Andrés Beiras

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

SummaryThe presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissners corpuscles.

Substantially altered expression pattern of cannabinoid receptor 2 and activated endocannabinoid system in patients with severe heart failure.

Animal studies suggest that the endocannabinoid system (ECS) plays a role in the regulation of myocardial contractility and in the pathogenesis of heart failure. The current study aimed to proof the existence of endocannabinoid receptors on human ventricular myocardium and to determine whether human chronic heart failure (CHF) is associated with changes in endocannabinoid receptor expression and distribution. Expression of cannabinoid receptor 1 (CB1) and cannabinoid receptor (CB2) on human heart was assessed by means of real-time PCR and immunohistochemistry. On healthy human left ventricular myocardium, mRNA transcripts of CB1 and CB2 receptors were expressed in an almost equal proportion. In patients with CHF, mRNA expression of CB1 receptors was shown to be downregulated 0.7-fold (0.7.+/-0.15, n=12, p<0.01), whereas expression of CB2 receptors was upregulated more than 11-fold (11.6+/-4.5; n=12; p<0.005). Corresponding results were obtained by immunohistochemistry. Blood levels of endocannabinoids were significantly elevated (anandamide 3.5-fold (p<0.001); 2-AG 7-fold (p=0.02)) in patients with CHF, as compared to healthy volunteers. Both CB1 and CB2 receptors are present on healthy human left ventricular myocardium in a balanced distribution. Patients suffering from CHF exhibit a shift of the CB1-CB2 receptor ratio towards expression of CB2 receptors combined with significantly elevated peripheral blood levels of endocannabinoids indicating an activation of the ECS. These results might open up new perspectives regarding the role of endocannabinoid signalling in CHF and its potential as a target for pharmacological modulation.

Cellular localization of orexin receptors in human adrenal gland, adrenocortical adenomas and pheochromocytomas

Orexin-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and related with the regulation of the energy balance and arousal. They act on G protein receptors named orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). In the present study, we used immunohistochemical techniques to detect the distribution of OXR in normal human adrenal gland and adrenal tumours (adrenocortical adenomas and pheochromocytomas). OX1R was expressed in the cortex of the normal human adrenal gland (glomerulosa, fasciculata and reticular zones) and OX2R was located in the medulla (epinephrine and norepinephrine cells). By the double immunofluorescence techniques, we demonstrated that virtually all medullar cells (epinephrine and norepinephrine cells) expressed OX2R. As was expected, according to the results obtained in normal tissues, cortical tumours (adrenocortical adenomas) were positive for OX1R but not for OX2R and conversely, medullar tumours (pheochromocytomas) expressed only OX2R.

Ghrelin localization in rat and human thyroid and parathyroid glands and tumours

Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is a 28 amino acid peptide originally isolated from rat stomach that has been primarily involved in the central neuroregulation of GH secretion and food intake. Previous studies demonstrated that ghrelin has a widespread expression in different normal cells and tissues, as well as in gastric, thyroid, testicular, breast and lung neoplasias. In the current study, we use molecular biology to detect ghrelin transcripts expression in rats, and immunohistochemical techniques to investigate the cellular distribution of this peptide in rat and human thyroid and parathyroid glands and tumours. Ghrelin was localized in thyroid C cells and in parathyroid cells. Thyroid carcinomas (medullar, follicular and papillary) and parathyroid adenomas also showed intense and diffuse immunostaining for ghrelin. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine fashion in the regulation of thyroid follicular cell function. The diffuse ghrelin immunostaining found in the parathyroid gland opens up the possibility of its secretion to the bloodstream or its involvement in the regulation of the parathyroid function. Overall, expression of ghrelin in human and rat thyroid and parathyroid glands is highly suggestive of a conserved role of this molecule in the regulation of thyroid and parathyroid cell function.

Increased expression of growth hormone and prolactin receptors in hepatocellular carcinomas

The liver is an essential target tissue for growth hormone (GH) and prolactin (PRL). The aim of this study was to determine the in situ expression of growth hormone receptor (GHR) and prolactin receptor (PRLR) in hepatocellular carcinomas and to compare the results with normal liver. For this purpose, in situ hybridization (ISH) and immunohistochemical techniques were performed and several tests were conducted to validate the results. By radioactive ISH, all the hepatocellular carcinomas studied showed labeling for GHR and PRLR mRNAs. Relative expression levels, determined by computer-assisted microdensity, were higher in hepatocellular carcinomas than in normal liver. Immunohistochemistry led us to confirm the constant expression of both receptor proteins in hepatocellular carcinomas and normal liver and to demonstrate their localization not only in the cytoplasm but also in the nucleus. These results confirm that the liver is a major GH and PRL target tissue and suggest that in hepatocellular carcinomas the proliferative effects of these hormones may be increased by a higher expression of their receptors.

Altered myocardial expression of ghrelin and its receptor (GHSR-1a) in patients with severe heart failure

Ghrelin is a peptide hormone mainly produced by the stomach, which strongly stimulates the release of growth hormone (GH) via the GH secretagogue receptor 1a (GHSR-1a) located in the hypothalamus. It has been reported to exert performance-enhancing effects on myocardial function, and as both ghrelin and GHSR-1a are expressed in myocardial tissues, the ghrelin system may have a direct GH-independent impact on cardiac function. We intended to investigate the expression of ghrelin and its receptor GHSR-1a in different myocardial areas of patients with chronic heart failure (CHF) as compared to heart-healthy subjects to better define the role of the ghrelin signaling system in the networks regulating cardiac function and its potential as a target for diagnosis and/or treatment of CHF. Myocardium biopsies of 12 patients undergoing heart transplantation and suffering from CHF were obtained. Expression of both ghrelin and GHSR-1a was assessed by means of immunohistochemistry and real-time PCR. Expression of ghrelin was significantly decreased in CHF hearts both in atrium and ventricles in comparison to the control hearts (p<0.05). The expression of the GHS-1a receptor was significantly increased in the CHF biopsies as compared to controls (p<0.05). No significant differences were found between the anatomical areas studied. Expression of myocardial ghrelin and GHSR-1a is directly associated with myocardial function: CHF hearts exhibit an impaired ghrelin production which might reflect maladaptive processes and an - probably compensatory - increase in GHSR-1a expression. These findings may open up new perspectives regarding the potential of ghrelin signaling as a target for pharmacological modulation.

Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy

Background: Type 2 familial partial lipodystrophy (FPLD2) is characterised by loss of fat in the limbs and buttocks and results from mutations in the LMNA gene. Aim: To evaluate the role of several genes involved in adipogenesis in order to better understand the underlying mechanisms of regional loss of subcutaneous adipose tissue (scAT) in patients with FPLD2. Methods: In total, 7 patients with FPLD2 and 10 healthy control participants were studied. A minimal model was used to calculate the insulin sensitivity (IS). scAT was obtained from abdomen and thigh by biopsy. Relative gene expression was quantified by real-time reverse transcription PCR in a thermal cycler. Prelamin A western blot analysis was carried out on scAT and prelamin A nuclear localisation was determined using immunofluorescence. Adipocyte nuclei were examined by electron microscopy. Results: Patients with FPLD2 were found to have significantly lower IS. The expression of LMNA was similar in both groups. The expression of PPARG2, RB1, CCND3 and LPL in thigh but not in abdomen scAT was significantly reduced (67%, 25%, 38% and 66% respectively) in patients with FPLD2. Significantly higher levels of prelamin A were found in peripheral scAT of patients with FPLD2. Defects in the peripheral heterochromatin and a nuclear fibrous dense lamina were present in the adipocytes of patients with FPLD2. Conclusions: In FPLD2 participants, prelamin A accumulation in peripheral scAT is associated with a reduced expression of several genes involved in adipogenesis, which could perturb the balance between proliferation and differentiation in adipocytes, leading to less efficient tissue regeneration.

Neural cell adhesion molecule immunoreactivity in Merkel cells and Merkel cell tumours

We have analysed the expression of the neural cell adhesion molecule (NCAM) in normal Merkel cells of pig and human skin, and in nine neuroendocrine carcinomas of the skin (Merkel cell carcinomas). NCAM immunoreactivity was observed in virtually all Merkel cells, both in epidermis and vibrissae of pig snout skin and in human epidermis. Immunostaining surrounded the entire surface of Merkel cells and was not restricted to the contact areas between Merkel cells and nerve terminals. All Merkel cell carcinomas studied were also positive for NCAM. The immunostaining pattern of the tumour cells was similar to that observed in normal Merkel cells; the immunoreactivity was confined to the cell membranes. These results suggest that NCAM may be used as an immunohistochemical marker for both Merkel cells and Merkel cell tumours.

Expression of growth hormone receptor in benign and malignant cutaneous proliferative entities

The skin has the necessary elements to respond to growth hormone (GH) and suffers clinical changes in the pathological circumstances of excess and deficiency of GH. The GH has been involved in the development of different types of human neoplasms. Based on these data, we have studied the GH receptor (GHR) expression in acrochordons, seborrheic keratosis, melanocytic nevi, histiocytomas, squamous cell carcinomas, basal cell carcinomas, and malignant melanomas by means of the immunohistochemistry with the monoclonal antibody MAb 263. All the entities showed immunoreactivity for GHR. In the histiocytomas, the expression of GHR in the keratinocytes of the hyperplastic epidermis coating the lesion showed a strong nuclear pattern, but the non‐hyperplastic epidermis of the edges of the histiocytomas expressed GHR with a cytoplasmic pattern. In the basal cell carcinoma and squamous cell carcinoma, the immunoreactivity was weaker than in normal skin. In the squamous cell carcinoma, the intensity of immunostaining correlated directly with the grade of cellular differentation. In conclusion, the GH may be involved in the development of different kinds of cutaneous neoplasms, and the intracellular localization of GHR may imply a functional significance, at least in the histiocytomas.

A new seipin-associated neurodegenerative syndrome

Background Seipin/BSCL2 mutations can cause type 2 congenital generalised lipodystrophy (BSCL) or dominant motor neurone diseases. Type 2 BSCL is frequently associated with some degree of intellectual impairment, but not to fatal neurodegeneration. In order to unveil the aetiology and pathogenetic mechanisms of a new neurodegenerative syndrome associated with a novel BSCL2 mutation, six children, four of them showing the BSCL features, were studied. Methods Mutational and splicing analyses of BSCL2 were performed. The brain of two of these children was examined postmortem. Relative expression of BSCL2 transcripts was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in different tissues of the index case and controls. Overexpressed mutated seipin in HeLa cells was analysed by immunofluorescence and western blotting. Results Two patients carried a novel homozygous c.985C>T mutation, which appeared in the other four patients in compound heterozygosity. Splicing analysis showed that the c.985C>T mutation causes an aberrant splicing site leading to skipping of exon 7. Expression of exon 7-skipping transcripts was very high with respect to that of the non-skipped transcripts in all the analysed tissues of the index case. Neuropathological studies showed severe neurone loss, astrogliosis and intranuclear ubiquitin(+) aggregates in neurones from multiple cortical regions and in the caudate nucleus. Conclusions Our results suggest that exon 7 skipping in the BSCL2 gene due to the c.985C>T mutation is responsible for a novel early onset, fatal neurodegenerative syndrome involving cerebral cortex and basal ganglia.