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The Human Variome Project (HVP) is an international effort aiming systematically to collect and share information on all human genetic variants. It has been working for years in collaboration with local scientific societies by establishing systems to collect every genetic variant reported in a country and to store these variants within a database repository: LOVD (Argentinian chapter: ar.lovd.org). Formally established in 2017 in the Argentinian Node, up to June 2019 we collected more than 25,000 genetic variants deposited by 17 different laboratories. Nowadays the HVP country nodes represent more than 30 countries. In Latin America there are four country nodes: Argentina, Brazil, Mexico and Venezuela; the first two interacted recently launching the LatinGen database. In the present work we want to share our experience in applying the HVP project focusing on its organization, rules and nomenclature to reach the goal of sharing genetic variants and depositing them in the Leiden Open Variation Database. Contributing laboratories are seeking to share variant data to gain access all over the country. It is one of our goals to stimulate the highest quality by organizing courses, applying current nomenclature rules, sponsoring lectures in national congresses, distributing newsletter to serve the Argentinian genomics community and to stimulate the interaction among Latin America countries. Key words: Data sharing, Argentinian chapter of LOVD, Human Variome Project (HVP)-Argentina

Giannini E, Risso D, Botta F, Romagnoli P, Malfatti F, Fumagalli A, Testa E, Podestà E, Chiarbonello B, Polegato S, Testa R (University of Genoa, Genoa, Italy). Prognosis of hepatocellular carcinoma in anti-HCV positive cirrhotic patients: a single-centre comparison amongst four different staging systems. J Intern Med 2004; 255; 399–408. Epidemiological studies have foreseen an increase in the incidence of hepatocellular carcinoma (HCC) in the near future and it is estimated that this trend will mostly affect hepatitis C virus (HCV) positive cirrhotic patients. Therefore, accuracy of HCC staging is an important clinical issue. To investigate the prognostic usefulness of a series of newly proposed HCC prognostic systems such as the Cancer of the Liver Italian Program (CLIP) score, the Groupe d'Etude et de Traitement du Carcinome Hépatocellulaire (GRETCH) model and the Barcelona Clinic Liver Cancer (BCLC) staging classification when compared with the usefulness of a known staging system such as the Okuda staging system in a group of anti-HCV positive cirrhotic patients with HCC seen at a single centre. Okuda stage, CLIP score, GRETCH model and BCLC stages were retrospectively computed in 81 anti-HCV positive cirrhotic patients with HCC. We evaluated and compared the ability of these methods to assess survival prognosis. As of December 2001, 51 patients had died and overall median survival was 18 months. All the staging systems were able to identify various patient subgroups with different survival. The CLIP score, the GRETCH model and the BCLC staging classification were better at characterizing the 1-year prognosis of the patients when compared with the Okuda staging system, whilst the 3-year prognostic evaluation was improved only by using the CLIP score or the BCLC staging classification. The prognostic value and usefulness of the CLIP score, the GRETCH model and the BCLC staging classification was reproduced in a single-centre analysis of anti-HCV positive HCC cirrhotic patients. These scores provided a prognostic assessment of our patients which is superior to what was obtained by the Okuda staging system. [ABSTRACT FROM AUTHOR]

The advent of biological therapies represented the beginning of a new era in the therapy of Rheumatoid Arthritis (RA), as demonstrated in several studies, but still many questions about their safety, especially in long term use, and correct administration time remain unanswered. Once remission is achieved, the orientation of clinicians regarding the maintenance of biological therapy or the switch to another immunosuppressive therapy is still uncertain. In our previous study 21 patients affected by RA who reached remission by the use of a combined therapy of anti-TNF drugs and methotrexate (MTX) underwent CyA-MTX combination therapy for maintaining remission state and were evaluated during a 6-month follow-up. The present study aims to investigate these data by a longer follow-up (12 months) and on a larger population. Fifty-three RA patients, with a disease duration of less than 3 years and DAS28<3.2 that reached a level of low disease activity within 6-8 months from the beginning of anti-TNF and methotrexate therapy, were enrolled in the study. By the suspension of anti-TNF therapy, patients underwent A-Cyclosporine (2-3 mg/kg/day) and methotrexate (15mg/week) therapy. DAS28, Pain VAS, Erythrosedimentation rate (ESR), C Reactive Protein (CRP) were all tested at time 0 and every 2 months after the interruption of the anti-TNF therapy and the beginning of A-Cyclosporine and methotrexate therapy, as well as liver and kidney profiles. Side effects were also recorded. Of 53 patients, 50 completed the study with a 12-month follow-up. Twenty-one (42%) patients maintained clinical parameters within low disease activity values at 12 months, while 29 (58%) patients showed an increase in DAS28 and other parameters: 16 (32%) patients at the 6-month control, 13 (26%) patients at the 12-month control. Our data show that 42% of the patients undergoing A-Cyclosporin and Methotrexate therapy maintained low disease activity parameters of rheumatoid arthritis, obtained after 6-8 months of anti-TNF therapy. Further studies on larger populations are necessary in order to confirm such results and identify predictor factors for different responses.

Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.

Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.

Vacuolar apical compartment (VAC) is a transient organelle originally observed in Madin-Darby canine kidney (MDCK) epithelial cells impaired from forming cell-cell contacts. VACs are large vacuoles which contain microvilli and apical plasma membrane markers (among others, a 184-kDa plasma membrane protein, AP2), but exclude basolateral membrane markers. Upon reestablishment of cell-cell contacts, VACs are rapidly (within 1 h) exocytosed toward intercellular spaces, after which the apical plasma membrane drifts toward its final destination (Vega-Salas, Salas, and Rodriguez-Boulan. 1988. J. Cell Biol. 107, 1717-1728). In this work, we studied the role of cAMP as a mediator for the exocytosis of VACs. We shifted confluent cells from low to normal calcium medium (thus reestablishing cell-cell contacts and causing VAC exocytosis), a shift which resulted in a significant rise of cellular levels of both total intracellular and protein-bound cAMP. The 8-Br analog of cAMP (8-Br-cAMP) (5-50 microM) caused externalization of the intracellular compartment of AP2 as measured by radioimmunoassay. A similar effect was observed with 3-isobutyl-1-methylxanthine. 8-Br-cAMP also caused the appearance of AP2-positive VAC images in nonpermeabilized cells, namely, VACs that become accessible to extracellular antibodies upon fusion with the plasma membrane. Lanthanum, which abolishes the peak of intracellular free calcium during a calcium switch, failed to block the exocytosis. On the other hand, 12-O-tetradecanoylphorbol-13-acetate induced only a modest exocytic response. Finally, 8-Br-cAMP induced VAC exocytosis in sparse MDCK cells grown in normal calcium medium. These data indicate that cAMP is a mediator between the extracellular signal provided by cell-cell contacts and VAC exocytosis.

The mitochondria, the microsomes and the cytosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.

A protocol has been applied to all our patients consulting for nocturnal enuresis who also presented daytime wetting or urge to void, to rule out obstructive uropathies. Our protocol includes voiding cystourethrogram, IVP, cystometry and endoscopy. 64 females and 46 males, 4 to 14 year-old, were studied between 1980 and 1983. Obstructive urethral uropathy was ruled out in 37 females (58%) and 32 males (69%). The most common treatments in these patients were endoscopic resection (Bugbee) of posterior urethral valves in boys and resection of the distal ring in girls. All other associated diseases or malformations were treated according to their etiology. Results, analyzed through a questionnaire sent to parents, were good in 77% of the patients treated for obstruction. In conclusion we think that daytime wetting and urge to void are very frequently the emerging symptoms of a hidden uropathy, in most cases obstructing proximal or distal urethra. Surgical treatment can lead to a good cure rate.

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.

ACTH in vivo induces the formation of several steroidogenic factors in both cytosol and extramitochondrial particulate fractions of rat adrenal. Cycloheximide prevents the formation of these factors. Here we show the presence of a cytosolic steroidogenic component (C1) which is cycloheximide-sensitive and not ACTH-dependent. C1 is able to solubilize an ACTH-dependent steroidogenic factor (C2) from particulate fractions resulting in the release of the rat-limiting constraint of mitochondrial steroidogenesis. The thermolabile and trypsin-resistant factor C1 has an apparent mol.wt of 28,000 Daltons. In contrast, the cycloheximide-sensitive factor C2 from extra-mitochondrial fractions of ACTH-treated rats comigrates on Sephadex G-10 with phospholipids. Endogenous phospholipids isolated from particulate adrenal fractions of ACTH-treated rats or exogenous phospholipids will also stimulate steroidogenesis in vitro. Indeed, cytosolic solubilizing factor C1 enhances the exogenous phospholipid effect 3-4-fold. The results taken together suggest that C1 may be very similar to a well defined phospholipid exchange protein and C2 is itself a phospholipid. Both factors seem to be obligatory for the ACTH-induced steroidogenesis.

Discrepancies between adenosine 3':5'-cyclic monophosphate (cAMP) and steroid production have been frequently observed in isolated target cells stimulated by low concentrations of trophic hormone. This dissociation is particularly marked in the interstitial cells of the testis, where testosterone production is elicited by gonadotropin concentrations in the picomolar range. Because of these observations, and a disparity between steroidogenesis and protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activation in Leydig cells, the role of cAMP as a mediator of the acute steroidogenic response has been questioned. This problem has been further analyzed by assay of free and occupied cAMP-binding sites of the regulatory subunit of protein kinase in basal and hormone-stimulated cells. Free sites were measured by a [(3)H]-cAMP-binding assay, and occupied sites were measured by radioimmunoassay of endogenous cAMP eluted from receptor protein. After stimulation of purified Leydig cells with 0.1-10 pM human chorionic gonadotropin, a dose-dependent decrease in available [(3)H]cAMP-binding sites was observed, with no change in binding affinity. The reduction in cAMP-binding sites was equivalent to the increase in occupancy of cAMP receptors by endogenous nucleotide formed during gonadotropin action. Fractional occupancy of cAMP receptors rose progressively from basal values of 0.2-0.40 to full saturation as intracellular cAMP rose 10- to 30-fold during hormone stimulation. The testosterone dose-response curve was coincident with the initial part of the cAMP-receptor occupancy curve. These changes in endogenous cAMP binding to the regulatory subunit were accompanied by a significant increase in protein kinase activity in gonadotropin-stimulated Leydig cells. These observations provide direct evidence for the role of cAMP and protein kinase during hormonal activation of steroidogenesis in the Leydig cell by low concentrations of gonadotropin.