Search

The distribution of endothelin-converting-enzyme-1 (ECE-1) messenger ribonucleic acid (mRNA) and its protein in the human kidney was assessed by implementing immunochemistry and hybridization at the site. The results revealed that ECE-1 is dispersed throughout the human kidney in vascular endothelial and tubular epithelial cells. The comparison made between the manner of distribution of ECE-1 and anti-von Willebrand factor had established the endothelial mechanism of the cells studied.

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. [ABSTRACT FROM AUTHOR]

Investigates the distribution of the endothelin-converting enzyme-1 (ECE-1) messenger ribonucleic acid (mRNA) and protein in human kidney. Detection of ECE-1 immunoreactivity in the cortex and medualla of the kidney; Distribution patterns of ECE-1 mRNA; Optical density of ECE-1 mRNA autoradiographic signals.

Introduction: Perchlorates are ionic inhibitors antagonizing iodine transport into thyrocytes, hampering thyroid hormone synthesis. Nevertheless, perchlorates are not considered as first-line treatment in hyperthyroidism and thyrotoxicosis as compared to other pharmacological and non-pharmacological interventions., Aim: Reassessing the therapeutic role of perchlorates in hyperthyroidism and thyrotoxicosis throughout a systematic review of the Literature., Methods: Guidelines were searched and examined to summarize current recommendations on the use of perchlorates in the management of hyperthyroidism. Randomized and non-randomized clinical trials were also searched and reviewed to summarize the efficacy/effectiveness and safety of perchlorates in hyperthyroidisms and thyrotoxicosis., Results: The management of specific forms of hyperthyroidism was considered, including Graves' disease (GD) in non-pregnant adults, hyperthyroidisms in pregnancy, iodine media contrast-induced hyperthyroidism, amiodarone-induced hyperthyroidisms, and thyroid storm. Most of the reported studies had remarkable limitations in terms of study design (non-controlled trials, lack of blinding), low number of participants, and the lack of clinically relevant endpoints, such as cardiovascular events, cardiovascular mortality, and teratogenicity. Overall, perchlorates could be considered a second-line treatment after thionamides, radioiodine, and total thyroidectomy in both GD and hyperthyroidisms in pregnancy. The therapeutic potential of perchlorates alone or in combination with other agents could be considered a second-line treatment of iodine-related hyperthyroidisms and thyroid storm., Conclusion: Despite the low level of evidence, perchlorates could be considered in such specific forms of thyroid disorders, including iodine-induced hyperthyroidism and thyroid storm., (© 2024. The Author(s).)

Objective: Incidental diagnosis of thyroid nodules, and therefore of thyroid cancer, has definitely increased in recent years, but the mortality rate for thyroid malignancies remains very low. Within this landscape of overdiagnosis, several nodule ultrasound scores (NUS) have been proposed to reduce unnecessary diagnostic procedures. Our aim was to verify the suitability of five main NUS., Methods: This single-center, retrospective, observational study analyzed a total number of 6474 valid cytologies. A full clinical and US description of the thyroid gland and nodules was performed. We retrospectively applied five available NUS: KTIRADS, ATA, AACE/ACE-AME, EUTIRADS, and ACRTIRADS. Thereafter, we calculated the sensitivity, specificity, PPV, and NPV, along with the number of possible fine-needle aspiration (FNA) sparing, according to each NUS algorithm and to clustering risk classes within three macro-groups (low, intermediate, and high risk)., Results: In a real-life setting of thyroid nodule management, available NUS scoring systems show good accuracy at ROC analysis (AUC up to 0.647) and higher NPV (up to 96%). The ability in FNA sparing ranges from 10 to 38% and reaches 44.2% of potential FNA economization in the low-risk macro-group. Considering our cohort, ACRTIRADS and AACE/ACE-AME scores provide the best compromise in terms of accuracy and spared cytology., Conclusions: Despite several limitations, available NUS do appear to assist physicians in clinical practice. In the context of a common disease, such as thyroid nodules, higher accuracy and NPV are desirable NUS features. Further improvements in NUS sensitivity and specificity are attainable future goals to optimize nodule management., Key Points: • Thyroid nodule ultrasound scores do assist clinicians in real practice. • Ultrasound scores reduce unnecessary diagnostic procedures, containing indolent thyroid microcarcinoma overdiagnosis. • The variable malignancy risk of the "indeterminate" category negatively influences score's performance in real-life management of thyroid lesions., (© 2021. The Author(s).)

Cell-free DNA (cfDNA) quantity and quality in plasma has been investigated as a non-invasive biomarker in cancer. Previous studies have demonstrated increased cfDNA amount and length in different types of cancer with respect to healthy controls. The present study aims to test the hypothesis that the presence of longer DNA strands circulating in plasma can be considered a biomarker for tumor presence in thyroid cancer. We adopted a quantitative real-time PCR (qPCR) approach based on the quantification of two amplicons of different length (67 and 180 bp respectively) to evaluate the integrity index 180/67. Cell-free DNA quantity and integrity were higher in patients affected by nodular thyroid diseases than in healthy controls. Importantly, cfDNA integrity index was higher in patients with cytological diagnosis of thyroid carcinoma (Thy4/Thy5) than in subjects with benign nodules (Thy2). Therefore, cfDNA integrity index 180/67 is a suitable parameter for monitoring cfDNA fragmentation in thyroid cancer patients and a promising circulating biomarker in the diagnosis of thyroid nodules., Competing Interests: The authors declare no conflict of interest.

The purpose of this study is to describe a case of concurrent medullary and papillary thyroid carcinoma (MTC and PTC) and cutaneous melanoma and to analyze BRAF(V600E) mutation in plasma and tissues. We report the clinical history and the laboratory, imaging, and histopathological findings of a 47-year-old man affected by multinodular goiter. BRAF(V600E)-mutated DNA was quantified in plasma samples and in cancer sections by quantitative real-time polymerase chain reaction (qPCR). At ultrasound examination, the dominant right nodule of the thyroid was weakly hyperechoic and hypervascularized, while the left one was hypoechoic without internal vascularization. Regional lymphadenomegalia was not detected. Basal plasma calcitonin was elevated, and the patient underwent total thyroidectomy and resection of central cervical lymph nodes. Histopathological examination identified two distinct foci of MTC and PTC and micrometastasis of well-differentiated carcinoma in one of the six resected lymph nodes. RET proto-oncogene germline mutations were not detected. Cutaneous melanoma of the thorax was subsequently diagnosed. BRAF(V600E) tissue DNA was detected in PTC and melanoma but not in MTC. The cell-free plasma percentage of BRAF(V600E) DNA was detected in pre-thyroidectomy peripheral blood and was drastically reduced after cancer treatments. This study confirms the occurrence of synchronous MTC and PTC and is the first evidence of the co-existence of melanoma and distinct thyroid cancers of different origin. BRAF(V600E) allele was detected in PTC and melanoma but not in MTC tissues. BRAF(V600E) molecular quantification in pre- and post-treatment blood supports our previous data, suggesting its possible role in diagnosis and follow-up of BRAF-positive tumors.

It has been previously shown IFN-α, -β, -γ and TNF-α (synergically with IFNs) dose-dependently induce the release of CXCL9 and CXCL10 chemokines by thyroid follicular cells, suggesting that this process may be related, at least in part, to the appearance of thyroid dysfunction during IFNs therapy. No study has evaluated the effect of IFN-α and -β on CXCL11 chemokine production in thyrocytes. The aims of this study were: (a) to test the effect of IFN-α, -β and -γ on the secretion of the Th1 chemokine CXCL11, in primary cultures of human thyroid follicular cells; (b) to assess the effect of PPAR-γ activation on CXCL11 secretion. In primary cultures of human thyroid follicular cells, CXCL11 was undetectable in the supernatant. IFN-γ, -α and -β dose dependently induced CXCL11 release. TNF-α alone had no effect. The combination of each of the IFNs with TNF-α had a significant synergistic effect on CXCL11 secretion. Treatment of primary cultures of human thyroid follicular cells with rosiglitazone dose dependently inhibited the IFNs stimulated CXCL11 release. Compared with IFN-α and -β, IFN-γ was the most potent stimulus of CXCL11 secretion. In conclusion, we first show that IFN-α, -β and -γ and TNF-α (synergically with IFNs) dose-dependently induce the release of CXCL11 by primary cultures of human thyroid follicular cells, suggesting that this process may be related to the appearance of thyroid dysfunction during IFNs therapy. Furthermore, PPAR-γ activation partially inhibits this process., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

A large majority of thyroid nodules are benign, and only 5% have malignant features on cytological examination. Unfortunately, fine-needle aspiration is inconclusive in approximately 30% of all thyroid biopsies, because the cytological features are indeterminate (suspicious for malignancy but not completely diagnostic or nondiagnostic). Wide panels of somatic mutations have been identified in thyroid cancers, and detection of genetic alterations in fine-needle aspirate has been demonstrated to improve diagnostic accuracy. Nevertheless, the relatively high number of genetic targets to be investigated, in comparison with the low percentage of malignant samples, makes the usual diagnostic protocol both time-consuming and expensive. We developed a reliable and sensitive protocol based on high-resolution melting analysis for the rapid screening of mutations of KRAS, HRAS, NRAS, and BRAF oncogenes in thyroid fine-needle aspirations. The entire procedure can be completed in approximately 48 hours, with a dramatic reduction in costs. The proposed protocol was applied to the analysis of 260 consecutive fine-needle aspiration biopsy (FNAB) samples. In 35 of 252 samples, 36 sequence variants were detected for BRAF (17 samples), NRAS (6 samples), HRAS (3 samples), KRAS codon 12 (9 samples), and KRAS codon 61 (1 sample)., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

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.

No data are present in the literature about the effect of cytokines on the prototype β chemokine (C-C motif) ligand 2 (CCL2) or of peroxisome proliferator-activated receptor α (PPARα (PPARA)) activation on CCL2 and CXCL10 chemokines secretion in fibroblasts or preadipocytes in Graves' ophthalmopathy (GO). We have tested the effect of interferon γ (IFNγ (IFNG)) and tumor necrosis factor α (TNFα) on CCL2, and for comparison on the prototype α chemokine (C-X-C motif) ligand 10 (CXCL10), and the possible modulatory role of PPARα activation on secretion of these chemokines in normal and GO fibroblasts or preadipocytes in primary cell cultures. This study shows that IFNγ alone, or in combination with TNFα, stimulates the secretion of CCL2 in primary orbital fibroblasts or preadipocytes from patients with GO at levels similar to those observed in controls. IFNγ and TNFα also stimulated CXCL10 chemokine secretion as expected. The presence of PPARα and PPARγ (PPARG) in primary fibroblasts or preadipocytes of patients with GO has been confirmed. PPARα activators were able to inhibit the secretion of CXCL10 and CCL2, while PPARγ activators were confirmed to be able to inhibit CXCL10 but had no effect on CCL2. PPARα activators were stronger inhibitors of chemokine secretions than PPARγ agonists. In conclusion, CCL2 and CXCL10 are modulated by IFNγ and TNFα in GO. PPARα activators inhibit the secretion of the main prototype α (CXCL10) and β (CCL2) chemokines in GO fibroblasts or preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.

Peroxisome proliferator-activated receptors (PPAR)α have been shown to exert immunomodulatory effects in autoimmune disorders; no study evaluated the effect of PPARα activation in Graves' ophthalmopathy (GO). We show the presence of PPARα, δ and γ in GO fibroblasts and preadipocytes. PPARα activators have a potent inhibitory action on the secretion of CXCL9 and CXCL11 chemokines (induced by IFNγ and TNFα) in fibroblasts and preadipocytes. The potency of the used PPARα agonists was maximum on the secretion of CXCL11 (67% inhibition by fenofibrate) in fibroblasts. The relative potency of the compounds in GO fibroblasts was different with each chemokine. PPARα agonists were stronger inhibitors of CXCL9 and CXCL11 (in GO fibroblasts and preadipocytes) than PPARγ activators. This study first shows that PPARα activators inhibit CXCL9 and CXCL11 chemokines in normal and GO fibroblasts and preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Until now, no data are present about the effect of peroxisome proliferator-activated receptor (PPAR)α activation on the prototype Th1 [chemokine (C-X-C motif) ligand (CXCL)10] (CXCL10) and Th2 [chemokine (C-C motif) ligand 2] (CCL2) chemokines secretion in thyroid cells. The role of PPARα and PPARγ activation on CXCL10 and CCL2 secretion was tested in Graves' disease (GD) and control primary thyrocytes stimulated with interferon (IFN)γ and tumor necrosis factor (TNF)α. IFNγ stimulated both CXCL10 and CCL2 secretion in primary GD and control thyrocytes. TNFα alone stimulated CCL2 secretion, while had no effect on CXCL10. The combination of IFNγ and TNFα had a synergistic effect both on CXCL10 and CCL2 chemokines in GD thyrocytes at levels comparable to those of controls. PPARα activators inhibited the secretion of both chemokines (stimulated with IFNγ and TNFα) at a level higher (for CXCL10, about 60-72%) than PPARγ agonists (about 25-35%), which were confirmed to inhibit CXCL10, but not CCL2. Our data show that CCL2 is modulated by IFNγ and TNFα in GD and normal thyrocytes. Furthermore we first show that PPARα activators inhibit the secretion of CXCL10 and CCL2 in thyrocytes, suggesting that PPARα may be involved in the modulation of the immune response in the thyroid., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: To our knowledge, no study has evaluated serum levels of interleukin-6 (IL-6), together with tumour necrosis factor-alpha (TNF-α), in a large series of patients with 'mixed cryoglobulinemia and HCV chronic infection' (MC+HCV) in relation to the presence of autoimmune thyroiditis (AT). The aims of the study were to evaluate serum levels of IL-6 in MC+HCV patients and to correlate this parameter with the presence of AT and with circulating levels of TNF-α., Methods: Serum IL-6 and TNF-α were assayed in 41 MC+HCV patients, in 41 MC+HCV patients with autoimmune thyroiditis (MC+AT), in 41 sex- and age-matched controls, and 20 AT patients., Results: MC+HCV patients showed significantly (p<0.01; Mann-Whitney U-test) higher IL-6 (median 8.1ng/l, range 0.7-651) serum levels than controls (median 0.6ng/l, range 0.5-41), or AT (median 2.8ng/l, range 0.5-67). MC+AT showed significantly (p<0.01; Mann-Whitney U-test) higher mean IL-6 (median 15.8ng/l, range 0.5-781) than controls, AT and MC+HCV. Serum TNF-α levels were significantly higher in MC+HCV (median 9.9ng/l, range 1.5-283) or MC+AT (median 11.2ng/l, range 1.6-412) than in controls (median 1.0ng/l, range 0.6-6.4), or AT (median 1.7ng/l, range 0.6-11.8) (p<0.01, for each comparison)., Conclusions: Our study demonstrates significantly higher serum levels of IL-6 and TNF-α in patients with MC+HCV and MC+AT compared to healthy controls. Furthermore, the study first shows a significant increase in circulating IL-6 observed in MC+AT patients with respect to MC+HCV. Future studies in larger patients' series will be needed to evaluate the relevance of serum IL-6 and TNF-α determination as clinico-prognostic markers of MC+HCV patients and its usefulness in the therapeutic approach to these patients.

Context: Peroxisome proliferator-activated receptor (PPAR)-α has been shown to exert immunomodulatory effects in autoimmune disorders. However, until now, no data were present in the literature about the effect of PPARα activation on CXCL9 and CXCL11 chemokines in general or on secretion of these chemokines in thyroid cells., Objective and Design: The presence of PPARα and PPARγ has been evaluated by real-time-PCR in Graves' disease (GD) and control cells in primary culture. Furthermore, we have tested the role of PPARα and PPARγ activation on CXCL9 and CXCL11 secretion in GD and control cells after stimulation of these chemokines secretion with IFNγ and TNFα., Results: This study shows the presence of PPARα and PPARγ in GD and control cells. A potent dose-dependent inhibition by PPARα-agonists was observed on the cytokines-stimulated secretion of CXCL9 and CXCL11 in GD and control cells. The potency of the PPARα agonists used was maximum on the secretion of CXCL9, reaching about 90% of inhibition by fenofibrate and 85% by ciprofibrate. The relative potency of the compounds was different with each chemokine; for example, gemfibrozil exerted a 55% inhibition on CXCL11, whereas it had a weaker activity on CXCL9 (40% inhibition). PPARα agonists were stronger (ANOVA, P<0.001) inhibitors of CXCL9 and CXCL11 secretion in thyrocytes than PPARγ agonists., Conclusions: Our study shows the presence of PPARα in GD and control thyrocytes. PPARα activators are potent inhibitors of the secretion of CXCL9 and CXCL11, suggesting that PPARα may be involved in the modulation of the immune response in the thyroid.

The aim of this study was to evaluate serum levels of interleukin-1β (IL-1β), chemokine (C-X-C motif) ligand 10 (CXCL10), and interferon-gamma (IFN-γ) in a series of patients with "mixed cryoglobulinemia and hepatitis C virus chronic infection" (MC + HCV) in the presence or absence of autoimmune thyroiditis (AT) and to relate them to the clinical phenotype of these patients. Serum IL-1β, IFN-γ, and CXCL10 were assayed in 30 patients with MC + HCV without AT, in 30 patients with MC + HCV and AT, and in 30 sex- and age-matched controls. Cryoglobulinemic patients showed significantly higher mean IL-1β and CXCL10 levels than controls (P < 0.01). Moreover, CXCL10 was significantly increased in patients with AT patients with respect to those without AT (P < 0.01). Serum IFN-γ levels were not significantly higher in MC + HCV patients than in controls. In conclusion, our study demonstrates significantly high serum levels of IL-1β in patients with MC + HCV with and without AT compared with healthy controls. Further, significantly high serum levels of CXCL10 in patients with MC + HCV compared with healthy controls were confirmed, overall in the presence of AT. Moreover, a pathophysiological association between high circulating levels of IL-1β and CXCL10 has been suggested. A possible therapeutic role of the anti-IL-1 receptor antagonist (Anakirna) in MC remains to be evaluated.

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.

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.

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.

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.

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.