A szarko/endoplazmatikus retikulum Ca2+ ATPáz 3 szerkezet-elemzése és a nem-izom típusú sejtek Ca2+ -transzport fehérje készletének változásai sejtdifferenciáció során = Structural study of the sarco/endoplasmic reticulum Ca2+ ATPase 3 proteins and the modulation of the machinery of Ca2+-transport proteins during the differentiation of non-muscle cells

A Ca2+ függő sejtélettani folyamatok szabályozásában fontos szerepet betöltő szarko/endoplazmás retikulum (SERCA) és plazmamembrán (PMCA) Ca2+ATPázokat vizsgáltuk. Endogén, valamint vad típusú és mutáns rekombináns SERCA3 fehérjék tripszines proteolízisét elemezve kimutattuk két, kinetikailag eltérő fragmentációs profil párhuzamos megjelenését. Irányított mutagenezissel azonosítottunk néhány tripszines hasítási helyet. Eredményeink arra utalnak, hogy natív membránkörnyezetben a fragmentációs profilok a SERCA3 fehérje eltérő konformációs állapotaihoz rendelhetőek. Korábbi munkáinkhoz kapcsolódva elemeztük a gyomor-/béltumorsejtek differenciációja, és a SERCA és PMCA fehérjék expressziója, funkciója közötti összefüggéseket. Számos differenciációs modellben igazoltuk, hogy az éretlen sejtekben alacsony szinten kifejeződő SERCA3 és PMCA4b izoformák expressziója erősen indukálódik a tumorsejtek differenciációja során. A SERCA fehérjék funkcionális gátlása elősegítette egyes béltumorsejtek differenciációját. A béltumorok kialakulásában fontos APC/b-katenin/TCF4 jelátviteli út gátlása indukálta a SERCA3 expressziót. Különböző stádiumú béltumorokból nyert szöveti metszetek immunhisztokémiai vizsgálatai szerint a SERCA3 expresszió defektusa már a tumorok kialakulásának korai szakaszában jelentkezik. Eredményeink alapján felvetjük annak lehetőségét, hogy a gyomor-/béltraktusban egyes Ca2+ transzporterek, mint markerfehérjék, segíthetik malignus elváltozások kialakulásának és fenotípusának meghatározását, és diagnosztikai fejlesztések potenciális célpontjai lehetnek. | We studied the sarco/endoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ATPases. The proper functions of these proteins are essential in the regulation of Ca2+-dependent cellular processes. Limited tryptic digestion of endogenously expressed or wild-type and mutant recombinant SERCA3 proteins resulted in two distinct fragmentation profiles. Using site-directed mutagenesis approach some of the tryptic sites were determined. Our data indicated that the SERCA3 fragmentation profiles were related to different conformational states of the enzyme. To further explore our previous work, we investigated the expression and function of various SERCA and PMCA isoforms during the differentiation of gastric/colon cancer cells. Using a wide range of model cells and differentiation protocols, strong induction of SERCA3 and PMCA4b expression were detected in differentiating cancer cells. Inhibition of SERCA function induced the maturation of colon cancer cells. Inhibition of the APC/b-catenin/TCF4 signaling pathway, essential during colon carcinogenesis, resulted in up-regulated SERCA3 expression. Immunohystochemical analysis of various tissue sections from colonic lesions, adenomas and adenocarcinomas showed that loss of SERCA3 expression is an early event during colon carcinogenesis. Our data suggest that some Ca2+ transport proteins could serve as new biomarkers for the analysis of the formation and phenotype of gastric/colon tumors, and should help in novel diagnostic development

Mennyiségi molekuláris genetikai vizsgálatok és nagy kapacitású sejtelválasztás együttes alkalmazása rosszindulatú myeloid megbetegedéskeben = Combined application of molecular genetic techniques and high capacity cell sorting in malignant myeloid diseases

Többféle molekuláris diagnosztikai módszer alkalmazásával vizsgáltuk új örökletes tényezők hatását a kórképek kialakulására és klinikai sajátosságaira rosszindulatú myeloid megbetegedésekben. Korábbi kutatásainkhoz kapcsolódóan elsőként fedeztük fel a vasanyagcsere örökletes oki tényezője és a krónikus myeloproliferatív szindróma (CMPD) közötti kapcsoltságot. Szintén CMPD-ben erősítettük meg és terjesztettük ki mások megfigyeléseit, hogy a JAK2 gén 46/1 haplotípusa fokozott gyakoriságot mutat CMPD-ben és elsőként írtuk le ugyanezt akut myeloid leukémiában. Így ez a genetikai sajátosság hajlamosító tényező lehet mindkét kórképben. Részletes klinikai adatgyűjtéssel kimutattuk, hogy a JAK2 gén 46/1 haplotípus jelenlétében gyakoribb a myelo-monocytás morfológiájú AML, a klinikai lefolyás során gyakoribb a fertőzéses halálozás, és kedvezőtlenebb a betegség-mentes, ill. összesített túlélés. Krónikus myeloid leukémiában (CML) vizsgáltuk a tirozin kináz inhibitor rezisztencia két fontos mechanizmusa a kiegészítő kromoszóma eltérések, illetve az ABL gén kináz domén mutációk jelentőségét és kapcsolatát. Szintén CML-ben, többféle molekuláris genetikai technikát, illetve bioinformatikai elemzést alkalmazva mutattuk ki, hogy az ABL gén 7. exon deléciója nem játszik szerepet az imatinib-rezisztencia kialakulásában. A pályázati alatt a témavezető, ill. helyettese utolsó és egy megosztott első szerzőségével 6 (IF:23,334), és további 15 társszerzős nemzetközi közlemény született (IF:43,928). | In the framework of the present project, effects of inherited factors were examined on the formation of certain disease types and clinical characteristics of patient cohorts with malignant myeloid diseases. Connected to earlier research, we first described the association between inherited factor of iron metabolism and chronic myeloproliferative syndrome (CMPD). Testing this CMPD cohort, we confirmed and extended recent results inasmuch as the frequency of 46/1 haplotype of the JAK2 gene is increased in CMPD and we first described this in acute myelogenous leukemia (AML). We found that in the presence of the JAK2 46/1 haplotype the myelo-monocyter morphology subtype of AML is more frequent, as well as the infection as cause of death, moreover the disease-free and the overall survival is poorer. In chronic myelogenous leukemia (CML) cohorts, the significance and relationship of two mechanisms of tyrosine kinase inhibitor resistance, namely the additional chromosome alterations and mutations of the kinase domain of the ABL gene were studied. Testing the same CML cohorts, we found that, the exon 7 deletion of the ABL gene does not play a role in the formation of imatinib-resistance. During the project, 6 international publications (IF:23,334) were published with senior and one shared first authorship positions of the project leader and his deputy in the immediate topic of the current project. In addition, 15 further co-author publications were also published (IF:43,928)

NFKB1 -94ins/delATTG polymorphism is a novel prognostic marker in first line-treated multiple myeloma

Nuclear factor kappa B (NFKB) plays an important role in multiple myeloma (MM), and bortezomib affects this pathway. We retrospectively analysed the effect of the NFKB1 -94ins/delATTG polymorphism on the survival of 295 MM patients treated at a single centre. The median progression-free survival (PFS) was 790 (659-921) d in patients with NFKB1 homozygous insertion genotype (I/I, n = 99) and 624 (515-733) d in deletion-carriers (I/D&D/D, n = 196, P = 0.013). In multivariate analysis, I/I carriers showed a favourable PFS compared to I/D&D/D with a hazard ratio of 0.622 (0.457-0.847), P = 0.003, in addition to international staging system (ISS) score, fluorescence in situ hybridization (FISH) risk score, age and bortezomib treatment. I/I patients benefited more from bortezomib treatment [PFS 902 (703-1101) and 580 (343-817), P = 0.008] than I/D&D/D patients [PFS 659 (487-831) and 488 (323-653), P = 0.531]; in addition the beneficial effect of low ISS score was not observed in the I/D&D/D group [PFS 639 (454-824) and 650 (458-842), P = 0.226], while it was clear in I/I patients [PFS 1140 (803-1477) and 580 (408-752), P < 0.001]. We conclude that homozygous carriers of the insertion allele of the NFKB1 -94ins/delATTG polymorphism have a better prognosis and probably benefit more from bortezomib treatment than MM patients carrying the deletion allele

Anti-microbial antibodies in celiac disease: trick or treat?

AIM: To determine the prevalence of a new set of anti-glycan and anti-outer membrane protein (anti-OMP) antibodies in a Hungarian cohort of adult Celiac disease (CD) patients. METHODS: 190 consecutive CD patients [M/F: 71/119, age:39.9 (SD:14.1) years], 100 healthy, and 48 gastrointestinal controls were tested for glycan anti-Saccharomyces cerevisiae (gASCA), anti-laminaribioside (ALCA), anti-chitobioside, anti-mannobioside, anti-OMP antibodies and major NOD2/CARD15 mutations. Thirty out of 82 CD patients enrolled at the time of diagnosis were re-evaluated for the same antibodies after longstanding gluten-free diet (GFD). RESULTS: 65.9% of the CD patients were positive for at least one of the tested antibodies at the time of the diagnosis. Except anti-OMP and ALCA, anti-microbial antibodies were exclusively seen in untreated CD; however, the overall sensitivity was low. Any glycan positivity (LR+: 3.13; 95% CI: 2.08-4.73) was associated with an increased likelihood ratio for diagnosing CD. Significant correlation was found between the levels of anti-glycan and anti-endomysial or anti-transglutaminase antibodies. Anti-glycan positivity was lost after longstanding GFD. Anti-glycan antibody titers were associated with symptoms at presentation, but not the presence of NOD2/CARD15 mutations. Patients with severe malabsorption more frequently had multiple antibodies at diagnosis (P = 0.019). CONCLUSION: The presence of anti-glycan antibodies in CD seems to be secondary to the impaired small bowel mucosa which can lead to increased antigen presentation. Furthermore, anti-glycan positivity may be considered an additional marker of CD and dietary adherence

Komplex molekuláris genetikai vizsgálati algoritmus myeloproliferativ neoplasiák diagnosztikájában

Introduction: Mutations in Janus kinase 2, calreticulin and thrombopoietin receptor genes have been identified in the genetic background of Philadelphia chromosome negative, "classic" myeloproliferative neoplasms. Aim: The aim of the authors was to identify driver mutations in a large myeloproliferative cohort of 949 patients. Method: A complex array of molecular techniques (qualitative and quantitative allele-specific polymerase chain reactions, fragment analyzes, high resolution melting and Sanger sequencing) was applied. Results: All 354 patients with polycythemia vera carried Janus kinase 2 mutations (V617F 98.6%, exon 12: 1.4%). In essential thrombocythemia (n = 468), the frequency of V617F was 61.3% (n = 287), that of calreticulin 25.2% (n = 118), and that of thrombopoietin receptor mutations 2.1% (n = 10), while 11.3% (n = 53) were triple-negative. Similar distribution was observed in primary myelofibrosis (n = 127): 58.3% (n = 74) V617F, 23.6% (n = 30) calreticulin, 6.3% (n = 8) thrombopoietin receptor mutation positive and 11.8% (n = 15) triple-negative. Conclusions: The recent discovery of calreticulin gene mutations led to definite molecular diagnostics in around 90% of clonal myeloproliferative cases. Orv. Hetil., 2014, 155(52), 2074-2081

Distinct clinical characteristics of myeloproliferative neoplasms with calreticulin mutations

Somatic insertions/deletions in the calreticulin gene have recently been discovered to be causative alterations in myeloproliferative neoplasms. A combination of qualitative and quantitative allele-specific polymerase chain reaction, fragment-sizing, high resolution melting and Sanger-sequencing was applied for the detection of three driver mutations (in Janus kinase 2, calreticulin and myeloproliferative leukemia virus oncogene genes) in 289 cases of essential thrombocythemia and 99 cases of primary myelofibrosis. In essential thrombocythemia, 154 (53%) Janus kinase 2 V617F, 96 (33%) calreticulin, 9 (3%) myeloproliferative leukemia virus oncogene gene mutation-positive and 30 triple-negative (11%) cases were identified, while in primary myelofibrosis 56 (57%) Janus kinase 2 V617F, 25 (25%) calreticulin, 7 (7%) myeloproliferative leukemia virus oncogene gene mutation-positive and 11 (11%) triple-negative cases were identified. Patients positive for the calreticulin mutation were younger and had higher platelet counts compared to Janus kinase 2 mutation-positive counterparts. Calreticulin mutation-positive patients with essential thrombocythemia showed a lower risk of developing venous thrombosis, but no difference in overall survival. Calreticulin mutation-positive patients with primary myelofibrosis had a better overall survival compared to that of the Janus kinase 2 mutation-positive (P=0.04) or triple-negative cases (P=0.01). Type 2 calreticulin mutation occurred more frequently in essential thrombocythemia than in primary myelofibrosis (P=0.049). In essential thrombocythemia, the calreticulin mutational load was higher than the Janus kinase 2 mutational load (P<0.001), and increased gradually in advanced stages. Calreticulin mutational load influenced blood counts even at the time point of diagnosis in essential thrombocythemia. We confirm that calreticulin mutation is associated with distinct clinical characteristics and explored relationships between mutation type, load and clinical outcome