjects. TRIAL REGISTRATION NUMBER: NCT00818948; results.
RESUMEN
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.
Asunto(s)
Aminopiridinas/síntesis química , Antiinflamatorios/síntesis química , Carbamatos/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirimidinas/síntesis química , Administración Oral , Aminopiridinas/química , Aminopiridinas/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Disponibilidad Biológica , Carbamatos/química , Carbamatos/farmacología , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Células Jurkat , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Estructura Molecular , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Benzamidas/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Quinazolinas/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Benzamidas/química , Benzamidas/farmacología , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Técnicas In Vitro , Interleucina-2/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Quinazolinas/química , Quinazolinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
OBJECTIVE: To assess the safety and immunologic impact of inhibiting interferon-γ (IFNγ) with AMG 811, a human IgG1 monoclonal antibody against IFNγ, in patients with systemic lupus erythematosus (SLE). METHODS: Twenty-six patients with mild-to-moderate, stable SLE were administered placebo or a single dose of AMG 811, ranging from 2 mg to 180 mg subcutaneously or 60 mg intravenously. RESULTS: Similar to results previously reported following inhibition of type I IFNs, treatment of SLE patients with AMG 811 led to a dose-dependent modulation of the expression of genes associated with IFN signaling, as assessed by microarray analysis of the whole blood. The list of impacted genes overlapped with that identified by stimulating human whole blood with IFNγ and with those gene sets reported in the literature to be differentially expressed in SLE patients. Serum levels of IFNγ-induced chemokines, including IFNγ-inducible protein 10 (IP-10), were found to be elevated at baseline in SLE patients as compared to healthy volunteers. In contrast to previously reported results from studies using type I IFN-blocking agents, treatment with AMG 811 led to dose-related reductions in the serum levels of CXCL10 (IP-10). CONCLUSION: The scope and nature of the biomarkers impacted by AMG 811 support targeting of IFNγ as a therapeutic strategy for SLE.
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Anticuerpos Monoclonales/farmacología , Quimiocina CXCL10/sangre , Quimiocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/antagonistas & inhibidores , Interferones/fisiología , Lupus Eritematoso Sistémico/metabolismo , Adulto , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados , Quimiocina CXCL10/genética , Quimiocina CXCL10/fisiología , Quimiocinas/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Interferón gamma/efectos de los fármacos , Interferón gamma/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/fisiopatología , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Resultado del TratamientoRESUMEN
BACKGROUND: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology. METHODOLOGY/PRINCIPAL FINDINGS: We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4. CONCLUSION: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.
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Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/fisiología , Regulación de la Expresión Génica , Células Th2/citología , Transcripción Genética , Animales , Células Presentadoras de Antígenos , Membrana Celular/metabolismo , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Proteína Coestimuladora de Linfocitos T Inducibles , Inflamación , Interleucina-4/metabolismo , Leucocitos Mononucleares/citología , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.
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Sistema Nervioso Central/metabolismo , Epitelio/metabolismo , Proteínas de la Membrana/metabolismo , Envejecimiento/fisiología , Animales , Línea Celular , Sistema Nervioso Central/citología , Clonación Molecular , Vía Clásica del Complemento , Eritrocitos/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Conos de Crecimiento/metabolismo , Hemólisis/efectos de los fármacos , Humanos , Hibridación in Situ , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/farmacología , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ovinos , SolubilidadRESUMEN
ICOS is expressed on activated T cells and particularly on CXCR5+ follicular Th cells in germinal centers (GC). Its deletion leads to a profound deficiency in memory B cell formation and switched Ab response in humans. Here, we show that in ICOS-deficient patients the generation of GCs is severely disturbed, and the numbers of circulating CXCR5+CD45RO+ memory CD4 T cells are significantly reduced, indicating an essential role of ICOS in the differentiation of CXCR5+CD4 T cells. The GC-specific CD57+CXCR5+ subpopulation is virtually absent. In ICOS-/- mice, the decrease of circulating CXCR5+CD4 T cells reflects the reduction of CXCR5+ follicular Th cells in lymph nodes and spleen. Therefore, in concurrence with the absence of CXCR5+ T cells in the blood of CD40L-deficient patients, these data support the hypothesis that circulating CD57+CXCR5+ T cells are GC derived and thus may serve as a surrogate marker for the presence of functional GCs in humans.
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Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Centro Germinal/citología , Receptores de Citocinas/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Centro Germinal/inmunología , Humanos , Memoria Inmunológica , Proteína Coestimuladora de Linfocitos T Inducibles , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores CXCR5 , Receptores de Quimiocina , Receptores de Citocinas/inmunologíaRESUMEN
T cell activation and tolerance are regulated by costimulatory molecules. Although PD-1 serves as a crucial negative regulator of T cells, the function of its ligands, PDL1 and PDL2, is still controversial. In this study, we created a PDL2-deficient mouse to characterize its function in T cell activation and tolerance. Antigen-presenting cells from PDL2-/- mice were found to be more potent in activation of T cells in vitro over the wild-type controls, which depended on PD-1. Upon immunization with chicken ovalbumin, PDL2-/- mice exhibited increased activation of CD4(+) and CD8(+) T cells in vivo when compared with WT animals. In addition, T cell tolerance to an oral antigen was abrogated by the lack of PDL2. Our results thus demonstrate that PDL2 negatively regulates T cells in immune responses and plays an essential role in immune tolerance.
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Tolerancia Inmunológica/fisiología , Activación de Linfocitos , Péptidos/metabolismo , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación/inmunología , Pollos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Péptidos/genética , Proteína 2 Ligando de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1RESUMEN
BACKGROUND: Inducible CO-stimulatory molecule (ICOS), the third member of the CD28/CTLA4 family, is expressed by activated T cells and interacts with its ligand (ICOSL, B7-related protein-1 [B7RP-1]) that is constitutively expressed by B cells. The interaction of ICOS with its ligand leads to terminal differentiation of B cells to plasma cells. ICOS-deficient mice fail to undergo immunoglobulin-class switch recombination (CSR) and germinal center formation, suggesting that ICOS could be a candidate gene for phenotypic hyper-IgM (HIGM) syndromes characterized by recurrent infections, low serum IgG and IgA, and normal or elevated IgM. Genetically, at least 4 distinct molecular defects have been identified that result in defective CSR and present as HIGM syndromes: defects of the CD40 ligand gene (CD40L; HIGM1, X-linked), the activation-induced cytidine deaminase gene (AID; HIGM2, autosomal recessive), the CD40 gene (HIGM3, autosomal recessive), and the nuclear factor-kappaB (NF-kappaB) essential modulator gene (NEMO, or IKK-gamma, X-linked). In a substantial subgroup of HIGM patients, these 4 genes are normal, and the genetic defect is unknown. OBJECTIVE: We sought to investigate the possibility that mutations of ICOS could account for some patients' belonging to this HIGM subgroup. METHODS: The expression of ICOS protein by activated peripheral blood mononuclear cells and/or interleukin-2-dependent T cell lines derived from these patients was estimated by flow cytometery after incubation of the cells with the ICOS ligand fusion protein B7RP-1 Fc. The coding region and exon-intron boundaries of ICOS were assessed by sequence analysis. RESULTS: We studied 33 HIGM patients from 30 families, selected from an original cohort of 136 patients from 113 families, by excluding mutations of CD40L, AID, CD40, and NEMO. Activated T cells from all 33 patients expressed ICOS normally, and sequence analysis of the coding region and exon-intron boundaries revealed only wild-type ICOS, predicting that the protein structure is normal in this patient population. CONCLUSION: These findings strongly suggest that ICOS does not belong to the group of genes that, if mutated, present clinically as the HIGM syndrome.