RESUMEN
The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3gamma-ITAM in T cell development, we created knock-in mice in which the CD3gamma chain of the TCR complex is replaced by a mutant signaling-deficient CD3gamma chain, lacking the CD3gamma-ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3gamma-deltaITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5-CD3gamma-deltaITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3gamma-deltaITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR-CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3gamma-ITAM in TCR-driven thymocyte selection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Complejo CD3/metabolismo , Diferenciación Celular/inmunología , Proteínas de la Membrana , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Timo/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3/genética , Complejo CD3/inmunología , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Proteínas Portadoras/metabolismo , Células Cultivadas , Cruzamientos Genéticos , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos , Lectinas Tipo C , Ligandos , Ratones , Ratones Mutantes , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citología , Timo/inmunología , Proteína Tirosina Quinasa ZAP-70RESUMEN
BACKGROUND & AIMS: Originally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn's disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis. METHODS: A CSF-1 pathway gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn's mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq. RESULTS: Both the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn's disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by â¼50%, reduced histological disease scores by â¼60%, and reduced F4/80+ mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in "M1" inflammatory and "M0" resident macrophages and in activated T cells. CONCLUSIONS: CSF-1 biology is activated in Crohn's disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease.
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Colitis/tratamiento farmacológico , Imidazoles/farmacología , Mucosa Intestinal/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Piridinas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Linfocitos T/patología , Animales , Colitis/inmunología , Colitis/patología , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Imidazoles/uso terapéutico , Inflamación/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacosRESUMEN
MOTIVATION: DNA microarrays are a well-known and established technology in biological and pharmaceutical research providing a wealth of information essential for understanding biological processes and aiding drug development. Protein microarrays are quickly emerging as a follow-up technology, which will also begin to experience rapid growth as the challenges in protein to spot methodologies are overcome. Like DNA microarrays, their protein counterparts produce large amounts of data that must be suitably analyzed in order to yield meaningful information that should eventually lead to novel drug targets and biomarkers. Although the statistical management of DNA microarray data has been well described, there is no available report that offers a successful consolidated approach to the analysis of high-throughput protein microarray data. We describe the novel application of a statistical methodology to analyze the data from an immune response profiling assay using human protein microarray with over 5000 proteins on each chip.
Asunto(s)
Análisis por Matrices de Proteínas/métodos , Autoanticuerpos/sangre , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/inmunología , Interpretación Estadística de Datos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas InformáticosRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) and its accepted animal model, murine collagen-induced arthritis (CIA), are classic autoimmune inflammatory diseases which require proinflammatory cytokine production for pathogenesis. We and others have previously used N, N-dimethylglycine (DMG) and extracts from the New Zealand green-lipped mussel Perna canaliculus (Perna) as potent immunomodulators to modify ongoing immune and/or inflammatory responses. METHODS: In our initial studies, we treated lipopolysaccahride (LPS) stimulated THP-1 monocytes in vitro with increasing concentrations of Perna extract or DMG. Additionally, we treated rat peripheral blood neutrophils with increasing concentrations of Perna extract and measured superoxide burst. In subsequent in vivo experiments, CIA was induced by administration of type II collagen; rats were prophylactically treated with either Perna or DMG, and then followed for disease severity. Finally, to test whether Perna and/or DMG could block or inhibit an ongoing pathologic disease process, we induced CIA in mice and treated them therapeutically with either of the two immunomodulators. RESULTS: Following LPS stimulation of THP-1 monocytes, we observed dose-dependent reductions in TNF-alpha and IL-12p40 production in Perna treated cultures. DMG treatment, however, showed significant increases in both of these cytokines in the range of 0.001-1 microM. We also demonstrate that in vitro neutrophil superoxide burst activity is dose-dependently reduced in the presence of Perna. Significant reductions in disease incidence, onset, and severity of CIA in rats were noted following prophylactic treatment with either of the two immunomodulators. More importantly, amelioration of mouse CIA was observed following therapeutic administration of Perna. In contrast, DMG appeared to have little effect in mice and may act in a species-specific manner. CONCLUSION: These data suggest that Perna, and perhaps DMG, may be useful supplements to the treatment of RA in humans.
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Antiinflamatorios/farmacología , Artritis Experimental/tratamiento farmacológico , Huesos/efectos de los fármacos , Inmunosupresores/farmacología , Perna , Sarcosina/análogos & derivados , Animales , Antiinflamatorios/administración & dosificación , Artritis Experimental/inmunología , Huesos/inmunología , Colágeno/inmunología , Colágeno/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inmunosupresores/administración & dosificación , Ratones , RatasRESUMEN
Biomarkers in the clinical oncology field can have tremendous therapeutic impact especially if the marker is detected before clinical symptoms. This impact can be extended to the evaluation of clinical oncology treatments allowing evaluation of potential compounds to determine their efficacy in the disease treatment. The discovery of clinical biomarkers can consume time, resources and costs. Therefore, it is important that the most effective strategies are employed to discover these biomarkers. These strategies may include the integration of available genomic, proteomic and histopathological technologies, which could reduce the costs and aid in the validation of the biomarker. Certainly the type of biomarker needed to address a particularly defined problem will drive the type of technology. However, a single biomarker to diagnose a specific cancer can be as elusive as relying on a single technology. This review examines some of the technologies used to discover biomarkers and presents the use of combinatorial technical synergies to discover and validate potential clinical oncology biomarkers.
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Biomarcadores de Tumor , Evaluación Preclínica de Medicamentos/métodos , Genómica/métodos , Histocitoquímica/métodos , Humanos , Proteómica/métodosRESUMEN
OBJECTIVE: To assess the efficacy and safety of JNJ-40346527, a selective inhibitor of colony-stimulating factor-1 (CSF-1) receptor kinase that acts to inhibit macrophage survival, proliferation, and differentiation in patients with active rheumatoid arthritis (RA) despite disease-modifying antirheumatic drug (DMARD) therapy. METHODS: In this randomized, double-blind, placebo-controlled, parallel group study, adults were randomized (2:1) to receive oral JNJ-40346527 100 mg or placebo twice daily through Week 12. Patients with RA had disease activity [≥ 6 swollen/≥ 6 tender joints, C-reactive protein (CRP) ≥ 0.8 mg/dl] despite DMARD therapy for ≥ 6 months. The primary endpoint was change from baseline at Week 12 in the 28-joint Disease Activity Score with CRP (DAS28-CRP). Pharmacokinetic/pharmacodynamic analyses were also performed, and safety was assessed through Week 16. RESULTS: Ninety-five patients were treated (63 JNJ-40346527, 32 placebo); 8 patients discontinued treatment (6 JNJ-40346527, 2 placebo) through Week 12. Mean improvements in DAS28-CRP from baseline to Week 12 were 1.15 for the JNJ-40346527 group and 1.42 for the placebo group (p = 0.30); thus, a statistically significant difference was not observed for the primary endpoint. Pharmacokinetic exposure to JNJ-40346527 and its active metabolites was above the projected concentration needed for pharmacologic activity, and effective target engagement and proof of activity were demonstrated by increased levels of CSF-1 and decreased CD16+ monocytes in JNJ-40346527-treated, but not placebo-treated, patients. Thirty-seven (58.7%) JNJ-40346527-treated and 16 (50.0%) placebo-treated patients reported ≥ 1 adverse event (AE); 1 (1.6%) JNJ-40346527-treated and 3 (9.4%) placebo-treated patients reported ≥ 1 serious AE. CONCLUSION: Although adequate exposure and effective peripheral target engagement were evident, JNJ-40346527 efficacy was not observed in patients with DMARD-refractory active RA. ClinicalTrials.gov identifier: NCT01597739. EudraCT Number: 2011-004529-28.
Asunto(s)
Antirreumáticos/administración & dosificación , Artritis Reumatoide/tratamiento farmacológico , Drogas en Investigación/administración & dosificación , Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Administración Oral , Adulto , Artritis Reumatoide/diagnóstico , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Quimioterapia Combinada , Drogas en Investigación/efectos adversos , Femenino , Estudios de Seguimiento , Humanos , Factor Estimulante de Colonias de Macrófagos/administración & dosificación , Masculino , Persona de Mediana Edad , Seguridad del Paciente , Medición de Riesgo , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
The emerging field of biomarkers has applications in the diagnosis, staging, prognosis and monitoring of disease progression, as well as in the monitoring of clinical responses to a therapeutic intervention and the development and delivery of personalized treatments to reduce attrition in clinical trials. Moreover, biomarkers have a positive impact on health economics. The word "biomarker" has been used extensively across therapeutic areas and many disciplines, and its nature takes into consideration clinical, physiological, biochemical, developmental, morphological and molecular measures. In drug trials, biomarkers have been proposed for use in efficacy determination and patient population stratification, in deducing pharmacokinetic-pharmacodynamic relationships and in safety monitoring. The interfacing and integration of different technologies for data collection and analysis are pivotal to biomarker identification, characterization, validation and application. "Integrative functional informatics" represents a novel direction in such technology integration.
Asunto(s)
Biomarcadores , Biología Computacional , Recolección de Datos , Genómica , Proteómica , HumanosRESUMEN
Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans.
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Quimasas/metabolismo , Hipersensibilidad/metabolismo , Proteínas Recombinantes/metabolismo , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Quimasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Hipersensibilidad/inmunologíaRESUMEN
Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.
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ADN Recombinante , Células Epiteliales/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Células Epiteliales/citología , Exones/genética , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción GenéticaRESUMEN
The enediyne moiety is a versatile functional group found in natural anticancer and anti-infective agents, undergoing the Bergman cyclization reaction to afford a diradical which cleaves double-stranded DNA. We have incorporated the enediyne group into 10- (4-10) and 12-membered ring (11) cyclic amino acids and dipeptides, respectively, and explored their relative reactivity toward cyclization, varying N-substitution in the case of the 10-membered ring substrate, which gave the expected cyclization products in good yields when using either thermal conditions in the presence or absence of microwave irradiation. The N-tosyl substituted derivative (4) was shown to nick double-stranded supercoiled DNA. N-Arylsulfonyl substitution on the ring promoted the cyclization, when compared to N-mesyl or acyl substitution, possibly because of a pi-pi stacking effect as an endo-relationship of the aryl group with the enediyne was demonstrated in both the solid state and in solution. The 12-membered ring enediyne dipeptide (11) was inert to the Bergman cyclization under a variety of conditions. When this substrate was irradiated with ultraviolet light, regio- and stereospecific reduction was observed in which one of the alkynes was reduced to a Z-olefin (47).
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Aminoácidos Cíclicos/química , Aminoácidos Cíclicos/farmacología , Aminoácidos/química , Aminoácidos/síntesis química , Benceno/química , Aminoácidos/farmacología , Animales , Bovinos , Cristalografía por Rayos X , Ciclización , Daño del ADN/efectos de los fármacos , Enediinos , Estructura Molecular , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/efectos de los fármacosRESUMEN
Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.
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Evaluación Preclínica de Medicamentos/métodos , Perfilación de la Expresión Génica/métodos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Industria Farmacéutica/métodos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Análisis por Matrices de Proteínas/métodos , Proteína Quinasa C/análisis , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Progression of immature CD4(-)CD8(-) thymocytes beyond the beta-selection checkpoint to the CD4(+)CD8(+) stage requires activation of the pre-TCR complex; however, few of the DNA-binding proteins that serve as molecular effectors of those pre-TCR signals have been identified. We demonstrate in this study that members of the early growth response (Egr) family of transcription factors are critical effectors of the signals that promote this developmental transition. Specifically, the induction of three Egr family members (Egr1, 2, and 3) correlates with pre-TCR activation and development of CD4(-)CD8(-) thymocytes beyond the beta-selection checkpoint. Enforced expression of each of these Egr factors is able to bypass the block in thymocyte development associated with defective pre-TCR function. However, Egr family members may play somewhat distinct roles in promoting thymocyte development, because there are differences in the genes modulated by enforced expression of particular Egr factors. Finally, interfering with Egr function using dominant-negative proteins disrupts thymocyte development from the CD4(-)CD8(-) to the CD4(+)CD8(+) stage. Taken together, these data demonstrate that the Egr proteins play an essential role in executing the differentiation program initiated by pre-TCR signaling.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Inmediatas-Precoces/fisiología , Timo/inmunología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz , Proteína 2 de la Respuesta de Crecimiento Precoz , Proteína 3 de la Respuesta de Crecimiento Precoz , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Proteínas Inmediatas-Precoces/genética , Inmunofenotipificación , Ratones , Técnicas de Cultivo de Órganos , ARN Mensajero/biosíntesis , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/clasificación , Subgrupos de Linfocitos T/inmunología , Timo/embriología , Factores de Transcripción/genética , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Pre-TCR complexes are thought to signal in a ligand-independent manner because they are constitutively targeted to lipid rafts. We report that ligand-independent signaling is not a unique capability of the pre-TCR complex. Indeed, the TCR alpha subunit restores development of pT alpha-deficient thymocytes to the CD4(+)CD8(+) stage even in the absence of conventional MHC class I and class II ligands. Moreover, we found that pre-TCR and alpha beta TCR complexes exhibit no appreciable difference in their association with lipid rafts, suggesting that ligand-independence is a function of the CD4(-)CD8(-) (DN) thymocytes in which pre-TCR signaling occurs. In agreement, we found that only CD44(-)CD25(+) DN thymocytes (DN3) enabled activation of extracellular signal-regulated kinases by the pre-TCR complex. DN thymocytes also exhibited a lower signaling threshold relative to CD4(+)CD8(+) thymocytes, which was associated with both the markedly elevated lipid raft content of their plasma membranes and more robust capacitative Ca(2+) entry. Taken together these data suggest that cell-autonomous, ligand-independent signaling is primarily a property of the thymocytes in which pre-TCR signaling occurs.