RESUMEN
PROBLEM: Normal pregnancy has been described as both a pro-inflammatory condition and a T helper (Th)2-dominated state. Deviations in the percentage of different subpopulations of circulating leukocytes have been detected, although with conflicting results. This study was designed to analyse further the phenotype of subpopulations of peripheral blood leukocytes in normal pregnant women. METHOD OF STUDY: Whole-blood flow cytometry was used to differentiate subsets of leukocytes using directly labeled monoclonal antibodies to specific cell surface antigens and to a panel of activation-associated markers in 33 normal pregnant women in their third trimester and in 26 non-pregnant controls. RESULTS: We found a significant increase in the proportion of granulocytes and of CD8+ T lymphocytes during pregnancy. Up-regulation of the expression of adhesion molecules was observed on granulocytes, monocytes and T lymphocytes. CONCLUSIONS: Pregnancy alters the representation of leukocyte subpopulations in the maternal circulation and is associated with systemic activation of leukocytes.
Asunto(s)
Leucocitos/inmunología , Activación de Linfocitos/inmunología , Embarazo/inmunología , Adulto , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/inmunología , Femenino , Citometría de Flujo , Granulocitos/inmunología , Humanos , Inmunofenotipificación , Monocitos/inmunologíaRESUMEN
Myoblast transplantation has been investigated as a therapy for muscle-related diseases and as a gene delivery vehicle for therapeutic recombinant proteins. Clinical successes involving muscle cell transplantation have been limited, in part because of poor donor cell survival, and the heterogeneous nature of myogenic donor cells has largely been ignored. We have previously reported an isolation technique, preplating, that results in purified myogenic cells that are capable of significantly higher rates of donor cell survival leading to enhanced gene transfer to skeletal muscle. Characterization of these purified cells revealed that they display markers common to stem cells and are capable of multilineage differentiation. This study was performed to phenotypically characterize, by flow cytometry, muscle-derived cell populations obtained by the preplate technique for the purpose of eventually developing a method to quickly identify and isolate viable muscle cells best suited for transplantation. Muscle cell cultures were analyzed for expression of the surface proteins Sca-1, c-Kit, and CD34. We found that the preplate technique purifies distinct myogenic cell subpopulations expressing CD34 alone (Sca-1 negative) and Sca-1 alone (CD34 negative), but that this expression is subject to change with time in culture. Isolation and transplantation of phenotypically pure Sca-1-positive myogenic cells, obtained by magnetic cell sorting, demonstrates the ability to quickly select viable myogenic cells capable of regenerating skeletal muscle and restoring dystrophin expression within dystrophic host skeletal muscle. Flow cytometric described phenotypes will aid in the rapid isolation of specific donor cell populations for muscle cell transplants and muscle cell-mediated gene therapies, thereby enhancing their future success.
Asunto(s)
Separación Inmunomagnética/métodos , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Regeneración/fisiología , Células Madre/citología , Células Madre/fisiología , Proteína de la Poliposis Adenomatosa del Colon , Animales , Antígenos CD/análisis , Antígenos Ly/análisis , Trasplante de Células , Proteínas del Citoesqueleto/análisis , Desmina/análisis , Distrofina/análisis , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas Fosfatasas/análisis , Proteínas Proto-Oncogénicas c-kit/análisis , Células Madre/efectos de los fármacosRESUMEN
Although the etiopathogenesis of idiopathic dilated cardiomyopathy (IDC) is still unclear, it is widely accepted that a complex interplay between viral infections and immune mechanisms is the basis of disease genesis. Previously, we showed that heart-infiltrating T cells of patients suffering from acute, fulminant Coxsackie virus B3+-IDC shared a preferential usage of three variable gene segments of the T cell receptor beta chain-(TCR-Vbeta) encoding families Vbeta3, 7 and 13.1. This indicated the possible presence of a superantigen-driven immune response. Here, we further investigated the IDC immunological scenario by analysing different phenotypes of heart-infiltrating cells: TCR repertoires, cytokine expression and presence of enterovirus-specific antigens. IDC patients who underwent heart transplantation at different times after the onset of heart failure were studied. A cardiac infiltrate of CD4+ and CD8+ T cells was present together with activated macrophages. Furthermore, the same Vbeta gene families, previously found to be skewed in hearts from fulminant cases of CVB3+-IDC, together with two additional Vbeta gene families, Vbeta1 and 5B, were increased. IL-1beta, IL-2, IL-6 and IFN-gamma were expressed in the myocardium while others, like IL-4 were not. In conclusion, an orchestrated complex of immune mechanisms seems to be the basis of IDC etiopathogenesis.
Asunto(s)
Cardiomiopatía Dilatada/inmunología , Citocinas/genética , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Antígenos Virales/análisis , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/virología , Enterovirus Humano B/genética , Enterovirus Humano B/inmunología , Expresión Génica , Antígenos HLA-DQ/clasificación , Cadenas alfa de HLA-DQ , Cadenas beta de HLA-DQ , Prueba de Histocompatibilidad , Humanos , Técnicas para Inmunoenzimas , Interferón gamma/genética , Interleucina-1/genética , Interleucina-2/genética , Interleucina-4/genética , Interleucina-6/genética , Leucocitos Mononucleares/inmunología , Miocarditis/inmunología , Miocardio/inmunología , Miocardio/patología , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS/HYPOTHESIS: High frequencies of T-cell receptor (TCR) V beta 7+ T cells were detected among the lymphocytes isolated from pancreatic islets of children at the onset of Type I (insulin-dependent) diabetes mellitus. We assessed whether a preferential expression of certain TCR V beta gene families could also be detected among the peripheral blood mononuclear cells from diabetic patients. METHODS: T-cell receptor repertoires were evaluated by using a semi-quantitative RT-PCR-based technique and confirmed by FACS analysis in peripheral blood mononuclear cells from diabetic patients before, at and after onset of the disease. These patients were also tested for exposure to enteroviruses by RT-PCR and by measuring titres of enterovirus-specific antibodies of the IgA, IgG, and IgM classes. RESULTS: T-cell receptor V beta 7 gene family values were higher in recently-diagnosed diabetic patients (10.5% +/- 3.7) than in age-matched non-diabetic control subjects (5.1% +/- 1.6) (p < 0.001). In a time-course analysis of people who developed diabetes during clinical monitoring (i.e., converters), T-cell receptor V beta 7 gene expression showed values consistently above 10% (p < 0.0005). Long-standing diabetic patients showed lower percentage of V beta 7 expression compared to values measured at disease onset. In the longitudinal study of the converters, multiple acute enterovirus infections were also detected. These infections appeared to be temporally related to increased percentage of V beta 7 gene transcripts. CONCLUSION/INTERPRETATION: The deviation in the T-cell receptor V beta repertoire among circulating T cells from diabetic patients seems to re-emphasize the importance of enterovirus infections in accelerating the progression of Type I diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Infecciones por Enterovirus/complicaciones , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Islotes Pancreáticos/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Niño , Preescolar , Diabetes Mellitus Tipo 1/virología , Infecciones por Enterovirus/inmunología , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Familia de Multigenes , Proyectos Piloto , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Linfocitos T/inmunologíaRESUMEN
FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liver in vivo and in vitro evaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/inmunología , Hematopoyesis Extramedular/efectos de los fármacos , Hematopoyesis Extramedular/inmunología , Proteínas de la Membrana/farmacología , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Dendríticas/efectos de los fármacos , Femenino , Inyecciones Subcutáneas , Ligandos , Proteínas de la Membrana/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLAsunto(s)
Células Dendríticas/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos CD40/metabolismo , Comunicación Celular , Reactivos de Enlaces Cruzados , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Técnicas In Vitro , Interferón-alfa/metabolismo , Interleucina-12/biosíntesis , Interleucina-12/metabolismo , Isoantígenos , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunologíaAsunto(s)
Interleucina-12/uso terapéutico , Animales , Linfocitos B/metabolismo , Células Dendríticas , Fibroblastos , Terapia Genética , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-12/administración & dosificación , Interleucina-12/fisiología , Interleucina-6/biosíntesis , Ratones , Retroviridae , Transducción GenéticaAsunto(s)
Trasplante de Médula Ósea/inmunología , Supervivencia de Injerto/efectos de la radiación , Quimera por Radiación/inmunología , Animales , Relación Dosis-Respuesta en la Radiación , Supervivencia de Injerto/inmunología , Tolerancia Inmunológica/inmunología , Tolerancia Inmunológica/efectos de la radiación , Linfocitos/inmunología , Linfocitos/efectos de la radiación , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas , Especificidad de la Especie , Trasplante Heterólogo/inmunología , Irradiación Corporal TotalAsunto(s)
Arginina Vasopresina/metabolismo , Hipotiroidismo/metabolismo , Riñón/fisiopatología , Agua/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Arginina Vasopresina/fisiología , Sangre , AMP Cíclico/metabolismo , Femenino , Médula Renal/enzimología , Neurofisinas/sangre , Concentración Osmolar , Ratas , Glándula Tiroides/fisiología , Orina , Equilibrio HidroelectrolíticoRESUMEN
Rat posterior pituitaries were extracted in acid and total rat neurophysins were isolated. Preparative disc gel electrophoresis separated the total neurophysins into three main peptides of differing electrophoretic mobility. Antisera raised in rabbits recognized a common antigenic site in the three peptides and identical radioimmunoassay standard curves were obtained with each of the isolated rat neurophysins. A homologous rat neurophysin radioimmunoassay was utilized to measure neurophysin in samples of unextracted rat plasma. Basal neurophysin levels, 3.7 +/- 0.2 ng/ml (mean +/- SEM), did not differ in samples collected by decapitation, carotid artery cannulation, or tail vein bleeding. Water-loading caused a significant reduction in neurophysin, 2.8 +/- 0.1 ng/ml, while hypertonic saline and dehydration caused a significant elevation, 10.4 +/- 2.1 and 8.0 +/- 1.4 ng/ml, respectively. A step-wise decrease in blood volume caused a step-wise increase in plasma neurophysin concentrations which returned to baseline with reinfusion of the withdrawn blood. A second hemorrhage caused an even greater release of neurophysin indicating large neurophysin reserve in the pituitary. In periodic tail vein samples over 23 days of pregnancy a rise in plasma neurophysin was found from day 14 continuing to parturition with a peak value of greater than 13 ng/ml by day 21. Two days postpartum the value was 4.6 +/- 0.3 ng/ml. With this homologous assay, the basal levels of plasma neurophysin are lower and the stimulated values higher than with previously reported heterologous assays. Therefore, the relative change with physiologic maneuvers is distinctly increased.
Asunto(s)
Neurofisinas/aislamiento & purificación , Neurofisinas/fisiología , Animales , Deshidratación/sangre , Dietilestilbestrol/farmacología , Electroforesis Discontinua , Éteres de Etila/farmacología , Femenino , Hemorragia/sangre , Neurofisinas/análisis , Embarazo , Radioinmunoensayo , Ratas , Agua/farmacologíaRESUMEN
Two human neurophysins, nicotine stimulated neurophysin (NSN), and estrogen stimulated neurophysin (ESN) were assayed during physiologic maneuvers and pathologic states in man. NSN is thought to be associated with vasopressin and was elevated in some subjects by volume depletion, surgical stress, hypotension and hypertonic saline infusion. Overnight dehydration did not elevate NSN in spite of urinary concentration. NSN was elevated in some subjects with the syndrome of inappropriate secretion of antidiuretic hormone and when tested was unresponsive to administered water, alcohol or nicotine. ESN was elevated during estrogen administration, in pregnancy, in newborns and in patients with cirrhosis. NSN was also acutely increased at parturition. These data support the association of NSN with vasopressin although changes in NSN were found only with potent stimuli for vasopressin release. ESN may be associated with oxytocin but demonstration of this awaits knowledge of oxytocin physiology in humans.