A központi idegrendszeri pajzsmirigyhormon aktiváció szabályozásának molekuláris biológiája = Molecular biology of the regulation of thyroid hormone activation in the central nervous system

A pajzsmirigyhormonok számos biológiai folyamat, így a fejlődés, a növekedés és az anyagcsere alapvető fontosságú tényezői. A tiroxin (T4) pro-hormon, melynek a pajzsmirigyhormon aktiváció során dejodációval T3-á kell alakulnia ahhoz, hogy a pajzsmirigyhormon magreceptorhoz kötődve biológiai hatásait kifejthesse. A T4 aktivációt az agyban a kettes-típusú dejodáz enzim (D2) katalizálja. A pályázat célkitűzései a központi idegrendszeri pajzsmirigyhormon aktiváció celluláris és molekuláris vonatkozásainak vizsgálatához kapcsolódtak. Az alacsony T3 szindróma molekuláris pathogenezisének vizsgálata során feltártuk az emberi dio2 gén NF-kB érzékenységét és leírtuk annak molekuláris hátterét. Jellemeztük a D2 fehérje ubikvitinációjában kulcsszerepet játszó fehérjék idegrendszeri eloszlását, továbbá a D2 enzim poszt-transzlációs szabályozásának szubcelluláris és konformációs feltételeit. Vizsgálataink során tanulmányoztuk a T3 képződés mechanizmusát a fejlődő idegrendszerben és kimutattuk a D2 expresszió megjelenését a fejlődő hipotalamusz tanicitáiban. Munkánk az idegrendszeri T3 függő génexpressziós profilok kialakulásért felelős szabályozó mechanizmusok feltérképezése révén járul hozzá a fejlődő és kifejlett agy működését befolyásoló tényezők jobb megértéséhez. | Thyroid hormone is a crucial factor of development, growth and metabolism. The pro-hormone thyroxine (T4) has to be converted to T3 via thyroid hormone activation in order to bind the thyroid hormone receptor to modulate thyroid hormone dependent pathways. Local T3 generation in the adult and developing brain is catalyzed by the type 2 deiodinase selenoenzyme (D2). The aim of the project was to better understand the cellular and molecular events underlying thyroid hormone activation in the central nervous system. We studied the molecular pathogenesis of the low T3 syndrome and described the molecular components ensuring NF-kB mediated induction of the human dio2 gene. We characterized the distribution of key proteins of D2 ubiquitination in the brain and determined the subcellular and conformational requirements of post-translational regulation of the D2 enzyme. We studied the mechanisms of T3 generation in the developing brain and determined the developmental expression of D2 in hypothalamic tanycytes. Our data contribute to the better understanding of factors regulating the function of the developing and adult brain via providing a deeper insight into mechanisms generating T3 dependent gene expression profiles

A szarvasmarha neonatalis Fc receptor (bFcRn) által mediált IgG katabolizmus és epithelialis transzport molekuláris szintű elemzése = Studies on the bovine FcRn mediated IgG catabolism and epithelial transport at molecular level

A pályázat révén jelentősen bővítettük a szarvasmarha FcRn (bFcRn) szerepéről alkotott ismereteinket arról hogyan szabályozza ez a receptor az IgG homeosztázisát. Egyik legfontosabb eredményünknek tartjuk, hogy tisztáztuk a bFcRn szerepét tőgy IgG transzport folyamatában. Felületi plazmon rezonancia elemzéseinkkel kimutattuk, hogy a receptor lényegesen nagyobb affinitással kötődik a bovin IgG2, mint az IgG1 izotípushoz. A tejmirigyben kifejeződő bFcRn transzgenikus egerek elemzésével pedig megállapítottuk, hogy a nagyobb mértékű receptor kifejeződés jelentősen növeli az állatok szérum IgG szintjét, és csak kismértékben a tej IgG koncentrációját. E két megfigyelés alapján kijelenthető, hogy a bFcRn a tőgyben nem az IgG1-et szekretálja, hanem az IgG2-t juttatja vissza a keringésbe, megakadályozza ennek az izotípusnak a tejbe történő kiürülését. Szarvasmarhában végzett IgG kiürülési vizsgálatainkkal megállapítottuk, hogy a bFcRn aktívan közreműködik az IgG lebomlásának szabályozásában. A bFcRn-t faj-specifikus, test szerte kifejeződő transzgenikus egerekben végzett elemzéseink kimutatták, hogy az FcRn kifejeződésének fokozása csökkenti az IgG lebomlását és immunizálást követően fokozza az antigén specifikus B limfociták termelődését. Ennek köszönhetően ezeknek az állatoknak az antigén specifikus ellenanyag termelése lényegesen meghaladja a hagyományos állatokét. Ez utóbbi felismerés gazdasági hasznosítására a kutatók létrehozták az ImmunoGenes Kft-et (www.immunogenes.com). | In the frame of this grant, we significantly extended our knowledge about the role of the bovine FcRn (bFcRn) in the IgG homeostasis. One of our most important results was to clarify the role of this receptor in the IgG transport during colostrum formation. By using surface plasmon resonance assay, we could show that the FcRn binds to bovine IgG2 at a much higher affinity as compared to the IgG1 isotype. Transgenic mice that express bFcRn exclusively in their mammary gland during lactation showed significantly higher serum IgG level, while the IgG concentration in the milk was only slightly increased. Based on these two observations, we concluded that the bFcRn recycles IgG2 to the blood from the mammary gland, instead of secreting IgG1 into colostrums/milk. Results on IgG clearance studies in cattle showed that the bFcRn plays an important role in IgG protection regulating its catabolism. Our other transgenic mice that express the bFcRn in species specific mode throughout the body showed reduced IgG catabolism and enhanced antigen specific B cell production upon immunization. Due to these two effects the antigen specific antibody production is significantly improved in these animals. Based on this latter observation researchers founded ImmunoGenes Kft (www.immunogenes.com) to execute a plan towards creating a profitable company

Chromogranin A and its role in the pathogenesis of diabetes mellitus

Chromogranin A is a member of the granin glycoprotein family that is expressed by the endocrine and neuroendocrine cells of different organs. Intracellularly, Chromogranin A contributes to the regulation of secretion, and gives several cleavage products after secretion. Some of its cleavage products modify the hormone functions in autocrine and paracrine ways, while the functions of others have not been fully understood yet. Serum Chromogranin A level is most prominently used in neuroendocrine tumor diagnostics. In addition, recent studies have suggested that Chromogranin A and some of its cleavage products, pancreastatin and WE-14, also play important roles in the pathogenesis of the various forms of diabetes mellitus, but their exact mechanisms still need to be clarified. Higher Chromogranin A, pancreastatin and WE-14 levels have been reported in type 1, type 2 and gestational diabetic patients compared to healthy controls. A notable connection has been inferred through the observation that type 1 diabetes mellitus is not at all or rarely developed in Chromogranin A gene-knockout, non-obese diabetic model mice compared to non-knockout, non-obese diabetic mice. Pancreastatin inhibits insulin release in various cell and animal models, and WE-14 serves as an autoantigen for both CD4+ and CD8+ beta cell-destructive diabetogenic T-cell clones in type 1 diabetes. Chromogranin A contributes to the pathogenesis of diabetes mellitus according to the available literature. The current findings facilitate further investigation to unravel the deeper relationships between this glycoprotein and diabetes

Szteroid-21-hidroxiláz-deficientia, a congenitalis adrenalis hyperplasia leggyakoribb oka

Abstract: Congenital adrenal hyperplasia is a group of genetic diseases due to the disablement of 7 genes; one of them is steroid 21-hydroxylase deficiency. The genes of congenital adrenal hyperplasia encode enzymes taking part in the steroidogenesis of adrenal gland. Steroid 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations of the steroid 21-hydroxylase gene. The mutations of steroid 21-hydroxylase gene cause 95% of the congenital adrenal hyperplasia cases. Although the non-classic steroid 21-hydroxylase deficiency with mild symptoms is seldom diagnosed, the classic steroid 21-hydroxylase deficiency may lead to life-threatening salt-wasting and adrenal crises due to the insufficient aldosterone and cortisol serum levels. The classic type requires life-long steroid replacement which may result in cushingoid side effects, and typical comorbidities may be also developed. The patients? quality of life is decreased, and their mortality is much higher than that of the population without steroid 21-hydroxylase deficiency. The diagnosis, consequences and the patients? life-long clinical care require a multidisciplinary approach: the specialists in pediatrics, internal medicine, endocrinology, laboratory medicine, genetic diagnostics, surgery, obstetrics-gynecology and psychology need to work together. Orv Hetil. 2018; 159(7): 269?277

MASP-1 Induces a Unique Cytokine Pattern in Endothelial Cells: A Novel Link between Complement System and Neutrophil Granulocytes

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms

Common Genetic Variants of the Human Steroid 21-Hydroxylase Gene (CYP21A2) Are Related to Differences in Circulating Hormone Levels

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Hungarian Scientific Research Fund (OTKA, PD100648 (AP)) Technology Innovation Fund, National Developmental Agency (KTIA-AIK-2012-12-1-0010). AP is the recipient of a “Lendület” grant from the Hungarian Academy of Sciences

Both Positive and Negative Selection Pressures Contribute to the Polymorphism Pattern of the Duplicated Human CYP21A2 Gene.

The human steroid 21-hydroxylase gene (CYP21A2) participates in cortisol and aldosterone biosynthesis, and resides together with its paralogous (duplicated) pseudogene in a multiallelic copy number variation (CNV), called RCCX CNV. Concerted evolution caused by non-allelic gene conversion has been described in great ape CYP21 genes, and the same conversion activity is responsible for a serious genetic disorder of CYP21A2, congenital adrenal hyperplasia (CAH). In the current study, 33 CYP21A2 haplotype variants encoding 6 protein variants were determined from a European population. CYP21A2 was shown to be one of the most diverse human genes (HHe=0.949), but the diversity of intron 2 was greater still. Contrary to previous findings, the evolution of intron 2 did not follow concerted evolution, although the remaining part of the gene did. Fixed sites (different fixed alleles of sites in human CYP21 paralogues) significantly accumulated in intron 2, indicating that the excess of fixed sites was connected to the lack of effective non-allelic conversion and concerted evolution. Furthermore, positive selection was presumably focused on intron 2, and possibly associated with the previous genetic features. However, the positive selection detected by several neutrality tests was discerned along the whole gene. In addition, the clear signature of negative selection was observed in the coding sequence. The maintenance of the CYP21 enzyme function is critical, and could lead to negative selection, whereas the presumed gene regulation altering steroid hormone levels via intron 2 might help fast adaptation, which broadly characterizes the genes of human CNVs responding to the environment

A szarvasmarha neonatális Fc receptor transzkripciós szabályozása

The neonatal Fc receptor (FcRn), like other MHC class I molecules, is composed of an α-chain and a β2-microglobulin (β2m). This receptor has been detected in the bovine mammary gland, small intestine, lower respiratory system, and endothelial cells. While epithelial FcRn is involved in IgG transport through these barriers, the FcRn expressed in capillary pendothelial cells is responsible for regulating the IgG catabolism. Due to these crucial immunological functions, the gene regualtion of the bovine FcRn (bFcRn) may contribute to the immune homeostasis. Although gene expression is controlled at multiple levels, one of the most important is transcriptional regulation. Accordingly the sequences of the human and mouse FcRn α-chain cis-regulatory region have been published and their preliminary examination has been achieved, but their transcriptional regulation has not been adequately unravelled. In order to reveal the regulation of the bFcRn transcription, the 5’-flanking sequence of the bFcRn α-chain gene was isolated, cloned and functionally examined. The bFcRn α-chain cis-regulatory region was induced by NFκB in the luciferase reporter gene assays of human and bovine cell models. Three functional κB binding sites were identified in the cis-regulatory region using site-directed mutagenesis accompanied by luciferase reporter gene assays, and it was verified that these κB sites were responsible for the complete NFκB responsiveness of the bFcRn cis-regulatory region. The κB binding sites were also tested in gel retardation assay verifying their binding ability to NFκB complex with p65 content. These in vitro findings indicated, in accordance with the present in vivo data, that the bFcRn was under the control of an important transcriptional pathway activated during infection and inflammation. The β2m is a chaperone of FcRn and other MHC class I (-like) proteins ensuring the appropriate function of these molecules. To fulfil this function, it is expressed ubiquitously under constitutive and cytokine-induced transcriptional controls. Transcriptional elements of the β2m cis-regulatory region have been experimentally well characterized in human, and it has been found that a κB and an ISRE sites were responsible for the cytokine-induced regulation. The 5’-flanking sequence of the bovine β2m (bβ2m) has been isolated and cloned in order to assess its cytokine-induced gene expression in relation to FcRn. Although the ISRE site was conserved in the cattle, there was a deletion in the bβ2m κB site compared to the human orthologue, and there was no NFκB responsiveness of the bβ2m cis-regulatory region in the luciferase reporter gene assays of human and bovine cell models. To the contrary, the bβ2m κB site did bind the NFκB complex with p65 content in gel retardation assay rendering these 6 in vitro results controversial. In vivo data about the mRNA level of the bβ2m upon LPS induction are also contradictory, therefore the NFκB inducibility of the bβ2m cis-regulatory region cannot be deduced from the present data. The functionality of the bβ2m ISRE site was confirmed in vitro by gel retardation and luciferase reporter gene assays, thus, the bβ2m ISRE site mediated the IFN-γ induction similarly to its human orthologue, and there were no differences in the ISRE-mediated transcriptional regulation of this gene in cattle. In order to establish a species-specific system that can be used to analyze gene regulation in bovine, the full length coding sequence of the bovine p65 (bp65) subunit of NFκB was isolated and cloned. The cloned bp65 was expressed in mammalian cells, and it induced the NFκB-specific luciferase reporter gene expression. Using gel retardation assay, it was demonstrated that the cloned bp65 bound to the consensus κB sequence. The comparison of the bp65 with its human and mouse orthologues at amino acid level showed high homology in both the DNA-binding domain, known as Rel homology domain (RHD) and the transactivation domain (TAD). The phylogenetic analysis at DNA level provided a new insight into the evolution of the NFκB family, and it was able to resolve the topology of the mammalian p65 molecules. Although, the RHD was conserved in vertebrates, the TAD sequences deviated from each other, and showed faster molecular evolution than RHD sequences

Szérum kromogranin A szint vizsgálata 2-es típusú cukorbetegségben [Serum chromogranin A level in patients with type 2 diabetes]

A kromogranin A (CgA) a granin fehérjecsaládba tartozó glükoprotein. E fehérje és a diabetes közötti szoros kapcsolatot 1-es típusú cukorbetegségben korábban több munkacsoport is igazolta. Jelen vizsgálatunk során a Semmelweis Egyetem, II. sz. Belgyógyászati Klinika Anyagcsere Ambulanciáján 2-es típusú cukorbetegek szérum kromogranin A szintjét határoztuk meg. Vizsgáltuk továbbá, hogy mely anamnesztikus és/vagy laboratóriumi paraméter függ össze a kromogranin A szinttel. Összesen 86 2-es típusú cukorbeteg személyt vontunk be a vizsgálatba. A betegekről felvett anamnesztikus adatok mellett éhgyomri vérvételt követően elemeztük a vérképet, a HbA1c-szintet, a vérzsírokat, a vesefunkciót, illetve a TSH-t. A szérum kromogranin A szint meghatározása radioimmunoassay módszerrel történt. A vizsgált személyek közül 6 főnél (6,98%) volt igazolható emelkedett szérum kromogranin A szint. A normális tartomány felső határértéke alapján 2 kohorszra osztottuk a betegeket (Normál CgA: 80 fő, szérum-CgA 50,43±21,73 ng/ml; Magas CgA: 6 fő, szérum-CgA 129,33±41,07 ng/ml; p<0,0001). A két vizsgálati kohorsz között sem a laboratóriumi paraméterekben, sem a HbA1c-értékekben, sem pedig az anamnesztikus adatokban nem igazoltunk különbséget. Eredményeink alapján 2-es típusú diabetesben a kromogranin A szint és a diabetes fennállási ideje, valamint az anyagcserehelyzet között nem igazolható összefüggés. | Chromogranin A (CgA) is a member of the granin glycoprotein family. Most important effects of the protein related to diabetes have been demonstrated in type 1 diabetes. In the present cohort study serum chromogranin A levels of patients with type 2 diabetes were analyzed, who attended the Metabolic Clinic of the 2nd Department of Internal Medicine, Semmelweis University. It was also investigated whether anamnestic or laboratory data has any relation to chromogranin A levels. Altogether 86 patients with type 2 diabetes were included. Anamnestic data were collected and fasting blood samples were taken. Complete blood count, HbA1c , lipids, renal function and TSH were analyzed. CgA values were measured via radioimmunoassay. At six of the 86 subjects (6,98%) had elevated serum chromogranin A levels. Patients were divided into two cohorts based on the serum CgA upper limit of normal (Normal CgA: 80 subjects, serum CgA 50,43±21,73 ng/ml; High CgA: 6 subjects, serum CgA 129,33±41,07 ng/ml; p<0,0001). No differences were found between study groups neither in laboratory parameters, nor in HbA1c values, nor in anamnestic data. Our results suggest that in type 2 diabetes there’s no connection between serum chromogranin A levels and the duration of diabetes, and it has no effect on glycemic control