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Summary Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD., We defined urinary stem cell (USC) RNA-seq signatures and provided evidence that they are a non-invasive, affordable cell source to study Duchenne muscular dystrophy (DMD), an X-linked disease, due to dystrophin gene mutations. Based on USCs’ RNA profiling and whole-genome sequencing (WGS), we identified three undetected atypical DMD mutations.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients’ plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

Europe is experiencing a third wave of COVID-19 due to the spread of highly transmissible SARS-CoV-2 variants. A number of positive and negative factors constantly shape the rates of COVID-19 infections, hospitalization, and mortality. Among these factors, the rise in increasingly transmissible variants on one side and the effect of vaccinations on the other side create a picture deeply different from that of the first pandemic wave. Starting from the observation that in several European countries the number of COVID-19 infections in the second and third pandemic wave increased without a proportional rise in disease severity and mortality, we hypothesize the existence of an additional factor influencing SARS-CoV-2 dynamics. This factor consists of an immune defence against severe COVID-19, provided by SARS-CoV-2-specific T cells progressively developing upon natural exposure to low virus doses present in populated environments. As suggested by recent studies, low-dose viral particles entering the respiratory and intestinal tracts may be able to induce T cell memory in the absence of inflammation, potentially resulting in different degrees of immunization. In this scenario, non-pharmaceutical interventions would play a double role, one in the short term by reducing the detrimental spreading of SARS-CoV-2 particles, and one in the long term by allowing the development of a widespread (although heterogeneous and uncontrollable) form of immune protection.

The aim was to assess 3-year longitudinal data using 6MWT in 26 ambulant boys affected by DMD carrying nonsense mutations and to compare their results to other small mutations. We also wished to establish, within the nonsense mutations group, patterns of change according to several variables. Patients with nonsense mutations were categorized according to the stop codon type newly created by the mutation and also including the adjacent 5' (upstream) and 3' (downstream) nucleotides. No significant difference was found between nonsense mutations and other small mutations (p 0.05) on the 6MWT. Within the nonsense mutations group, there was no difference in 6MWT when the patients were subdivided according to: Type of stop codon, frame status of exons involved, protein domain affected. In contrast, there was a difference when the stop codon together with the 3' adjacent nucleotide ("stop+4 model") was considered (p 0.05) with patients with stop codon TGA and 3' adjacent nucleotide G (TGAG) having a more rapid decline. Our finding suggest that the stop+4 model may help in predicting functional changes. This data will be useful at the time of interpreting the long term follow up of patients treated with Ataluren that are becoming increasingly available.

Background Management of immunocompromised COVID-19 patients is the object of current debate. Accumulating evidence suggest that treatment with high-titer COVID-19 convalescent plasma (CCP) may be effective in this characteristic clinical scenario. Case report A 52-years old immunocompromised female patient, previously treated with rituximab for low grade B-cell lymphoma, showed prolonged SARS-CoV-2 shedding and a long-term course of signs of severe COVID-19. A first cycle of treatment with remdesivir, a nucleotide analogue prodrug effective in inhibiting SARS-CoV-2 replication, did not provide fully and sustained clinical remission. A second hospitalization was deemed necessary after 10 days from the first hospital discharge due to recrudescence of symptoms of severe COVID-19 and the evidence of bilateral interstitial pneumonia at the chest-CT scan. Clinical and radiological findings completely disappeared after CCP administration. The viral culture confirmed the absence of SARS-CoV-2-related cytopathic effect. The clinical evaluation, performed two months after hospital discharge, was unremarkable. Results Findings from our case report suggest that the host T-cell specific response to SARS-CoV-2 is not sufficient to reduce viral load in the absence of neutralizing antibodies. Acquired immune antibodies and/or related components passively infused with CCP might help in boosting the plasma recipient response to the virus and promoting complete viral clearance. Conclusions Independently from negative results in immunocompetent individuals, the potential effectiveness of CCP infusion in selected cohorts of patients with primary or secondary impaired immune response should be tested. Further research about mechanisms of host response in immunocompromised patients with SARS-CoV-2 infection is required.

Blooms of the toxic dinoflagellates Ostreopsis have become common along rocky shores of the Mediterranean Sea. In addition to health problems for beach-goers, Ostreopsis toxins may accumulate in benthic marine animals used for human consumption, which however at times have shown signals of stress and even mortality. In order to elucidate the actual relationships between Ostreopsis and benthic invertebrates, we exposed mussels Mytilus galloprovincialis and sea urchins Paracentrotus lividus from the Gulf of Naples to cultures and natural material of O. cf. ovata and assessed feeding and adverse effects on the animals, along with their acquired toxicity. Mussels exposed to O. cf. ovata for 24 hours filtered the microalgae at different rates, depending on both mussel size and microalgal density, and became weakly toxic in some cases. Under longer exposure most animals died and all survivors were toxic. Detoxification of a naturally toxic mussel populations from an area affected by O . cf. ovata blooms took more than two weeks. Sea urchins fed with the red alga Asparagopsis taxiformis epiphytised by O. cf. ovata did not show damages and became mildly toxic in some cases. However, the direct exposure of sea urchins to O. cf. ovata cultures caused the partial or total loss of the spines in a density-dependent way, with the death of the animals at the highest microalgal concentrations. Milder effects were registered with sonicated cultures or toxin extracts. Our results indicate that the balance between toxicity and animal health in these invertebrates depends on the mode and intensity of exposure to the toxic microalga, while the response varies between the two species but also within the same species. This scenario matches the variety of responses of benthic populations recorded in the natural environment in areas affected by O. cf. ovata blooms.

Severe coronavirus disease 2019 (COVID-19) causes an uncontrolled activation of the innate immune response, resulting in acute respiratory distress syndrome and systemic inflammation. The effects of COVID-19–induced inflammation on cancer cells and their microenvironment are yet to be elucidated. Here, we formulate the hypothesis that COVID-19–associated inflammation may generate a microenvironment favorable to tumor cell proliferation and particularly to the reawakening of dormant cancer cells (DCCs). DCCs often survive treatment of primary tumors and populate premetastatic niches in the lungs and other organs, retaining the potential for metastatic outgrowth. DCCs reawakening may be promoted by several events associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, including activation of neutrophils and monocytes/macrophages, lymphopenia and an uncontrolled production of pro-inflammatory cytokines. Among pro-inflammatory factors produced during COVID-19, neutrophil extracellular traps (NETs) released by activated neutrophils have been specifically shown to activate premetastatic cancer cells disseminated in the lungs, suggesting they may be involved in DCCs reawakening in COVID-19 patients. If confirmed by further studies, the links between COVID-19, DCCs reactivation and tumor relapse may support the use of specific anti-inflammatory and anti-metastatic therapies in patients with COVID-19 and an active or previous cancer.

Background: Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment. Methods and findings: We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in the TNFRSF10A gene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N = 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results. Conclusion: We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker. The EU BIO-NMD project no. 241665 and the SOLVE-RD project (H2020 ID: 779257) were supported this study. FM was supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. The support of the Muscular Dystrophy UK to the Dubowitz Neuromuscular Centre and of the MRC Neuromuscular Centre and BRC Biobank is also gratefully acknowledged. Part of this study was also supported by the NIHR grant “Rare Diseases Translational Research Collaboration” on Duchenne muscular dystrophy to FM. HL receives support from the Canadian Institutes of Health Research (Foundation Grant FDN-167281), the Canadian Institutes of Health Research and Muscular Dystrophy Canada (Network Catalyst Grant for NMD4C), the Canada Foundation for Innovation (CFI-JELF 38412), and the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, 950-232279). The EUROBIOBANK, European Network of DNA, Cell and Tissue Banks for Rare Diseases was also acknowledged.

ObjectiveGenetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries.MethodsWe performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers.ResultsWe identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations.ConclusionsOur data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

We report here the first families carrying recessive variants in the MSTO1 gene: compound heterozygous mutations were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia. Human MSTO1 is a poorly studied protein, suggested to have mitochondrial localization and to regulate morphology and distribution of mitochondria. As for other mutations affecting genes involved in mitochondrial dynamics, no biochemical defects typical of mitochondrial disorders were reported. Studies in patients’ fibroblasts revealed that MSTO1 protein levels were strongly reduced, the mitochondrial network was fragmented, and the fusion events among mitochondria were decreased, confirming the deleterious effect of the identified variants and the role of MSTO1 in modulating mitochondrial dynamics. We also found that MSTO1 is mainly a cytosolic protein. These findings indicate recessive mutations in MSTO1 as a new cause for inherited neuromuscular disorders with multisystem features.

Bethlem myopathy represents the milder form of the spectrum of Collagen VI-related dystrophies, which are characterized by a clinical continuum between the two extremities, the Bethlem myopathy and the Ullrich congenital muscular dystrophy, and include less defined intermediate phenotypes. Bethlem myopathy is mainly an autosomal dominant disorder and the causing mutations occur in the COL6A genes encoding for the α1 (COL6A1), α2 (COL6A2) and α3 (COL6A3) chains. However, few cases of recessive inheritance have been also reported. We here describe clinical, genetic and functional findings in a recessive Bethlem myopathy family harbouring two novel pathogenic mutations in the COL6A2 gene. Two adult siblings presented with muscle weakness and wasting, elbows and Achilles tendon retractions, lumbar hyperlordosis, waddling gait and positive Gowers' sign. Muscle biopsy showed a dystrophic pattern. Molecular analysis of the COL6A2 gene revealed the novel paternally-inherited nonsense p.Gln889* mutation and the maternally-inherited p.Pro260_Lys261insProPro small insertion. Fibroblast studies in both affected patients showed the concomitant reduction in the amount of normal Collagen VI (p.Gln889*) and impairment of Collagen VI secretion and assembly (p.Pro260_Lys261insProPro). Each of the two variants behave as a recessive mutation as shown by the asymptomatic heterozygous parents, while their concomitant effects determined a relatively mild Bethlem myopathy phenotype. This study confirms the occurrence of recessive inherited Bethlem myopathy and expands the genetic heterogeneity of this group of muscle diseases.

Duchenne muscular dystrophy (DMD) is the most common childhood muscular dystrophy affecting ~1:5000 live male births. Following the identification of pathogenic variations in the dystrophin gene in 1986, the underlining genotype/phenotype correlations emerged and the role of the dystrophin protein was elucidated in skeletal, smooth, and cardiac muscles, as well as in the brain. When the dystrophin protein is absent or quantitatively or qualitatively modified, the muscle cannot sustain the stress of repeated contractions. Dystrophin acts as a bridging and anchoring protein between the sarcomere and the sarcolemma, and its absence or reduction leads to severe muscle damage that eventually cannot be repaired, with its ultimate substitution by connective tissue and fat. The advances of an understanding of the molecular pathways affected in DMD have led to the development of many therapeutic strategies that tackle different aspects of disease etiopathogenesis, which have recently led to the first successful approved orphan drugs for this condition. The therapeutic advances in this field have progressed exponentially, with second-generation drugs now entering in clinical trials as gene therapy, potentially providing a further effective approach to the condition.

Azaspiracids (AZAs) are marine biotoxins including a variety of analogues. Recently, novel AZAs produced by the Mediterranean dinoflagellate Azadinium dexteroporum were discovered (AZA-54, AZA-55, 3-epi-AZA-7, AZA-56, AZA-57 and AZA-58) and their biological effects have not been investigated yet. This study aimed to identify the biological responses (biomarkers) induced in mussels Mytilus galloprovincialis after the bioaccumulation of AZAs from A. dexteroporum. Organisms were fed with A. dexteroporum for 21 days and subsequently subjected to a recovery period (normal diet) of 21 days. Exposed organisms accumulated AZA-54, 3-epi-AZA-7 and AZA-55, predominantly in the digestive gland. Mussels&rsquo, haemocytes showed inhibition of phagocytosis activity, modulation of the composition of haemocytic subpopulation and damage to lysosomal membranes, the digestive tissue displayed thinned tubule walls, consumption of storage lipids and accumulation of lipofuscin. Slight genotoxic damage was also observed. No clear occurrence of oxidative stress and alteration of nervous activity was detected in AZA-accumulating mussels. Most of the altered parameters returned to control levels after the recovery phase. The toxic effects detected in M. galloprovincialis demonstrate a clear biological impact of the AZAs produced by A. dexteroporum, and could be used as early indicators of contamination associated with the ingestion of seafood.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015&ndash, 2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV, 8.9%), norovirus GI (NoVGI, 10.8%) and GII (NoVGII, 39.7%), rotavirus (RV, 9.0%), astrovirus (AsV, 20.8%), sapovirus (SaV, 18.8%), aichivirus-1 (AiV-1, 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Algal biotoxins, chemical compounds produced by some microscopic algae, constitute the phytoplankton. The mussels, feeding on phytoplankton, can accumulate these compounds to become themselves toxic. There have been several cases of food poisoning by consumption of contaminated shellfish. Such food poisoning have pushed our health care system to provide monitoring of shellfish in the framework of the monitoring plans carried out by AASSLL. In this paper we report the results obtained monitoring the presence of ASP (Amnesic Shellfish Poisoning) biotoxins, like domoic acid (DA) and its isomers, produced by Pseudonitzschia algae. The analysis were carried out by using both the HPLC-UV official method and an experimental method performed with a Time of Flight mass spectrometer (ESI-TOF). The 100% of samples analysed by the official method have always been below the limits of sensitivity (except one sample), the 65% of samples analysed by ESI-TOF, showed the presence of domoic acid.

A ready source of autologous myogenic cells is of vital importance for drug screening and functional genetic studies in Duchenne muscular dystrophy (DMD), a rare disease caused by a variety of dystrophin gene mutations. As stem cells (SCs) can be easily and noninvasively obtained from urine specimens, we set out to determine whether they could be myogenically induced and useful in DMD research. To this end, we isolated stem cells from the urine of two healthy donors and from one patient with DMD, and performed surface marker characterization, myogenic differentiation (MyoD), and then transfection with antisense oligoribonucleotides to test for exon skipping and protein restoration. We demonstrated that native urine-derived stem cells express the full-length dystrophin transcript, and that the dystrophin mutation was retained in the cells of the patient with DMD, although the dystrophin protein was detected solely in control cells after myogenic transformation according to the phenotype. Notably, we also showed that treatment with antisense oligoribonucleotide against dystrophin exon 44 induced skipping in both native and MyoD-transformed urine-derived stem cells in DMD, with a therapeutic transcript-reframing effect, as well as visible protein restoration in the latter. Hence MyoD-transformed cells may be a good myogenic model for studying dystrophin gene expression, and native urine stem cells could be used to study the dystrophin transcript, and both diagnostic procedures and splicing modulation therapies in both patients and control subjects, without invasive and costly collection methods. New, bankable bioproducts from urine stem cells, useful for prescreening studies and therapeutic applications alike, are also foreseeable after further, more in-depth characterization.

Evidence for the expression of the canonic androgen receptor (AR) in human adrenal cortex has not been provided so far. The aim of the present study was to demonstrate the expression of the AR gene in normal and neoplastic adrenocortical human tissues and in the human adrenocortical cancer cell line, NCI-H295, and then to evaluate the effect of dihydrotestosterone (DHT) on human adrenocortical cell growth. An AR cDNA fragment with the expected size of 262 bp was detected by using reverse transcription (RT)-PCR in normal and neoplastic adrenocortical human tissues and in the neoplastic cell line, demonstrating that the gene for AR is indeed expressed in human adrenal cells. In the human adrenocortical cancer cell line NCI-H295, DHT at physiological concentrations produced a significant reduction in cell proliferation and inhibition of colony formation in soft agar. The inhibitory effect on adrenocortical cell growth was evident after both 24 and 48 h of treatment. The antiandrogens, cyproterone acetate and hydroxyflutamide, were capable of reversing the effects exerted by DHT. The androgen-induced growth inhibitory effect was also detected in primary culture of three non-functioning adrenocortical adenomas. These findings show that the canonic AR is present in human adrenocortical cells and that androgens may have a role in the adrenal cortex by reducing cell proliferation.

Germline mutations of aryl-hydrocarbon-receptor interacting protein (AIP) are associated with pituitary adenoma predisposition. They occur in 20 % of familial isolated pituitary adenoma (FIPA) and in about 3-5 % of sporadic pituitary adenomas, especially in early onset somatotropinomas and prolactinomas. Our aim was to evaluate the clinical and genetic features of a large Italian FIPA family, where an AIP variant was identified. AIP direct sequencing from genomic DNA was carried out in 16 available family members. AIP R16H carriers also underwent magnetic resonance imaging and hormonal assessments. AIP mutations were also searched in 16 patients with sporadic growth hormone-secreting pituitary adenoma and in 6 unrelated patients in whom pituitary adenoma was excluded. We found an AIP R16H variation in two family members harbouring a pituitary adenoma and in 6 unaffected family members. No AIP mutation was found neither in growth hormone-secreting pituitary adenoma patients, nor in the unrelated patients without pituitary adenoma. We report a FIPA family harbouring an AIP R16H change, supporting the hypothesis that the latter represents a variant of unknown significance.

Androgen-binding activity has been identified in normal and pathological thyroids, but evidence for the expression of the canonic androgen receptor (AR) in the thyroid has not been provided so far. In this study we have used reverse transcription (RT)-PCR to examine RNA expression of the canonic AR gene in human thyroid tissues, in primary cultures of human thyrocytes and in a variety of neoplastic thyroid cell lines (NPA, TPC and WRO). An AR cDNA fragment with the expected size of 262 bp was detected in normal tissues and cultured thyrocytes as well as in neoplastic cell lines, demonstrating that the gene for AR is indeed expressed in thyroid follicular cells. Immunocytochemical analysis revealed the presence of the AR protein in cancer cell lines and androgen treatment increased nuclear positivity to AR. In a survey of 35 thyroid tissues AR cDNA was detected in all the non-neoplastic samples (6 normal and 3 goitrous) and in 19 of 26 neoplastic samples. AR cDNA was not detected in 4 of the 9 follicular adenomas and in 3 of the 12 papillary carcinomas. AR was revealed by immunohistochemistry in 1 of 2 normal thyroids, in 1 goiter and in 1 of 2 neoplastic thyroids. These findings show the presence of the canonic AR in the human thyroid. Journal of Endocrinology (1996) 148, 77–85

degli Uberti EC, Ambrosio MR, Bondanelli M, Trasforini G, Valentini A, Rossi R, Margutti A, Campo M. Effect of human galanin on the response of circulating catecholamines to hypoglycemia in man. Eur J Endocrinol 1995;133:723–8. ISSN 0804–4643 Human galanin (hGAL) is a neuropeptide with 30 amino acid residues that has been found in the peripheral and central nervous system, where it often co-exists with catecholamines. In order to clarify the possible role of hGAL in the regulation of sympathoadrenomedullary function, the effect of a 60 min infusion of hGAL (80 pmol·kg−1 · min−1) on plasma epinephrine and norepinephrine responses to insulin-induced hypoglycemia in nine healthy subjects was investigated. Human GAL administration significantly reduced both the release of basal norepinephrine and the response to insulin-induced hypoglycemia, whereas it attenuated the epinephrine response by 26%, with the hGAL-induced decrease in epinephrine release failing to achieve statistical significance. Human GAL significantly increased the heart rate in resting conditions and clearly exaggerated the heart rate response to insulin-induced hypoglycemia, whereas it had no effect on the blood pressure. We conclude that GAL receptor stimulation exerts an inhibitory effect on basal and insulin-induced hypoglycemia-stimulated release of norepinephrine. These findings provide further evidence that GAL may modulate sympathetic nerve activity in man but that it does not play an important role in the regulation of adrenal medullary function. Ettore C degli Uberti, Chair of Endocrinology, University of Ferrara, Via Savonarola 9, I-44100 Ferrara, Italy

In order to clarify the effect of retinoids on thyroid cell growth and function, the presence of retinoic acid receptors (RARs) and the action of retinoic acid (RA) on DNA synthesis and on thyroid peroxidase (TPO) and thyroglobulin (TGB) mRNA expression were investigated in primary cultures of human thyroid follicular cells. A time and dose-dependent reduction in 3H-thymidine (3H-thy) incorporation was found in cells exposed for 48 h to all-trans-RA up to 1 microM. A cytotoxic effect was found only with the higher dose of 50 microM. The RA-induced decrease of 3H-thy incorporation was reflected by parallel change in DNA content of cell monolayers. The inhibitory effect of 1 microM RA on 3H-thy incorporation ranged from 28.5 +/- 4.6% in normal cells to 42 +/- 3.2% in adenomatous cells. In addition, 1 microM RA significantly reduced basal and TSH-induced TPO mRNA levels in normal, goitrous and adenomatous cells, but did not alter TGB mRNA levels. Furthermore, in these cells the study of RAR alpha and beta mRNA showed the presence of two major RAR alpha mRNA transcripts of approximately 3.5 and 2.8 Kb in size, whereas RAR beta mRNA was undetectable. Overall, our data indicate that RAR alpha gene is expressed in human thyrocytes and that RA may be involved in the regulation of the human thyroid by reducing proliferation and function of follicular cells.

Calcitonin gene-related peptide (CGRP) has positive chronotropic and inotropic effects in animals and humans, and produces the most potent vasodilation known for an endogenous peptide. Yet, a physiological role for CGRP in the regulation of vascular tone and blood pressure has not been demonstrated. We studied the effects of 1) assumption of the upright position and 2) iv infusion of angiotensin-II (sequential doses of 8, 16, and 32 ng/kg.min, each dose for 20 min) in eight normal subjects (four men). Serial venous blood samples were taken to determine the plasma CGRP, epinephrine, norepinephrine, and aldosterone levels and PRA. Blood pressure and heart rate were continuously monitored at the finger with a Finapres 2300 instrument. After assumption of the upright posture, a quick rise in plasma CGRP levels was observed together with the expected increases in plasma norepinephrine and aldosterone and PRA. A transient increment was also observed for diastolic blood pressure and heart rate. Angiotensin-II infusion caused dose-dependent increases in plasma CGRP and aldosterone concentrations, already significant at the lowest infusion rate and parallel with the blood pressure rise. Plasma catecholamines significantly increased only at higher infusion rates. Our data demonstrate that modifications of plasma CGRP concentrations are part of the normal response to postural and vasomotor changes. These findings suggest a physiological role for CGRP in regulation of the peripheral vascular tone and possibly blood pressure in man.

To assess the existence of an altered circulating pattern of calcitonin gene-related peptide (CGRP) in hypertension.The 24 h variation in plasma CGRP was measured and compared in 10 patients affected by uncomplicated essential hypertension and in nine age- and sex-matched healthy volunteers. The diurnal variations in blood pressure, atrial natriuretic peptide (ANP), plasma renin activity (PRA), plasma aldosterone and plasma cortisol were also assessed.Recumbency studies were performed under standardized, drug-free conditions beginning at 0800 h. Venous samples were drawn every 4 h for 24 h and hormone levels were assessed with specific radioimmunoassays. The blood pressure was measured every 15 min with a SpaceLabs 90207 monitor.The mean 24-h plasma CGRP concentrations were significantly lower in the hypertensive group than in the control group. In both groups a circadian rhythm was present with the same pattern, but at a lower level in hypertension. A temporal sequence starting with the nocturnal rise in plasma CGRP concentrations and progressing with the elevations of ANP, PRA, and plasma aldosterone and cortisol was apparent in both groups. The nocturnal rise in the CGRP and ANP concentrations coincided with the blood pressure and the heart rate falls.Our data show that CGRP is lower than normal but maintains its circadian variability and its relationship with the diurnal variations in blood pressure and other hormones known to be active on the cardiovascular system.

To determine the role of delta-opioid receptors in the modulation of hypothalamic-pituitary-adrenal activity, we studied in normal subjects the effect of the highly selective delta-opioid receptor agonist deltorphin (DT) on the secretion of ACTH, cortisol, and arginine vasopressin in response to insulin-induced hypoglycemia. In an attempt to clarify the site of opiate modulation of ACTH secretion, we also studied in normal subjects the effect of DT on the ACTH response to ovine CRH-41. DT blunted the ACTH, cortisol, and arginine vasopressin responses to insulin-induced hypoglycemia, whereas it had no effect on the ACTH and cortisol responses to CRH. We conclude that DT-induced activation of delta-opioid receptors exerts an inhibitory influence on hypoglycemia-stimulated ACTH secretion. Based on the lack of an effect of DT on the ACTH response to CRH, we postulate that DT may modulate the secretion of ACTH through suprapituitary mechanisms.

The action of somatostatin (SRIH) on 3H-thymidine (thy) incorporation and on c-myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10(-7) M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH-stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent.

The intravenous (IV) infusion of angiotensin II (AII) was administered to seven healthy male volunteers in a randomized placebo-controlled study. As expected, All induced a significant increase in blood pressure and plasma aldosterone concentrations. All caused a significant increase in corticotropin (ACTH) and growth hormone (GH) release, but had no effect on the release of thyrotropin (TSH) and prolactin (PRL). These findings suggest that peripherally circulating All might influence ACTH and GH secretion in humans.

Sex steroid-binding activities have been identified by several authors in normal and pathological thyroids and the expression of the canonic androgen receptor (AR) has recently been demonstrated in human thyroid follicular cells. In order to assess what influence, if any, androgen exposure has on thyroid cell growth, the effect of dihydrotestosterone (DHT) on [3H]thymidine (thy) incorporation and cell proliferation was investigated in thyroid follicular cells in vitro. In a primary culture of goitrous cells, DHT induced a significant reduction of [3H]thy incorporation at concentrations ranging from 10−12 to 10−8 m, with a more pronounced effect at 10−9 m. At this concentration, the inhibitory effect was evident after both 24 and 48 h of treatment and in various types of primary thyroid cell cultures. In goitrous cells, the DHT-induced decrease of [3H]thy was associated with a reduction of expression of the proliferation-associated nuclear Ki-67 antigen, a protein commonly used to assess cell growth fraction. In TPC cells, an AR-positive thyroid papillary carcinoma cell line, DHT at concentrations between 10−12 and 10−8 m significantly decreased the growth rate. DHT (10−9 m) produced an approximately 50–60% inhibition of cell proliferation and the antiandrogen cyproterone acetate was capable of reversing such effects. The DHT-induced reduction of TPC cell proliferation was associated with a significant reduction of c-myc RNA levels. Thyroperoxidase mRNA levels and thyroglobulin production were not reduced by androgen in primary cultures of goitrous cells. In conclusion, our results indicated that androgens may have a role in this gland by reducing the proliferation, but not the function, of follicular cells. Journal of Endocrinology (1996) 151, 185–194

The neuropeptide galanin (GAL) is widely distributed in the peripheral and central nervous systems, where it often coexists with catecholamines. To gain insight into the action of human GAL on sympathetic nervous system activity in man, we investigated the effects of a 60-min infusion of human (h) GAL (80 pmol/kg.min) or saline on peripheral norepinephrine (NE) and epinephrine concentrations, heart rate (HR), and systolic and diastolic blood pressure (BP) in the supine position as well as after assumption of the upright posture (UP) in eight healthy male volunteers. hGAL depressed supine plasma NE (0.84 +/- 0.06 vs. 0.33 +/- 0.02 nmol/L) and blunted the NE response to assumption of the UP (1.68 +/- 0.03 vs. 0.44 +/- 0.03 nmol/L), but caused a significant enhancement of the epinephrine response to assumption of the UP (0.22 +/- 0.02 vs. 0.65 +/- 0.06 nmol/L). hGAL significantly increased supine HR (70 +/- 2 vs. 99 +/- 4 beats/min) and potentiated the HR response to assumption of the UP (82 +/- 3 vs. 107 +/- 4 beats/min). hGAL did not alter supine systolic and diastolic BP, but caused a significant decrease in the systolic (121 +/- 3 vs. 98 +/- 2 mm Hg) and diastolic (74 +/- 2 vs. 62 +/- 2 mm Hg) BP responses to assumption of the UP. Our data show that hGAL decreases supine position- and UP-stimulated release of NE, suggesting an inhibitory modulation of hGAL on sympathetic outflow in man. The finding that hGAL induces an increase in HR, both in the supine position and after UP, and an inhibition of the systolic and diastolic BP response to UP provides further support for an involvement of hGAL in regulation of the cardiovascular and autonomic nervous systems in man.