Functional Variant in Complement C3 Gene Promoter and Genetic Susceptibility to Temporal Lobe Epilepsy and Febrile Seizures

BACKGROUND: Human mesial temporal lobe epilepsies (MTLE) represent the most frequent form of partial epilepsies and are frequently preceded by febrile seizures (FS) in infancy and early childhood. Genetic associations of several complement genes including its central component C3 with disorders of the central nervous system, and the existence of C3 dysregulation in the epilepsies and in the MTLE particularly, make it the C3 gene a good candidate for human MTLE. METHODOLOGY/PRINCIPAL FINDINGS: A case-control association study of the C3 gene was performed in a first series of 122 patients with MTLE and 196 controls. Four haplotypes (HAP1 to 4) comprising GF100472, a newly discovered dinucleotide repeat polymorphism [(CA)8 to (CA)15] in the C3 promoter region showed significant association after Bonferroni correction, in the subgroup of MTLE patients having a personal history of FS (MTLE-FS+). Replication analysis in independent patients and controls confirmed that the rare HAP4 haplotype comprising the minimal length allele of GF100472 [(CA)8], protected against MTLE-FS+. A fifth haplotype (HAP5) with medium-size (CA)11 allele of GF100472 displayed four times higher frequency in controls than in the first cohort of MTLE-FS+ and showed a protective effect against FS through a high statistical significance in an independent population of 97 pure FS. Consistently, (CA)11 allele by its own protected against pure FS in a second group of 148 FS patients. Reporter gene assays showed that GF100472 significantly influenced C3 promoter activity (the higher the number of repeats, the lower the transcriptional activity). Taken together, the consistent genetic data and the functional analysis presented here indicate that a newly-identified and functional polymorphism in the promoter of the complement C3 gene might participate in the genetic susceptibility to human MTLE with a history of FS, and to pure FS. CONCLUSIONS/SIGNIFICANCE: The present study provides important data suggesting for the first time the involvement of the complement system in the genetic susceptibility to epileptic seizures and to epilepsy

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

The TRI 2007 and CMP 2013 Surveys of Members of Parliament

The study was conducted in two waves of interviews among members of parliament in two old and five new democracies as part of a larger comparative study of democratic development. While the TRI 2007 elite survey did not only include members of parliament, but also media elites, the integrated data file includes parliamentarians only. During the survey period 04.11.2006 to 16.12.2013, members of parliament were interviewed in face-to-face interviews and telephone interviews on the following topics: democracy, religion and the church, and the impact of the global financial and economic crisis. Respondents were selected by random sampling (Chile: full survey (Total universe/Complete enumeration).Satisfaction with life; trusting most people vs. need to be very careful; groups of people the respondent would not like to have as neighbors (people of a different race, people who have AIDS, immigrants or foreign workers, people of a different religion); estimated proportion of the population who would not want immigrants or foreign workers as neighbors; estimated proportion of voters or supporters of own party who would not want immigrants or foreign workers as neighbors; no freedom of choice vs. great deal of control over own life; people taking advantage of you if the got a chance vs. people try to be fair; country’s most important aim (first choice and second choice: maintaining order in the nation, giving people mor say in important government decisions, fighting rising prices, protecting freedom of speech); respondent`s position on a Left-right scale; estimated position of respondents party on a Left-right scale; estimated position of party`s voters on a Left-right scale; estimated position of population as a whole on a Left-right scale; income equality vs. income differences as incentives for individual effort; private vs. government ownership of business and industry; private vs. government responsibility for provision; competition is good and stimulates people to work hard vs. competition is harmful and brings out the worst in people; confidence in different organizations (religious communities, armed forces, press, television, labor unions, police, courts, government, political parties, parliament, the civil service, banks, environmental organizations, women`s organizations, charitable or humanitarian organizations, country specific regional organization (e.g. African Union, EU), United Nations, Constitutional Court; rating of various types of political systems (having a strong leader who does not have to bother with parliament and elections, having experts, not government, make decisions according to what they think is best for the country, having the army rule, having a democratic political system); essential characteristics of democracy (governments tax the rich and subsidize the poor, the individual human right to dignity (e.g. respect of others) is upheld, people choose their leaders in free elections, People receive state aid for unemployment, Civil rights protect people’s liberty against oppression, the economy is prospering, women have the same rights as men, complete freedom for anyone to criticize the government, equality of the vote in elections, basic necessities like shelter, food and water for everyone, jobs for everyone, equal opportunity in education, minority rights); rating of the current governments performance in handling the above mentioned matters; agreement with the following statement: Although democracy has many shortcomings, it is still better than any other political system; extend to which the country is democratically governed today; satisfaction with the development of democracy in the country; extent of respect for individual human rights nowadays in the country; likelihood that authorities could enforce the law if a person committed a serious crime and if a person did not pay tax on some of the earned income; likelihood that court will punish elected public office holders, civil servants and managers of private businesses who take bribes; prevalence of corruption in the country amongst elected public office holders, amongst civil servants, and amongst managers of private businesses; agreement with the following statements about the current political system (people have freedom of speech and association, nobody needs to be afraid of arbitrary arrest, people have the freedom to choose who to vote for without being pressured); rating of the communication between various political groups (governing and opposition parties, governing parties and mass media, opposition parties and mass media, governing parties and NGOs, and between the opposition parties and NGOs). Frequency of thinking about the meaning of life; religion or religious denomination; frequency of attending religious services; self-evaluation as religious person; opinion on churches (are giving adequate answers to the moral problems and needs of the individual, to the problems of family life, to people`s spiritual needs and to the social problems facing the society); importance of God in life; justification of homosexuality, of prostitution, of abortion, of divorce and of death penalty; proud to be citizen of the country. Global financial and economic crisis: evaluation of the current state of economy; evaluation of the state of economy compared to 12 months ago; evaluation of the state of economy over the next 12 months; impact of financial and economic crisis on the country in general, on country´s economy, on country´s banking system, on the people´s standard of living, and on the quality of democracy in the country; rating of the performance of different actors in handling the impact of the crisis (national government, parties (country specific), International Monetary Fund (IMF), G20, and the EU); personal involvement in government decision-making dealing with the crisis; parties that benefited or suffered from the crisis (country specific); crisis as a consequence of neo-liberal economic policies; impact of the crisis has already reached its peak vs. the worst is yet to come; position of different parties (country specific) on economic and welfare policies; respondents position on welfare state dealing with the crisis; rating of the contribution of national policy measures to overcoming the crisis; fairness of income distribution in the country; impact of the crisis on income distribution ; frequency of: public procurement contracts for most favorable kickbacks, public sector employees treat some groups unfairly, public sectors favor applicants with whom the have strong personal contacts, public sector employees act impartially); economic policy should be run by politicians or experts; economic policy in the country is run by politicians or experts; evaluation of globalization; more important in a democracy: equality or freedom; closer to own vision of state: guaranteeing political freedoms or equalizing citizens` welfare; perceived conflict between different groups in society (between the rich and the poor, young and old, religious believers and non-believers (atheists), women and men, employees and employers, public and private sector employees, educated and uneducated, working and middle classes, and between farmers and urban population). Demography: country of birth; highest level of formal education, father´s and mother´s highest level of formal education, party of respondent (country specific). Additionally coded was: survey wave; unique ID, case ID; country of interview; date of interview; weighting factors.Die Studie wurde in zwei Befragungswellen unter Parlamentsmitgliedern in zwei alten und fünf neuen Demokratien im Rahmen einer größeren vergleichenden Studie zur demokratischen Entwicklung durchgeführt. Während in der Elitenerhebung des TRI 2007 nicht nur Parlamentarier, sondern auch Medieneliten erfasst wurden, sind im integrierte Datensatz nur die Daten der Parlamentarier enthalten. Im Erhebungszeitraum 04.11.2006 bis 16.12.2013 wurden Parlamentsmitglieder in persönlichen und telefonischen Interviews zu folgenden Themen befragt: Demokratie, Religion und Kirche sowie Einfluss der weltweiten Finanz- und Wirtschaftskrise. Die Auswahl der Befragten erfolgte durch Wahrscheinlichkeitsauswahl (Chile: Vollerhebung).Lebenszufriedenheit; Vertrauen in die meisten Menschen vs. Notwendigkeit, sehr vorsichtig zu sein; Personengruppen, die der Befragte ungern als Nachbarn hätte (Menschen anderer Hautfarbe, Menschen mit AIDS, Immigranten/Gastarbeiter, Menschen anderer Religion); geschätzter Anteil der Bevölkerung, der Immigranten/Gastarbeiter als Nachbarn ablehnt; geschätzter Anteil der Wähler oder Anhänger der eigenen Partei, die Immigranten/Gastarbeiter als Nachbarn ablehnen; keine Wahlfreiheit vs. viel Kontrolle über das eigene Leben; Menschen, die einen ausnutzen, wenn sie die Chance dazu haben vs. Menschen versuchen, fair zu sein; wichtigstes Ziel des Landes (erste und zweite Wahl: Aufrechterhaltung der Ordnung im Land, mehr Mitsprache bei wichtigen Regierungsentscheidungen, Kampf gegen steigende Preise, Schutz der Redefreiheit); Position des Befragten auf einer Links-Rechts-Skala; geschätzte Position der Partei des Befragten auf einer Links-Rechts-Skala; geschätzte Position der Wähler der Partei auf einer Links-Rechts-Skala; geschätzte Position der Bevölkerung insgesamt auf einer Links-Rechts-Skala; Einkommensgleichheit vs. Einkommensunterschiede als Anreiz für individuelle Anstrengungen; privates vs. staatliches Eigentum an Unternehmen und Industrie; private vs. staatliche Verantwortung für die Versorgung; Wettbewerb ist gut und stimuliert die Menschen zu harter Arbeit vs. Wettbewerb ist schädlich und bringt das Schlechteste im Menschen hervor; Vertrauen in verschiedene Organisationen (Religionsgemeinschaften, Streitkräfte, Presse, Fernsehen, Gewerkschaften, Polizei, Gerichte, Regierung, politische Parteien, Parlament, öffentlicher Dienst, Banken, Umweltorganisationen, Frauenorganisationen, karitative oder humanitäre Organisationen, länderspezifische regionale Organisationen (z. B. Afrikanische Union, Europäische Union), Vereinte Nationen, Verfassungsgericht; Bewertung verschiedener Arten politischer Systeme (ein starker Führer, der sich nicht um Parlament und Wahlen kümmern muss, Experten, nicht die Regierung, die Entscheidungen treffen, die sie am besten für das Land halten, die Armee als Herrscher, ein demokratisches politisches System); wesentliche Merkmale der Demokratie (Regierungen besteuern Reiche und subventionieren Arme, das individuelle Menschenrecht auf Würde (z. B. Respekt vor anderen) wird gewahrt, die Menschen wählen ihre Führer in freien Wahlen, die Menschen erhalten staatliche Hilfe bei Arbeitslosigkeit, die Bürgerrechte schützen die Freiheit der Menschen vor Unterdrückung, die Wirtschaft floriert, Frauen haben die gleichen Rechte wie Männer, völlige Freiheit für jeden, die Regierung zu kritisieren, Gleichheit der Stimmen bei Wahlen, Grundbedürfnisse wie Unterkunft, Nahrung und Wasser für alle, Arbeitsplätze für alle, Chancengleichheit im Bildungswesen, Minderheitenrechte); Bewertung der Leistung der gegenwärtigen Regierung im Umgang mit den oben genannten Merkmalen; Zustimmung zu folgender Aussage: Obwohl die Demokratie viele Mängel hat, ist sie immer noch besser als jedes andere politische System; Ausmaß, in dem das Land heute demokratisch regiert wird; Zufriedenheit mit der Entwicklung der Demokratie im Land; Ausmaß der Achtung der individuellen Menschenrechte heutzutage im Land; Wahrscheinlichkeit, dass die Behörden das Gesetz durchsetzen können, wenn eine Person ein schweres Verbrechen begangen hat und wenn eine Person einen Teil ihres Einkommens nicht versteuert hat; Wahrscheinlichkeit, dass Gerichte gewählte Amtsträger, Beamte und Manager von Privatunternehmen bestrafen, die Bestechungsgelder annehmen; Verbreitung von Korruption im Land unter gewählten Amtsträgern, unter Beamten und unter Managern von Privatunternehmen; Zustimmung zu den folgenden Aussagen über das derzeitige politische System (die Menschen haben Rede- und Vereinigungsfreiheit, niemand muss Angst vor willkürlicher Verhaftung haben, die Menschen haben die Freiheit zu wählen, wen sie wählen wollen, ohne unter Druck gesetzt zu werden); Bewertung der Kommunikation zwischen verschiedenen politischen Gruppen (Regierungs- und Oppositionsparteien, Regierungsparteien und Massenmedien, Oppositionsparteien und Massenmedien, Regierungsparteien und NRO sowie zwischen den Oppositionsparteien und NRO). Häufigkeit des Nachdenkens über den Sinn des Lebens; Religion oder Konfession; Häufigkeit des Besuchs von Gottesdiensten; Selbsteinschätzung als religiöse Person; Meinung über Kirchen (geben angemessene Antworten auf die moralischen Probleme und Bedürfnisse des Einzelnen, auf die Probleme des Familienlebens, auf die spirituellen Bedürfnisse der Menschen und auf die sozialen Probleme der Gesellschaft); Bedeutung von Gott im Leben; Rechtfertigung von Homosexualität, Prostitution, Abtreibung, Scheidung und Todesstrafe; Stolz darauf, Bürger des Landes zu sein. Globale Finanz- und Wirtschaftskrise: Bewertung der aktuellen Wirtschaftslage; Bewertung der Wirtschaftslage im Vergleich zu vor 12 Monaten; Bewertung der Wirtschaftslage in den nächsten 12 Monaten; Auswirkungen der Finanz- und Wirtschaftskrise auf das Land im Allgemeinen, auf die Wirtschaft des Landes, auf das Bankensystem des Landes, auf den Lebensstandard der Bevölkerung und auf die Qualität der Demokratie im Land; Bewertung der Leistung der verschiedenen Akteure bei der Bewältigung der Auswirkungen der Krise (nationale Regierung, Parteien (länderspezifisch), Internationaler Währungsfonds (IWF), G20 und EU); Persönliche Beteiligung an der Entscheidungsfindung der Regierung im Umgang mit der Krise; Parteien, die von der Krise profitiert oder darunter gelitten haben (länderspezifisch); Krise als Folge der neoliberalen Wirtschaftspolitik; Auswirkungen der Krise haben ihren Höhepunkt bereits erreicht bzw. das Schlimmste steht noch bevor; Position der verschiedenen Parteien (länderspezifisch) zur Wirtschafts- und Sozialpolitik; Position der Befragten zum Umgang des Sozialstaates mit der Krise; Bewertung des Beitrags nationaler politischer Maßnahmen zur Überwindung der Krise; Gerechtigkeit der Einkommensverteilung im Land; Auswirkungen der Krise auf die Einkommensverteilung; Häufigkeit von: Vergabe öffentlicher Aufträge gegen Schmiergelder, Angestellte des öffentlichen Sektors behandeln einige Gruppen unfair, Angestellte des öffentlichen Sektors bevorzugen Bewerber, zu denen sie starke persönliche Kontakte haben, Angestellte des öffentlichen Sektors handeln unparteiisch; Wirtschaftspolitik sollte von Politikern oder Experten betrieben werden; Wirtschaftspolitik im Land wird von Politikern oder Experten betrieben; Bewertung der Globalisierung; wichtiger in einer Demokratie: Gleichheit oder Freiheit; näher an der eigenen Vorstellung vom Staat: Gewährleistung politischer Freiheiten oder Ausgleich des Wohlergehens der Bürger; wahrgenommener Konflikt zwischen verschiedenen Gruppen in der Gesellschaft (zwischen Reichen und Armen, Jungen und Alten, Gläubigen und Nicht-Gläubigen (Atheisten), Frauen und Männern, Arbeitnehmern und Arbeitgebern, Angestellten des öffentlichen und privaten Sektors, Gebildeten und Ungebildeten, Arbeitern und Mittelschicht sowie zwischen Landwirten und städtischer Bevölkerung). Demografie: Geburtsland; höchster formaler Bildungsgrad; höchster formaler Bildungsgrad von Vater und Mutter; Partei des Befragten (länderspezifisch). Zusätzlich kodiert wurden: Erhebungswelle; eindeutige ID; Fall-ID; Land der Befragung; Interviewdatum; Gewichtungsfaktoren

Upregulation of SIRPα upon differentiation of t(15;17) NB4 cells and induction of cell death following its triggering.

<p>(A) NB4 cells were exposed to 1 µM ATRA and granulocytic differentiation of the cells was examined by cell surface expression of the common myeloid marker, CD11b. (B) SIRPα protein expression, determined by western blotting, is upregulated in ATRA-incubated NB4 cells. β-actin is used as a loading control. (C) Flow cytometric analysis of chSIRPα surface expression is determined by using ED9 mAb in transduced NB4 empty vector and chSIRPα expressing cells. (D) 24 hrs following ED9 (10 µg/ml) incubation, the percentage of cell death in chSIRPα and EV transduced NB4 cells was quantified by APC-Annexin V and PE-7AAD FACS staining. (E) Percentage of apoptosis after exposure to 1 µM ATRA is shown in combination with 10 µg/ml of ED9.</p

SIRPα-derived signal synergizes with different antileukemic drugs.

<p>Inhibition of cell growth is depicted by combination of ED9 mAb (10 µg/ml) with (A) Ara-C and DNR in NB4 cells expressing chSIRPα (B) Ara-C, DNR, VP16, DAC and imatinib in Kasumi-1 cells expressing chSIRPα. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052143#s3" target="_blank">Results</a> are based on means of 3 experiments and are calculated using Calcusyn.</p

SIRPα protein expression in AML cell lines and patients.

<p>Western blot analysis was performed in (A) cell lines and (B) 20 pediatric AML patient samples. β-actin staining was used as loading control. (C) SIRPα expression is quantified relative to β-actin expression.</p

SIRPα upregulation in t(8;21) Kasumi-1 cells following treatment with inhibitors of epigenetic gene silencing.

<p>Kasumi-1 cells were incubated with 1 µM Decitabine, 0.5 mM valproic acid, 1 mM Butyrate and 300 nM Trichostatin. Endogenous SIRPα protein level, determined by Western blotting was upregulated at indicated time points. β-actin staining was used as a loading control.</p

Ligation of chSIRPα induces caspase 3-independent PCD in Kasumi-1 cells.

<p>(A) Flow cytometric analysis of SIRPα expression was performed by using ED9 mAb in stable Kasumi-1 cells expressing chSIRPα and EV. (B) Kasumi-1 chSIRPα and EV cells were incubated with 10 µg/ml ED9 mAb and the percentage of cell death was determined after 24 hrs. Annexin V and 7-AAD FACS staining defined that ligation of chSIRPα resulted in increased cell death in chSIRPα Kasumi-1 cells compared to EV control cells. Data are means ± SD calculated from 3 independent experiments using triplicate samples (*: significant difference <i>p</i><0.05). (C) Kasumi-1 cells expressing chSIRPα or EV were treated with 10 µg/ml ED9 for mentioned time points. Caspase 3 staining shows no cleavage of the p32 subunit. As a positive control for caspase 3 cleavage, human neutrophils (PMN) were incubated at room temperature for 0 and 24 hours.</p

Engagement of SIRP alpha Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

<p>Background: Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRP alpha) on macrophages. Although AML cells express SIRP alpha, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRP alpha in acute myeloid leukemia.</p><p>Design and Methods: We analyzed the expression of SIRP alpha, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRP alpha on two low SIRP alpha expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRP alpha expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.</p><p>Results: By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRP alpha is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0-M3. Interestingly, AML patients with high SIRP alpha expression had a poor prognosis. Our results also showed that SIRP alpha is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRP alpha with an agonistic antibody in the cells stably expressing chimeric SIRP alpha, led to inhibition of growth and induction of programmed cell death. Finally, the SIRP alpha-derived signaling synergized with the activity of established antileukemic drugs.</p><p>Conclusions: Our data indicate that triggering of SIRP alpha has antileukemic effect and may function as a potential therapeutic target in AML.</p>