K+ csatornák és a T sejt receptor jelátvitelének kapcsolata = Relationship between K+ channels and T cell receptor signaling

A T limfociták aktivációjának szabályzásában a feszültség-kapuzott Kv1.3 K+ csatornák központi szerepet játszanak. A pályázat megvalósítása a során kimutattuk, hogy a Kv1.3 csatornák immunológiai szinapszisba (IS) történő átrendeződése az IS funkciójának és a csatornák aktivitásának kölcsönös szabályozási lehetőségét jelentik. E kölcsönös kapcsolat elemei közül kiemelendő, hogy a Kv1.3 csatornák kapuzási kinetikájának jelentős változását mutattuk ki a sejtmembrán lipid összetételének változása ill. a csatornák IS-be történő akkumulációja során. A csatornák IS-beli akkumulációja a T sejt membránpotenciál szabályozásának egy új formáját, a membránpotenciál oszcillációját okozta, mely Kv1.3 gátlószerekkel modulálható volt. E kísérletekhez szorosan kapcsolódott a nagy affinitású és szelektivitású Kv1.3 gátlószerek azonosítása és jellemzése. Leírtunk öt új Kv1.3 gátló skorpió toxint [?-KTx2.8 (Ce1), ?-KTx2.9 (Ce2), ?-KTx2.11 (Ce4), ?- KTx2.13 (Css20) és ?- KTx4.6 (Tst26)], és meghatároztuk azokat a kritikus aminosavakat melyek a toxinok Kv1.3 és Kv1.2 csatornák közötti szelektivitásáért felelősek. Kísérleteink során olyan egyedülálló módszert dolgoztunk ki, mely alkalmas olyan kis-molekula gátlószerek azonosítására, melyek szelektíven kötődnek a csatornák inaktivált állapotához. A kutatómunka során kapott eredmények közelebb vittek a Kv1.3 csatornák élettani funkciójának megértéséhez ill. terápiásan alkalmazható immunoszuppresszív peptidek előállításához. | Voltage-gated Kv1.3 channels play a distinguished role in regulation of T cell activation. During the research program we showed that recruitment of Kv1.3 channels into the immunological synapse (IS) results in the reciprocal regulation of the function of the IS and the activity of Kv1.3 channels. Among several aspects of this regulation we showed that the gating kinetics of Kv1.3 channels is significantly altered upon recruitment of the channels in to the IS and that the kinetics is sensitive to the lipid composition of the plasma membrane. Recruitment of Kv1.3 into the IS allows a novel type of the membrane potential regulation in T cells by periodic membrane potential oscillations, which was sensitive to Kv1.3 inhibitors. These results motivated the search for novel high affinity and selectivity Kv1.3 inhibitors. We have described five Kv1.3 inhibitor scorpion toxins [?-KTx2.8 (Ce1), ?-KTx2.9 (Ce2), ?-KTx2.11 (Ce4), ?- KTx2.13 (Css20) and ?- KTx4.6 (Tst26)], and determined critical amino acid residues responsible for the selectivity between Kv1.3 and Kv1.2 channels. We have also developed a unique method for studying K+ channels blockers having selective affinity for the inactivated channels. Results obtained during the completion of the research program led to a better understanding of the physiological role of Kv1.3 channels in T cells and to the generation therapeutic immunosuppressor peptides

A T-sejt aktiváció/celluláris immunválasz szabályozása: membrán mikrodomén-függő immunmoduláció és az ösztrogén közvetített nem-genomiális hormonális hatások vizsgálata = Regulation of the T-cell mediated immune response: investigation of membrane microdomain-dependent immunomodulation and estrogen-mediated nongenomial hormonal effects

A T49696 OTKA projekt (3 év) támogatásával elért főbb eredmények: Kimutattuk, hogy a sejtmembrán "tutajok" szfingolipid és koleszterin alkotóelemei fontos szabályozói a T sejtek aktiváció/sejthalál egyensúlyának, valamint a polarizált helper T sejtek válaszképességének. Megmutattuk ezen folyamatokban a ceramidok, a Kv és Cav ioncsatornák alapvető szerepét is. Az általunk leírt szignálintegrációs modell újabb immunmodulációs lehetőségeket kínál. Kimutattuk, hogy az ösztrogén steroid hormonok gyors, nem-genomiális jeleket indukálnak T és B limfocitákon (foszforiláció, szelektív kalcium szignál, stb.), egy még nem azonosított membránreceptoron keresztül, és fokozzák a T sejt-függő, antigén-indukált ellenanyagtermelést. Eredményeink elősegíthetik az ösztrogénszint és egyes autoimmunbetegségek közötti összefüggések hátterének mélyebb megértését. Új, koleszterin-specifikus IgG monoklonális ellenanyagokat (AC1, AC8) állítottunk elő, melyeknek megmutattuk celluláris-koleszterin diagnosztikai célokra történő alkalmazhatóságát. Ezen ellenanyagok képesek a HIV-infekció/ termelés gátlására monocita-makrofág és T sejteken in vitro, elsősorban a célsejtek plazmamembránjának (lipid tutajok, HIV receptorok) molekuláris átrendezése révén. A projekt eredményeit felhasználva 2 PhD tudományos fokozat született; 6 referált tudományos közlemény nemzetközi folyóiratban; 4 új kongresszusi absztrakt, melyekből folyamatos a publikálás (3 publikáció-kész közlemény) | Main results of the project T49696 supported by OTKA (3 years): We have shown that two lipid constituents of rafts (sphingolipids or cholesterol) are critical in regulating the balance of activation/cell death signaling in T cells or the functional responses of polarized Th1 or Th2 cells. Specific, important contribution of ceramides, Kv and Cav ion channels was also shown in these processes, offering new possibilites of immunomodulation. Rapid, non-genomial signals (selective phosphorylation and Ca2+ signals or NFκB nuclear translocation) of estrogen steroid hormones have been shown in both T and B lymphocytes, that may be partly responsible for augmentation of the T-dependent humoral immune response observed in in vivo mice studies. These effects, mediated by a yet unidentified E2 membrane receptor, may help us to reveal and understand the relationship between estrogen levels and several autoimmune diseases. We generated novel cholesterol-specific IgG antibodies (AC1 and AC8), that may serve as diagnostic markers of clustered cholesterol (cell-free or cellular). As one of their most intriguing biological activity - inhibition of HIV entry/production in vitro - has been elucidated and shown that the major mechanism of action is remodeling of the target cells' plasma membrane (rafts and HIV receptors). 2 PhD degrees were received based on these results; 6 reviewed research articles; 4 new Congress Abstracts (publication is continuous: 3 publication-ready manuscripts

Trends in educational differences in adolescent daily smoking across Europe, 2002-10

Background: Across Europe, tobacco use is more prevalent among secondary school students attending vocational tracks compared with students attending academic tracks. The purpose of the present study is to describe trends in social inequality in daily smoking among adolescents between 2002 and 2010 by addressing both absolute social inequality (prevalence difference between vocational and academic tracks) and relative social inequality (prevalence ratio) in seven European countries. Methods: Analyses were based on data from 15-year-olds who participated in the Health Behaviour in School-aged Children study in 2002, 2006 and 2010 in Belgium, Croatia, France, Germany, Hungary, Italy and The Netherlands (total N = 32 867). Results: Overall, daily smoking decreased between 2002 and 2010 in Belgium, France, Germany and The Netherlands, increased in Croatia and remained stable in Hungary and Italy. Considerable differences in daily smoking according to educational track existed in all countries. Absolute educational inequalities increased dramatically in Croatia and Italy, while relative inequalities showed a tendency to increase in all countries (significant in Belgium and The Netherlands). Conclusions: Conclusions on social inequality in adolescent smoking may appear differently when described by absolute and relative measures. Especially the large increase in absolute educational inequalities in daily smoking in Croatia and Italy are worrisome and warrant attention from the public health domain. The findings underline the need for appropriate smoking policies and interventions in vocational schools across Europ

Ioncsatornák természetes környezetben: dendritikus sejtek és endotélsejtek ioncsatornáinak karakterizálása

Cells rapidly adjust their gene and ion channel expression upon the change of extracellular environment. To obtain valid measurements it is important to use models closest to the in vivo systems. In this work we focused on the characterization of ion channels on two different cell types, the human dendritic cells (DC) and the endothelial cells (EC) of the arteria mesenterica superior in rats in the most physiologic circumstances. We also used a DC model cell line (KG-1) during our electrophysiological studies to compare the obtained currents to that of DC. In the immune system VGPC, Kir, and KCa channels have been described to play a major role in controlling the membrane potential and regulating intracellular Ca2+ signaling pathways required for proliferation and differentiation. In this study for the first time we described that immature monocyte-derived DC express voltage-gated Na+ channels (Nav1.7). Transition from the immature to a mature state in DC however was accompanied by the down-regulation of Nav1.7 expression and the up-regulation of voltage-gated Kv1.3 K+ channels. The presence of Kv1.3 is common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential. Both unstimulated and stimulated KG-1 cells expressed KCa only, which makes them not an ideal model for electrophysiological studies on DC. EC function could be considerably altered during the process of isolation and cell culture. Previous electrophysiological studies on EC were conducted on isolated or cultured cells, ignoring the complex and fine network of EC and vascular smooth muscle. We developed a method that allows identifying and characterizing the ion channels of EC in their native environment. Rat mesenteric arteries mounted as ring preparations in a microvascular myograph for recording whole cell currents under ‘blind’ patch clamp technique. Neurobiotin staining demonstrated that intact EC are electrically coupled through gap junctions and 18β- gly gap junction blocker decreased the outward and inward currents registered. We observed Kir currents sensitive to BaCl2, KCa currents of small (SKCa), intermediate (IKCa1) and high conductance (BKCa) that were sensitive to apamin, TRAM 34 and IbTx, respectively. Moreover, Ach increased outwardly directed K+ currents that were sensitive to TEA. Under physiological circumstances Kir current is involved in maintaining the resting membrane potential, where SKCa and IKCa1 are mainly responsible for membrane hyperpolarization. The BKCa current reported in this study may arise from the vascular smooth muscle layer and potentially influence EC membrane potential via myoendothelial transfer of current.N

Prognostic value of miliary versus non-miliary sub-staging in advanced ovarian cancer

WOS: 000404945600010PubMed ID: 28495239Objective. The presence of miliary disease during initial ovarian cancer debulking may reflect a distinct mode of peritoneal spread independent from size-based tumor staging and may explain variation in response to treatment and survival outcomes. To infer the prevalence, presentation and clinical implications of miliary disease we reviewed existing surgical records. Methods. Reports were available for 1008 primary debulking surgeries for ovarian, primary peritoneal or fallopian tube cancer between 2001 and 2015 (685 reports from 2005 to 2015). Clinical outcome data was available for 938 patients. We analyzed a high-stage sub-cohort for survival (N = 436). Results. Most records were evaluable for miliary disease (761/938); for these, the miliary phenotype was highly prevalent (249/761, 32.7%) and often accompanied by ascites (185/249, 74%). While optimal debulking rates were unaffected by miliary disease, total resection (RO) rates were poorer. Liver, stomach, spleen or bladder appeared to be sporadically involved while the omentum, mesentery, bowel, peritoneum and diaphragm were affected simultaneously (Spearman rho > 0.5). Overall, miliary disease was associated with worse progression free survival, overall survival, and time from relapse to death independent of stage. Survival effects were particularly strong for Stage IV disease where median overall survival varied by over 30 months (log-rank p = 0.002). Conclusions. Miliary disease is an identifiable surgical phenotype reflecting a distinct clinical trajectory that adds prognostic information to standard disease burden-based staging. These findings should permit further retrospective investigation in a wider cohort and prompt the consideration of prospective structured operative reporting standards and treatment strategies. (C) 2017 The Authors. Published by Elsevier Inc.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [K01LM012100, T32CA108456, P30CA016056]; RPCI-UPCI Ovarian Cancer SPORE [P50CA159981-01A1]; Roswell Park Alliance Foundation; Roswell Park Cancer InstituteThis work was supported by the National Institutes of Health (K01LM012100, T32CA108456, P30CA016056), RPCI-UPCI Ovarian Cancer SPORE (P50CA159981-01A1) and the Roswell Park Alliance Foundation, Roswell Park Cancer Institute. The authors report no conflicts of interest

Estrogen Receptor-Beta2 (ERβ2)–Mutant p53–FOXM1 Axis: A Novel Driver of Proliferation, Chemoresistance, and Disease Progression in High Grade Serous Ovarian Cancer (HGSOC)

High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of epithelial ovarian cancer. Prevalence (~96%) of mutant p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERβ) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unsettled. However, whether there is functional interaction between ERβ and mutant p53 in HGSOC is unknown. ERβ1 and ERβ2 mRNA and protein analysis in HGSOC cell lines demonstrated that ERβ2 is the predominant isoform in HGSOC. Specificity of ERβ2 antibody was ascertained using cells depleted of ERβ2 and ERβ1 separately with isoform-specific siRNAs. ERβ2-mutant p53 interaction in cell lines was confirmed by co-immunoprecipitation and in situ proximity ligation assay (PLA). Expression levels of ERβ2, ERα, p53, and FOXM1 proteins and ERβ2-mutant p53 interaction in patient tumors were determined by immunohistochemistry (IHC) and PLA, respectively. ERβ2 levels correlate positively with FOXM1 levels and negatively with progression-free survival (PFS) and overall survival (OS). Quantitative chromatin immunoprecipitation (qChIP) and mRNA expression analysis revealed that ERβ2 and mutant p53 co-dependently regulated FOXM1 gene transcription. The combination of ERβ2-specific siRNA and PRIMA-1MET that converts mutant p53 to wild type conformation increased apoptosis. Our work provides the first evidence for a novel ERβ2-mutant p53-FOXM1 axis that can be exploited for new therapeutic strategies against HGSOC

Prognostic impact of adjuvant chemotherapy treatment intensity for ovarian cancer.

OBJECTIVE:We aimed to investigate the prognostic impact of duration of first-line chemotherapy administration in patients with epithelial ovarian cancer (EOC). METHODS:Chemotherapy records were abstracted from the electronic medical record. Patients with on-time completion (105 days) were compared to patients finishing early (4 weeks. For 222 women with stage IIIC/IV, stage-stratified estimates of progression-free survival (PFS) and overall survival (OS) were compared. A delay sub-study was performed with outliers removed. Each week of delay was correlated with the change in PFS and OS to identify time points associated with change in outcome. RESULTS:Most women had on-time completion of chemotherapy (23.6%) or a treatment delay of ≤4 weeks (21.8%); 21.6% of women experienced a delay longer than 4 weeks. R0 resection at initial debulking (OR = 1.99, 95%CI: 1.18-3.36, p = 0.010) and RECIST complete response (OR = 4.88, 95%CI: 2.47-10.63, p1 month delay had decreased median survival of 18.1 months (14.7-24.9 months), while women with short intervals survived 35.0 months (95%CI: 21.8-49.8 months). Short-term delays lead to progressively decreasing OS. This was significantly different from the on-schedule survival estimate after 6 weeks of delay. CONCLUSIONS:On-time completion of chemotherapy correlates with increased survival and higher complete response rates. Increasing delays in chemotherapy completion were associated with decreased survival

Induction of cell death in ovarian cancer cells by doxorubicin and oncolytic vaccinia virus is associated with CREB3L1 activation

We have demonstrated that oncolytic vaccinia virus synergizes with doxorubicin (DOX) in inducing immunogenic cell death in platinum-resistant ovarian cancer cells and increases survival in syngeneic and xenograft tumor models. However, the mechanisms underlying the virus- and doxorubicin-mediated cancer cell death remain unknown. In this study, we investigated the effect of the oncolytic virus and doxorubicin used alone or in combination on activation of the cytoplasmic transcription factor CREB3L1 (cyclic AMP [cAMP] response element-binding protein 3-like 1) in ovarian cancer cell lines and clinical specimens. We demonstrated that doxorubicin-mediated cell death in ovarian cancer cell lines was associated with nuclear translocation of CREB3L1 and that the effect was augmented by infection with oncolytic vaccinia virus or treatment with recombinant interferon (IFN)-β used as a viral surrogate. This combination treatment was also effective in mediating nuclear translocation of CREB3L1 in cancer cells isolated from ovarian tumor biopsies at different stages of disease progression. The measurement of CREB3L1 expression in clinical specimens of ovarian cancer revealed lack of correlation with the stage of disease progression, suggesting that understanding the mechanisms of nuclear accumulation of CREB3L1 after doxorubicin treatment alone or in combination with oncolytic virotherapy may lead to the development of more effective treatment strategies against ovarian cancer