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CONTEXT: Cell-free nucleic acids circulating in plasma are considered a promising noninvasive tool for cancer monitoring. BRAF(V600E) mutation in cell-free DNA (cfDNA) could represent an appropriate marker for papillary thyroid carcinoma (PTC). OBJECTIVE: Our aim is to investigate the role of BRAF(V600E)-mutated allele in cfDNA as a marker for the diagnosis and follow-up of PTC. STUDY DESIGN: BRAF(V600E) allele was detected and quantified by an allele-specific real-time quantitative PCR assay in plasma from 103 patients affected by nodular goiter. As control populations, we enrolled 49 healthy subjects and 16 patients with non-nodular thyroid diseases. RESULTS: The percentage of circulating BRAF(V600E) was significantly different between patients and controls and throughout different cytological categories of ultrasound-assisted fine-needle aspiration. Patients with a histopathological diagnosis of PTC showed a higher percentage of circulating BRAF(V600E) (P = .035) compared to those with benign histology. In 19 patients, a second blood draw, taken 3-6 months after surgery, showed a lower percentage of BRAF(V600E) in cfDNA than the presurgical sample (P < .001). The diagnostic performance of circulating BRAF(V600E) was assessed by receiver operating characteristic curve analysis resulting in an area under the curve of 0.797. A cutoff value was chosen corresponding to maximum specificity (65%) and sensitivity (80%). On this basis, we evaluated the predictive value of BRAF(V600E) in Thy 3 patients with a resulting positive predictive value of 33% and a negative predictive value of 80%. CONCLUSIONS: The results of the present study provide encouraging data supporting the possibility to take advantage of circulating BRAF(V600E) in the management of PTC.
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Carcinoma/diagnóstico , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/sangre , Carcinoma/genética , Carcinoma/patología , Carcinoma Papilar , ADN/análisis , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas B-raf/sangre , Curva ROC , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
BACKGROUND: Cytological examination of fine needle aspirates (FNA) is the standard procedure for discriminating potentially malignant thyroid nodules to be referred to surgery. In a fraction of cases, ultrasound (US) examination could provide information theoretically sufficient to avoid FNA, when typical US features suggesting malignancies are lacking. AIM: The aim of this study was to construct a simple US score predicting malignant nodules so as to reduce the number of unnecessary FNA. SUBJECTS AND METHODS: In a series of 1632 consecutive patients undergoing US-guided FNA (1812 nodules), echostructure, echogenicity, margins, halo, microcalcification, and vascularization were assessed. RESULTS: At multivariate analysis, the following parameters showed a strong predictive value for positive cytology (Thy 4 and Thy 5, suspicious and diagnostic for malignancy, respectively, according to the Thyroid British Association): solid echostructure, irregular margins and hypoechogenicity [adjusted odd ratio (OR) 5.13 (1.58-16.66), 3.03 (1.70-5.39), 2.05 (1.17-3.57), respectively]. A 10-point Thyroid Risk Ultrasound Score (TRUS) was constructed on the basis of the adjusted OR. A TRUS≥6 identified malignant nodules with sensitivity and specificity of 73% and 65%, respectively. Among the patients with follicular lesions (Thy 3) and final diagnosis of carcinoma, about 65% had a TRUS≥6.0. CONCLUSIONS: The sensitivity of TRUS, although higher than that of other scores, could still be insufficient for the identification of patients who could avoid FNA in routine clinical practice, whereas its predictive value for Thy 3 lesions deserves further investigations.
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Nódulo Tiroideo/diagnóstico por imagen , Nódulo Tiroideo/patología , Ultrasonografía Doppler en Color , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina , Citodiagnóstico , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Adulto JovenRESUMEN
AIMS: Cell-mediated immunity and pro-inflammatory cytokines are implicated in the pathogenesis of Type 1 diabetes. The aim of this study was to investigate whether circulating chemokines involved in T-helper 1 (CXCL10) and T-helper 2 (CCL2) autoimmunity are increased in children with Type 1 diabetes at onset and follow-up. METHODS: Serum CXCL10 and CCL2 were measured in 96 children with newly diagnosed Type 1 diabetes, 59 age-matched first-degree relatives of diabetic children and 40 age-matched non-diabetic children with no family history of diabetes. In the diabetic children, an additional serum sample was obtained a median of 16 months after diagnosis. RESULTS: Serum CXCL10 levels were significantly higher in Type 1 children than in relatives or control children (P < 0.001); 44.7% of patients had a serum CXCL10 level >or= 2 standard deviation above the mean value of the control group vs. 3.4% of relatives (P < 0.0001). In contrast, serum CCL2 levels were similar in patients, relatives and control subjects. In the Type 1 diabetic patients at follow-up, CXCL10 was significantly reduced vs. baseline (P = 0.01), while CCL2 did not change. CONCLUSIONS: In children with newly diagnosed Type 1 diabetes, raised serum CXCL10 and normal CCL2 concentrations signal a predominant T-helper 1-driven autoimmune process, which shifts toward T-helper 2 immunity over the first 1-2 years from diagnosis.
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Quimiocina CCL2/inmunología , Citocinas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Interleucina-10/inmunología , Receptores de Quimiocina/inmunología , Células TH1/inmunología , Quimiocina CCL2/sangre , Quimiocina CCL2/metabolismo , Niño , Citocinas/sangre , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Progresión de la Enfermedad , Femenino , Humanos , Interleucina-10/sangre , Estudios Longitudinales , Masculino , Curva ROC , Receptores de Quimiocina/sangre , Factores de TiempoRESUMEN
AIMS: To test for anti-CD38 autoimmunity in children with newly-diagnosed type 1 diabetes mellitus (DM1). METHODS: Serum anti-CD38 autoantibodies were detected by Western blot in 270 children (130 girls, 140 boys, mean age 8 +/- 4 years) with newly-diagnosed DM1 and 179 gender- and age-matched non-diabetic children. In 126 diabetic children, another blood sample was obtained 15 +/- 4 months after the diagnosis. RESULTS: Anti-CD38 autoantibody titers at least 3 SD above the mean value for the control group were found in 4.4% of children with DM1 vs 0.6% of controls (chi2 = 5.8, p <0.016). No statistical differences were observed between anti-CD38 positive and negative patients in terms of phenotype. At follow-up, of six diabetic children who were positive for anti-CD38 antibodies, two were new cases. A positive correlation was found between the antibody titer of diabetic sera at diagnosis and follow up (r = 0.46, p <0.0001). CONCLUSION: An autoimmune reaction against CD38, a protein expressed in human islets, is associated with newly-diagnosed DM1. In children with DM1, CD38 autoimmunity increases with time and persists.
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ADP-Ribosil Ciclasa 1/inmunología , Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Autoanticuerpos/sangre , Niño , Preescolar , Femenino , Humanos , MasculinoRESUMEN
Autoantibodies directed against human CD38 (an enzyme catalysing the interconversion of NAD(+) and cyclic ADP-ribose) have been demonstrated recently in patients with type 2 diabetes. We tested 220 consecutive Caucasian patients with autoimmune chronic thyroiditis, 104 patients with Graves' disease, 220 subjects from the general population (control I) and 78 healthy control subjects not affected by thyroid autoimmune disorders (control II) for the presence of anti-CD38 autoimmunity. Using Western blot analysis and optical densitometry, a specific band corresponding to human recombinant CD38 was identified in the serum of several subjects. By defining anti-CD38 positivity as a standardized optical reading > 3 s.d. higher than the mean value of control I, 10.4% of patients with thyroiditis and 7.7% of Graves' patients were anti-CD38 positive (P = 0.0009 versus 1.8% of control I). Similarly, 13.1% of patients with thyroiditis and 10.5% of Graves' patients had a standardized optical reading > 3 s.d. higher than the mean value of the subjects not affected by thyroid autoimmune disorders (P = 0.002 versus 1.2% of control II). Anti-CD38 autoimmunity did not differ between euthyroid, hyperthyroid or hypothyroid patients or between patients with or without thyroid hypoechogenicity. Anti-CD38 autoantibodies were associated with higher levels of circulating antithyroid-peroxidase antibodies (P = 0.03) and they were more frequent in Graves' patients with ophthalmopathy (P < 0.05). Anti-CD38 autoantibodies are a new autoimmune marker in chronic autoimmune thyroiditis and Graves' disease. The specific role of CD38 and its autoantibodies in the modulation of thyroid cell function or growth remains to be investigated.
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Antígenos CD , Antígenos de Diferenciación/inmunología , Autoanticuerpos/sangre , Enfermedad de Graves/inmunología , NAD+ Nucleosidasa/inmunología , Tiroiditis Autoinmune/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adulto , Anciano , Autoinmunidad , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Humanos , Yoduro Peroxidasa/inmunología , Masculino , Glicoproteínas de Membrana , Persona de Mediana EdadRESUMEN
CD38 is involved in transmembrane signaling in many cell types; anti-CD38 autoantibodies have been described in diabetic patients. We tested whether human anti-CD38 antibodies possess signaling properties by measuring their ability to raise intracellular calcium ([Ca2+]i) using the fluo-3-acetoxymethyl ester method in a human-derived T-cell line (Jurkat T-cells, expressing high levels of surface CD38) and in dispersed human islet cells from normal donors. In Jurkat T-cells, 11 of 19 anti-CD38-positive sera raised [Ca2+]i (by > or =20% of baseline), whereas no [Ca2+]i-mobilizing activity was found in 27 anti-CD38-negative sera (chi2 = 20.5, P < 0.0001). In dispersed human islet cells, 5 of 11 anti-CD38-positive sera (and none of three anti-CD38-negative sera) raised [Ca2+]i significantly. When preincubated with Staphylococcus aureus protein A to remove IgG, anti-CD38-positive sera showed a 70 +/- 5% reduction in [Ca2+]i-mobilizing activity. Preincubation with CD38-transfected NIH-3T3 fibroblasts, but not with mock-transfected NIH-3T3 cells, abolished [Ca2+]i mobilization. In blocking experiments, preincubation with nonagonistic anti-CD38 monoclonal antibodies also prevented [Ca2+]i mobilization. In cultured human islets, anti-CD38-positive sera exhibiting [Ca2+]i-mobilizing activity in Jurkat T-cells (n = 6) significantly stimulated insulin release at 3.3 mmol/l glucose (median [interquartile range] 738 microU/ml [234], P = 0.0001 vs. 320 [52] microU/ml of control), whereas 6 anti-CD38-positive sera without [Ca2+]i-mobilizing activity and 10 anti-CD38-negative did not. In further incubations, the five anti-CD38-positive sera displaying [Ca2+]i-mobilizing activity in dispersed islet cells significantly stimulated insulin release at both 3.3 mmol/l glucose (2.2 +/- 0.3% of insulin islet content, P < 0.002 vs. 1.2 +/- 0.1% of control) and 16.7 mmol/l glucose (3.7 +/- 0.3 vs. 2.3 +/- 0.3%, P < 0.002). We conclude that human anti-CD38 autoantibodies with agonistic properties on the CD38 effector system occur in nature; in human islets, their [Ca2+]i-mobilizing activity is coupled with the ability to stimulate insulin release.
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Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Autoanticuerpos/farmacología , Señalización del Calcio/fisiología , Calcio/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/fisiología , NAD+ Nucleosidasa/inmunología , Células 3T3 , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Anticuerpos Monoclonales/farmacología , Autoanticuerpos/sangre , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Enterotoxinas/farmacología , Humanos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Células Jurkat , Glicoproteínas de Membrana , Ratones , Proteínas Recombinantes/inmunología , Superantígenos/farmacología , Linfocitos T/inmunología , TransfecciónRESUMEN
In a previous report, we demonstrated that in FNC-B4 cells, derived and characterized from a human fetal olfactory epithelium, both sex steroids and odorants regulate GnRH secretion. We now report the presence and biological activity of endothelin (ET)-1 in this GnRH-secreting neuronal cell. By in situ hybridization and immunohistochemistry, we found gene and protein expression of ET-1 and its converting enzyme ECE-1 in both fetal olfactory mucosa and FNC-B4 cells. The presence of authentic ET-1 in the conditioned media of FNC-B4 cells was further supported by combined RIAs and high-performance liquid chromatography studies. Experiments with radiolabeled ET-1 and ET-3 strongly indicated the presence of two classes of binding sites, corresponding to the ETA (16,500 sites/cell) and the ETB receptors (8,700 sites/cell). Functional studies, using selective analogs, indicated that these two classes of receptors subserve distinct functions in human GnRH-secreting cells. The ETA receptor subtype mediated an increase in intracellular calcium and GnRH secretion. Conversely, stimulation of the ETB subtype induced DNA synthesis and mitogen-activated protein kinase p44ERK1 expression. This is the first demonstration, in a human in vitro model, of a neuroendocrine role for ET-1 as regulator of GnRH-secreting neuron activity.
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Endotelina-1/genética , Endotelina-1/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/metabolismo , Mucosa Olfatoria/citología , Calcio/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , Embrión de Mamíferos , Endotelina-1/análisis , Endotelina-3/metabolismo , Humanos , Hibridación in Situ , Neuronas/química , Neuronas/efectos de los fármacos , Mucosa Olfatoria/embriología , Mucosa Olfatoria/metabolismo , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/análisis , Receptores de Endotelina/metabolismoRESUMEN
The mechanisms responsible for mesangial cell proliferation in proliferative glomerulonephritis are only partially understood. This article reports the results of an immunohistochemical study showing high expression of the chemokine receptor CXCR3 by mesangial cells of patients with IgA nephropathy, membranoproliferative glomerulonephritis, or rapidly progressive glomerulonephritis. CXCR3 was also detectable by flow cytometry in cultured human mesangial cells, in which it appeared to be functionally active, as determined by the ability of its ligand, the (interferon-gamma)-inducible protein of 10 kD (IP-10) to induce intracellular Ca2+ influx. Both IP-10 and the monokine induced by interferon-gamma (Mig) were also effective in inducing proliferation of human mesangial cells. These data suggest that in patients with proliferative glomerulonephritis, the chemokines IP-10 and/or Mig not only may act as chemoattractants for infiltrating mononuclear cells in the inflamed tissue, but also may directly induce the proliferation of mesangial cells.
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Quimiocinas CXC/metabolismo , Glomerulonefritis/inmunología , Receptores de Quimiocina/metabolismo , Adulto , Anciano , Calcio/metabolismo , Estudios de Casos y Controles , División Celular , Células Cultivadas , Quimiocina CXCL10 , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/metabolismo , Glomerulonefritis por IGA/patología , Glomerulonefritis Membranoproliferativa/inmunología , Glomerulonefritis Membranoproliferativa/metabolismo , Glomerulonefritis Membranoproliferativa/patología , Humanos , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Persona de Mediana Edad , Receptores CXCR3RESUMEN
The type II transmembrane glycoprotein CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) has been proposed as a mediator of insulin secretion from pancreatic beta-cells and as a candidate for autoimmune reactions in type 2 diabetes. We evaluated the presence of anti-CD38 autoantibodies in Caucasian patients with diabetes and investigated the effect of these antibodies on insulin secretion from isolated human pancreatic islets. The presence of anti-CD38 autoantibodies was evaluated by using Western blot analysis in 236 patients with type 2 diabetes (mean age 63 years), in 160 patients with type 1 diabetes (mean age 38 years), and in 159 nondiabetic subjects. Anti-CD38 autoantibody titers at least 3 SD above the mean value of the control group were found in 9.7% of type 2 diabetic patients and in 13.1% of type 1 diabetic patients (chi2 = 15.9, P = 0.0003 vs. 1.3% of control subjects). No significant differences were observed in sex distribution, current age, age at diabetes onset, BMI, fasting serum glucose, or glycemic control between anti-CD38+ and anti-CD38-diabetic patients in either the type 2 or type 1 diabetic groups. The effect of 23 anti-CD38- and 13 anti-CD38+ sera on insulin secretion at low (3.3 mmol/l) or high (16.7 mmol/l) medium glucose concentrations was evaluated in isolated human pancreatic islets. Data are medians (interquartile range). The anti-CD38+ sera potentiated insulin release both at low [95 (64) vs. 23 (12) microU/ml of control incubations, respectively, P < 0.0001] and high [271 (336) vs. a control of 55 (37) microU/ml, respectively, P = 0.001] medium glucose concentrations, whereas the anti-CD38- sera did not. Furthermore, in the pooled data from all 36 tested sera, insulin levels in the islet incubation medium were directly related to the anti-CD38 antibody titer. We conclude that autoantibodies to CD38 are associated with both type 1 and type 2 diabetes in Caucasian subjects. These autoantibodies exert a stimulatory effect on insulin secretion by cultured human islets. The role of this autoimmune reaction in the pathogenesis of diabetes remains to be elucidated.
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Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , NAD+ Nucleosidasa/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adulto , Edad de Inicio , Autoanticuerpos/farmacología , Células Cultivadas , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Secreción de Insulina , Islotes Pancreáticos/inmunología , Italia , Masculino , Glicoproteínas de Membrana , Persona de Mediana Edad , Análisis de Regresión , Población BlancaRESUMEN
Dopamine receptors type 2 (D2)-like receptor blockers cause an increase in the norepinephrine response to intense physical exercise. However, during intense physical exercise, D2-like antagonists also cause an increase in the epinephrine response, which itself might cause an increase in plasma norepinephrine through the activation of beta2 presynaptic receptors. Therefore, we evaluated the effect of domperidone, a D2-like antagonist, on the norepinephrine response to physical exercise in 6 Addison patients (3 were adrenalectomized and 3 had adrenal tuberculosis). In these patients, the norepinephrine increase observed during exercise was significantly higher after the administration of domperidone than a placebo (F=4,328; P<0.001). Because peripheral plasma norepinephrine does not reflect the sympathetic tone to the heart accurately, we evaluated the effect of domperidone administration (20 mg orally) on the sympathovagal balance, which was measured by the ratio between the high- and low-frequency components of heart rate variability, in 9 normal volunteers in the supine and sitting positions. When compared with placebo, domperidone caused a significant increase in the low/high frequency ratio (P<0.05) in the sitting position without modifying basal and stimulated norepinephrine plasma levels or blood pressure. These data support a role for endogenous dopamine in modulating norepinephrine release by human sympathetic nerves in vivo.
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Domperidona/farmacología , Antagonistas de Dopamina/farmacología , Dopamina/fisiología , Receptores de Dopamina D2/metabolismo , Sistema Nervioso Simpático/fisiología , Enfermedades de las Glándulas Suprarrenales/metabolismo , Enfermedades de las Glándulas Suprarrenales/fisiopatología , Adulto , Estudios Cruzados , Dopamina/sangre , Antagonistas de los Receptores de Dopamina D2 , Método Doble Ciego , Femenino , Frecuencia Cardíaca , Humanos , Masculino , Persona de Mediana Edad , Norepinefrina/sangre , Esfuerzo Físico/fisiología , Valores de ReferenciaRESUMEN
Nitric oxide (NO) plays an important role in the cytotoxic mechanisms responsible for acute renal allograft rejection, where macrophages produce high levels of inducible nitric oxide synthase (iNOS). By contrast, both the source and the role of NO in chronic allograft nephropathy (CAN) are still unclear. In this study, the expression of iNOS mRNA and protein was assessed in the kidneys of patients with graft failure due to chronic rejection. As controls, kidney specimens were obtained from patients undergoing nephrectomies for primary renal tumours, and from patients suffering from IgA nephropathy or mesangial-proliferative glomerulonephritis. In normal kidneys, iNOS production was absent or limited to a low signal, while it was found only in the inflammatory infiltrate of kidneys affected by glomerulonephritis, as assessed by immunohistochemistry and in situ hybridization. In contrast, in CAN, iNOS protein was localized not only in inflammatory cells, but also in vascular, glomerular, and, more rarely, tubular structures. Accordingly, in situ hybridization localized iNOS mRNA in both macrophages and lymphocytes, as well as in vascular structures and glomeruli. Double immunostaining for iNOS and a-smooth muscle actin (a-SMA) or von Willebrand factor (vWf) revealed that smooth muscle cells were the main vascular source of iNOS, while both mesangial and inflammatory cells were immunostained at the glomerular level. These data demonstrate that macrophages and lymphocytes are not the only source of iNOS mRNA and protein in human CAN. Vascular smooth muscle and mesangial cells also synthesize iNOS, raising the question of heterogeneous regulation and function of iNOS in this disease.
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Rechazo de Injerto/enzimología , Glomérulos Renales/enzimología , Trasplante de Riñón/fisiología , Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa/metabolismo , Adulto , Enfermedad Crónica , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa/genética , ARN Mensajero/genéticaRESUMEN
The aim of the present study was to evaluate the role of angiotensin II (AngII) in regulating both the gene expression and secretion of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) in human mesangial cells (HMC) in culture. Densitometric analysis of Northern blot experiments demonstrated that AngII increases VPF/VEGF mRNA in a dose-dependent manner. The levels of VPF/VEGF mRNA in HMC exposed for 3 h to 10 nM, 100 nM, and 1 microM AngII were, respectively, 1.5-, 2.3-, and 1.6-fold higher than control cells (P < 0.05, P < 0.0001, and P < 0.05, respectively). This effect was blocked by the pretreatment with losartan (1 microM) (P < 0.005), a selective antagonist of the AngII AT1 receptor. Reverse transcription-PCR performed in HMC using oligonucleotide primers specific for all VPF/VEGF mRNA splicing variants detected three bands corresponding to VEGF 189, 165, and 121. Exposure of the cells to 100 nM AngII resulted in an increase of all the mRNA transcripts. Furthermore, in situ hybridization experiments showed that the levels of hybridization signals for VPF/VEGF mRNA resulted consistently higher in HMC exposed for 3 h to AngII (100 nM) than in control cells. The effects of AngII on the secretion of VPF/VEGF peptide in the culture medium of HMC were assessed using an enzyme-linked immunosorbent assay method. When different concentrations of AngII were tested in 3-h stimulation periods, the percentage of increase in the levels of released VPF/VEGF was significantly higher than control cells for AngII concentrations of 100 nM (62 +/- 11% mean +/- SD, P < 0.0001) and 1 microM (17.3 +/- 10.9%, P < 0.01). The pretreatment of HMC with losartan (1 microM) prevented the increase of VPF/VEGF secretion induced by AngII (100 nM) (AngII 54.7 +/- 3.9 pg/microg DNA versus AngII + losartan 37.8 +/- 3.6 pg/microg DNA, mean +/- SD, P < 0.005). VPF/VEGF protein was time dependently released in the culture medium under basal, steady-state conditions. Compared with control cells, AngII (100 nM) caused a significant increase in the levels of released VPF/VEGF after 3 and 6 h (control 33.8 +/- 1.7 pg/microg DNA at 3 h, 42.1 +/- 1.1 at 6 h, and 117.7 +/- 10 at 24 h; AngII 54.7 +/- 3.9 at 3 h, P < 0.0001, 61.6 +/- 8.7 at 6 h, P < 0.05, and 144.7 +/- 22.7 at 24 h, NS; mean +/- SD). According to the results obtained from enzyme-linked immunosorbent assay experiments, Western blot analysis showed that the intensity of the 19-kD band corresponding to VPF/VEGF was 1.5-fold higher in AngII (100 nM)-treated HMC than in control cells. Similarly, immunocytochemistry on HMC demonstrated an increase in intracellular VPF/VEGF immunostaining in response to AngII treatment (100 nM) compared with control cells. This study demonstrated that in HMC, AngII augmented the levels of VPF/VEGF gene expression and stimulated the synthesis and secretion of its peptide by activating AT1 receptors. Through these mechanisms, AngII may affect the functions of endothelial cells during the development of renal diseases involving the glomerulus.
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Angiotensina II/farmacología , Factores de Crecimiento Endotelial/metabolismo , Mesangio Glomerular/metabolismo , Linfocinas/metabolismo , Northern Blotting , Western Blotting , Células Cultivadas , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/genética , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Mesangio Glomerular/citología , Humanos , Inmunohistoquímica , Hibridación in Situ , Linfocinas/biosíntesis , Linfocinas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The human alpha-chemokine receptor fusin/CXCR4 is an important cofactor for entry of T lymphocyte-tropic HIV-1 strains. We investigated the possible regulatory role of T cell cytokine patterns on CXCR4 as well as HIV expression by using in vitro models of both secondary and primary immune responses. Antigen-specific memory CD4+ T cells infected with a T-tropic HIV-1 strain showed significantly higher CXCR4 and HIV-1 expression in Th0/2-oriented responses in comparison with Th1-oriented responses. Similarly, in naive CD4+ T cells activated in the presence of IL-4 or IL-12 and infected with the same T-tropic strain, IL-4 up-regulated whereas IL-12 down-regulated both CXCR4 and HIV-1 expression. The down-regulatory effect of IL-12 on CXCR4 expression was found to be dependent on its capacity to induce IFN-gamma production. These observations can account for the higher risk of progression in HIV-1-infected individuals undergoing Th0/2-oriented immune responses.
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VIH-1/inmunología , Interferón gamma/inmunología , Interleucina-4/inmunología , Receptores CXCR4/inmunología , Células Th2/inmunología , Células Cultivadas , Regulación hacia Abajo/inmunología , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Memoria Inmunológica , Células TH1/inmunología , Células TH1/virología , Células Th2/virología , Regulación hacia Arriba/inmunologíaRESUMEN
We report the case of a 49-yr-old man affected by coma and hypoglycemia unawareness following repetitive hypoglycemic episodes due to dumping syndrome. The dumping syndrome, which was due to partial gastrectomy and vagotomy performed for recurrent peptic ulcer, was responsible for reactive hyperinsulinemia as demonstrated by an oral glucose tolerance test. While the glucose counterregulatory hormones were all normally sensitive to specific stimulation tests, insulin-induced hypoglycemia failed to induce an adequate counterregulatory response, causing no response in plasma norepinephrine, a slight and short increase in plasma cortisol, ACTH and glucagon and an insufficient increase in plasma epinephrine and GH. This case demonstrates that hypoglycemia unawareness has to be taken into account not only in patients affected by IDDM or insulinoma but also in any case of reactive hypoglycemia.
Asunto(s)
Síndrome de Vaciamiento Rápido/fisiopatología , Hipoglucemia/fisiopatología , Hormona Adrenocorticotrópica , Glucemia/metabolismo , Péptido C/sangre , Coma/etiología , Hormona Liberadora de Corticotropina , Síndrome de Vaciamiento Rápido/sangre , Síndrome de Vaciamiento Rápido/etiología , Gastrectomía/efectos adversos , Prueba de Tolerancia a la Glucosa , Hormonas/sangre , Humanos , Hipoglucemia/sangre , Hipoglucemia/etiología , Insulina/sangre , Masculino , Persona de Mediana Edad , Percepción , Pruebas de Función Hipofisaria , VagotomíaRESUMEN
In a previous study, we reported the presence of endothelin-1 and endothelin receptors in the human testis. We have now extended our investigations to the human epididymis. Since sperm appear to be immotile during their transit through the epididymis, it is conceivable that specific local factors promote smooth muscle contraction, enabling sperm transport. In this paper, we show that endothelin-1 mRNA and protein are readily detectable in the epithelial compartment of the human epididymis, and that endothelin converting enzyme- 1, which converts the precursor pro-endothelin-1 into active endothelin-1, is expressed in the epididymis, consistent with active processing of the prohormone. In addition, two classes of endothelin receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the previously characterized endothelinA and endothelinB receptor. These receptors appear to be biologically active and mediate the contractile activity of the epididymis in vitro. Our data suggest that endothelin-1, via a paracrine mode of action, may be responsible for sperm progression through this organ.
Asunto(s)
Endotelina-1/biosíntesis , Epidídimo/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
CD30 is a member of tumor necrosis factor (TNF) receptor superfamily that is expressed by activated T cells in the presence of interleukin-4 (IL-4). Although CD30 can mediate a variety of signals, CD30-deficient mice have impaired negative selection of T cells, suggesting that at least in the context of murine thymus, CD30 is a cell death-mediating molecule. The ligand for CD30 (CD30L) is a membrane-associated glycoprotein related to TNF, which is known to be expressed mainly by activated T cells and other leukocytes. However, the nature of CD30L-expressing cells involved in the interaction with CD30+ thymocytes is unclear. We report here that in postnatal human thymus the great majority of CD30+ cells are double positive (CD4+CD8+), activated, IL-4 receptor-expressing T cells which selectively localize in the medullary areas. Moreover, many medullary epithelial cells and Hassal's corpuscles in the same thymus specimens showed unusually high expression of CD30L in comparison with other lymphoid or nonlymphoid tissues. These findings provide additional information on the nature and localization of CD30+ thymocytes and show that epithelial cells are the major holder of CD30L in the thymic medulla.
Asunto(s)
Células Epiteliales/inmunología , Antígeno Ki-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Subgrupos de Linfocitos T/inmunología , Timo/inmunología , Ligando CD30 , Diferenciación Celular , Citometría de Flujo , Humanos , Hibridación in Situ , Queratinas/metabolismo , Activación de Linfocitos , Receptores de Interleucina-4/metabolismo , Timo/citología , Distribución TisularAsunto(s)
Hormona Adrenocorticotrópica/deficiencia , Síndrome de Cushing/sangre , Epinefrina/sangre , Hidrocortisona/sangre , Norepinefrina/sangre , Feniletanolamina N-Metiltransferasa/metabolismo , Adenoma/sangre , Adenoma/cirugía , Enfermedades de la Corteza Suprarrenal/sangre , Enfermedades de la Corteza Suprarrenal/cirugía , Neoplasias de las Glándulas Suprarrenales/sangre , Neoplasias de las Glándulas Suprarrenales/cirugía , Médula Suprarrenal/metabolismo , Adrenalectomía , Adulto , Síndrome de Cushing/enzimología , Epinefrina/metabolismo , Femenino , Glucagón , Humanos , Hiperplasia , MasculinoRESUMEN
The distribution of endothelin-converting enzyme-1 (ECE-1) mRNA and protein was investigated in human kidney excised because of renal tumors. ECE-1 immunoreactivity was detected by immunohistochemistry throughout the different areas of the kidney in the vascular and tubular structures. In the cortex, ECE-1 immunostaining was present in the endothelial surface of arcuate and interlobular arteries and in arterioles. Weak specific immunoreactivity was present over some proximal and distal tubules. Few endothelial glomerular cells contained ECE-1 protein. In the medulla, ECE-1 immunoreactivity was observed in the vasa recta bundles and capillaries. ECE-1 immunostaining was also detected in the outer and inner medullary collecting ducts and thin limbs of Henle's loops. Immunohistochemical detection of the von Willebrand factor on adjacent sections confirmed the endothelial nature of the vascular cells that exhibited ECE-1 immunostaining. The distribution patterns of ECE-1 mRNA, investigated by in situ hybridization, appeared similar to that obtained by immunohistochemistry in the cortical and medullary vasculature and in different portions of the nephron. Northern blot and densitometric analyses demonstrated that ECE-1 mRNA levels were quantitatively similar in both the renal cortex and medulla. These results demonstrate that vascular endothelial and tubular epithelial cells in the cortex and medulla of the human kidney synthesize ECE-1, which, in turn, may play an important role in regulating endothelin production in physiological and pathological conditions.
Asunto(s)
Arteriolas/enzimología , Ácido Aspártico Endopeptidasas/análisis , Endotelio Vascular/enzimología , Riñón/enzimología , Adulto , Anciano , Enzimas Convertidoras de Endotelina , Femenino , Humanos , Inmunohistoquímica , Riñón/irrigación sanguínea , Riñón/patología , Corteza Renal/irrigación sanguínea , Médula Renal/irrigación sanguínea , Médula Renal/enzimología , Neoplasias Renales/patología , Neoplasias Renales/cirugía , Masculino , Metaloendopeptidasas , Persona de Mediana Edad , Sondas ARN , Arteria Renal/enzimologíaRESUMEN
The pattern of cytokine production of skin-infiltrating T cells from patients with progressive systemic sclerosis was investigated. Most CD4+ T-cell clones generated from skin biopsy specimens showed a type 2 helper (Th2) cytokine profile (production of interleukin-4, but no interferon (IFN)-gamma). High interleukin-4 but little or no IFN-gamma mRNA expression was found by in situ hybridization in skin perivascular mononuclear cell infiltrates. The immunohistochemical analysis revealed CD30 expression by high numbers of CD4+ T cells in the same specimens. Finally, the great majority of patients with diffuse disease had elevated levels of soluble CD30 in their sera. These data suggest the existence in patients with progressive systemic sclerosis of a predominant activation of Th2-like T cells, which may account for the major alterations (endothelial cell injury, fibrosis, and autoantibody production) occurring in this disease.
Asunto(s)
Antígeno Ki-1/biosíntesis , Esclerodermia Sistémica/inmunología , Células Th2/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Antígeno Ki-1/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/inmunología , Piel/patología , Células Th2/metabolismoRESUMEN
We have previously reported the presence of endothelin-1 (ET-1) and its receptors in the human testis. In the present study we extended our investigations to human epididymis. The rationale of our study originated from the fact that sperm appear to be immotile during their transit through the epididymis. Hence, it is conceivable that specific factors, unknown to date, are present in this organ, capable of inducing smooth muscle contractions, thus forcing sperm transport. In this paper it is shown that ET-1 messenger ribonucleic acid and protein are readily detectable in the epithelial compartment of the human epididymis, and that ET-converting enzyme-1, which converts the precursor pro-ET-1 into the active peptide ET-1, is expressed in the epididymis, thus indicating an active processing of the prohormone. In addition, two classes of ET receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the ETA and ETB receptors previously characterized. These receptors mediate the contractile activity of the epididymis in vitro, thus suggesting that ET-1 can be responsible of sperm progression through this organ, acting via a paracrine mode of action.