RESUMEN
A single-dose study was conducted to characterize the safety, pharmacokinetic, immunogenicity, and pharmacodynamic activity of highly purified Staphylococcal protein A (SPA), a native bacterial protein with immune-modulatory activity. Twenty healthy adults received a single intravenous dose of either 0.3 µg/kg (n = 8) or 0.45 µg/kg (n = 8) of SPA or placebo (n = 4). Changes in C-reactive protein and neopterin were used as markers of immune activation. All treatment-related AEs were of mild severity. Twelve of 16 active-dosed subjects developed detectable anti-protein A antibodies after dosing. These subjects had notably more rapid plasma clearance of SPA even prior to development of detectable titers. A transient post-dose decrease in circulating lymphocytes was observed as a notable pharmacodynamic effect, but was not correlated with plasma clearance or AUC. In peripheral blood mononuclear cells, SPA dosing increased transcription of multiple genes regulated by type-1 interferons, and up-regulation of several of these genes correlated with the degree of lymphopenia seen 24 hours after dosing. This study demonstrates the safety and tolerability of small intravenous doses of SPA and delineates acute and transient pharmacodynamic effects not previously reported.
Asunto(s)
Factores Inmunológicos/administración & dosificación , Proteína Estafilocócica A/administración & dosificación , Anticuerpos Antibacterianos/sangre , Proteína C-Reactiva/análisis , Método Doble Ciego , Perfilación de la Expresión Génica , Humanos , Factores Inmunológicos/sangre , Factores Inmunológicos/farmacocinética , Inyecciones Intravenosas , Leucocitos Mononucleares , Neopterin/sangre , Proteína Estafilocócica A/sangre , Proteína Estafilocócica A/inmunología , Staphylococcus/inmunologíaRESUMEN
CD7 is a cell-surface molecule, expressed on T lymphocytes and NK cells, which functions as a costimulatory receptor for T cell proliferation. SECTM1 has been proposed as a ligand for CD7. However, the expression pattern of this molecule in human immune cells and role in human T cell function remain unclear. In the present study, using human rSECTM1, we demonstrate that SECTM1 strongly costimulates CD4 and CD8 T cell proliferation and induces IFN-γ production, likely via a CD7-dependent mechanism. In addition, SECTM1 synergizes with suboptimal anti-CD28 to strongly augment T cell functions. We found a robust induction of IL-2 production when SECTM1 and anti-CD28 signals were present with TCR ligation. Furthermore, addition of SECTM1 into a MLR significantly enhanced proliferation of alloantigen-activated T cells, whereas blockade of SECTM1 inhibited T cell proliferation in a two-way MLR assay. Simultaneously blocking the effect of SECTM1, along with CTLA-4/Fc, diminishes two-way MLR. Finally, we demonstrated that expression of SECTM1 is not detected in monocytes and imMoDCs at the protein level. However, it is strongly induced by IFN-γ in monocytes and imMoDCs, and this induction is STAT1-dependent. These results indicate that SECTM1 is a broadly expressed, IFN-γ-inducible molecule, which functions as a potent costimulatory ligand for T cell activation and is synergistic with anti-CD28.
Asunto(s)
Antígenos CD28/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Linfocitos T/metabolismo , Antígenos CD7/metabolismo , Secuencia de Bases , Antígenos CD28/inmunología , Antígeno CTLA-4/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Interferón gamma/farmacología , Células Jurkat , Ligandos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Proteínas de la Membrana/genética , Motivos de Nucleótidos , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
An adherent cell differentiation and cytotoxicity (ACDC) assay was developed using pluripotent J1 mouse embryonic stem cells (mESCs). Adherent mESCs were used to evaluate chemical-induced effects on both stem cell viability and differentiation using an in-cell western technique after a 9-day culture. DRAQ5/Sapphire700 stains were used to quantify cell number. Myosin heavy chain protein was used as a marker of cardiomyocyte differentiation and was corrected for cell number, thereby separating cytotoxicity and effects on differentiation. Acetic acid, 5-fluorouracil and bromochloroacetic acid were evaluated using the embryonic stem cell test and ACDC assay. Both systems distinguish the relative potencies of these compounds. TaqMan low-density arrays were used to characterize the time course of differentiation and effects of chemical exposure on multiple differentiation gene markers. The ACDC assay is a technique that can be used to evaluate the effects of xenobiotics on mESC differentiation and cell number using a single assay.
Asunto(s)
Alternativas a las Pruebas en Animales , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Pruebas de Toxicidad , Acetatos/toxicidad , Ácido Acético/toxicidad , Animales , Western Blotting , Diferenciación Celular/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/patología , Fluorouracilo/toxicidad , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Contracción Miocárdica/genética , Miocitos Cardíacos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/patología , Reacción en Cadena de la Polimerasa , Medición de Riesgo , Factores de TiempoRESUMEN
BACKGROUND: Preterm delivery (PTD) is a significant public health problem associated with greater risk of mortality and morbidity in infants and mothers. Pathophysiologic processes that may lead to PTD start early in pregnancy. We investigated early pregnancy peripheral blood global gene expression and PTD risk. METHODS: As part of a prospective study, ribonucleic acid was extracted from blood samples (collected at 16 weeks gestational age) from 14 women who had PTD (cases) and 16 women who delivered at term (controls). Gene expressions were measured using the GeneChip(R) Human Genome U133 Plus 2.0 Array. Student's T-test and fold change analysis were used to identify differentially expressed genes. We used hierarchical clustering and principle components analysis to characterize signature gene expression patterns among cases and controls. Pathway and promoter sequence analyses were used to investigate functions and functional relationships as well as regulatory regions of differentially expressed genes. RESULTS: A total of 209 genes, including potential candidate genes (e.g. PTGDS, prostaglandin D2 synthase 21 kDa), were differentially expressed. A set of these genes achieved accurate pre-diagnostic separation of cases and controls. These genes participate in functions related to immune system and inflammation, organ development, metabolism (lipid, carbohydrate and amino acid) and cell signaling. Binding sites of putative transcription factors such as EGR1 (early growth response 1), TFAP2A (transcription factor AP2A), Sp1 (specificity protein 1) and Sp3 (specificity protein 3) were over represented in promoter regions of differentially expressed genes. Real-time PCR confirmed microarray expression measurements of selected genes. CONCLUSIONS: PTD is associated with maternal early pregnancy peripheral blood gene expression changes. Maternal early pregnancy peripheral blood gene expression patterns may be useful for better understanding of PTD pathophysiology and PTD risk prediction.