Genetic Variants in Matrix Metalloproteinase-9 Gene Modify Metalloproteinase-9 Levels in Black Subjects

Altered matrix metalloproteinases (MMPs) levels are involved in cardiovascular diseases and increased MMP-9 levels enhance the cardiovascular risk in apparently healthy subjects. We investigated the effects of MMP-9 gene polymorphisms and haplotypes on the circulating MMP-9 levels in healthy black subjects and the effects of an MMP-2 polymorphism on the plasma MMP-2 concentrations. We studied 190 healthy subjects, nonsmokers, self-reported as blacks (18-63 years). Genotypes for the MMP-2 C-1306T polymorphism and the MMP-9 C-1562T, 90(CA)(14-24) and Q279R polymorphisms (rs243865, rs3918242, rs2234681, and rs17576, respectively) were determined by TaqMan (R) Allele Discrimination assay and real-time polymerase chain reaction or restriction fragment length polymorphism. Alleles for the 90(CA)(14-24) polymorphism were grouped as low (L) when there were < 21 and high (H) when there were >= 21 CA repeats. The plasma levels of MMP-2 and MMP-9 were determined by gelatin zymography. The software PHASE 2.1 was used to estimate the haplotypes frequencies. Although we found no effects of the MMP-9 C-1562T or the Q279R polymorphisms on MMP-9 levels, higher MMP-9 levels were associated with the HH genotype for the -90(CA)(14-24) polymorphism compared with the HL or LL genotypes. Lower MMP-9 levels were found in carriers of the CRL haplotype (combining the C, R, and L alleles for the MMP-9 polymorphisms) compared with the CRH haplotype. Consistent with this finding, the CRL haplotype was more commonly found in subjects with low MMP-9 levels. The MMP-2 C-1306T polymorphism had no effects on the plasma MMP-2 levels. Our results show that MMP-9 genetic variations modify MMP-9 levels in black subjects and may offer biochemical evidence implicating MMP-9 in the pathogenesis of cardiovascular diseases in blacks.

Thioredoxin interacting protein genetic variation is associated with diabetes and hypertension in the Brazilian general population

Objective: To investigate the relationship between TXNIP polymorphisms, diabetes and hypertension phenotypes in the Brazilian general population. Methods: Five hundred seventy-six individuals randomly selected from the general urban population according to the MONICA-WHO project guidelines were phenotyped for cardiovascular risk factors. A second, independent, sample composed of 487 family-trios from a different site was also selected. Nine TXNIP polymorphisms were studied. The potential association between TXNIP variability and glucose-phenotypes in children was also explored. TXNIP expression was quantified by real-time PCR in 53 samples from human smooth muscle cells primary culture. Results: TXNIP rs7211 and rs7212 polymorphisms were significantly associated with glucose and blood pressure related phenotypes. In multivariate logistic regression models the studied markers remained associated with diabetes even after adjustment for covariates. TXNIP rs7211 T/rs7212 G haplotype (present in approximately 17% of individuals) was significantly associated to diabetes in both samples. In children, the TXNIP rs7211 T/rs7212 G haplotype was associated with fasting insulin concentrations. Finally, cells harboring TXNIP rs7212 G allele presented higher TXNIP expression levels compared with carriers of TXNIP rs7212 CC genotype (p = 0.02). Conclusion: Carriers of TXNIP genetic variants presented higher TXNIP expression, early signs of glucose homeostasis derangement and increased susceptibility to chronic metabolic conditions such as diabetes and hypertension. Our data suggest that genetic variation in the TXNIP gene may act as a "common ground" modulator of both traits: diabetes and hypertension. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background: The mechanisms by which humans regulate pro-and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. Methodology/Principal Findings: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-alpha receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-alpha, IL-10/interferon (IFN)-gamma, IL-10/IL-6 and sTNFRII/TNF-alpha ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-alpha receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. Conclusions: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.

Polymorphisms in antithrombin and in tissue factor pathway inhibitor genes are associated with recurrent pregnancy loss

Recurrent pregnancy loss (RPL) is a multifactorial condition. The effect of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) single nucleotide polymorphisms (SNPs) on the risk of RPL is thus far unknown. Our objective was to determine the association of SNPs in the above mentioned genes with RPL. We included 117 non-pregnant women with three or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to viability, and 264 healthy fertile non-pregnant women who had at least two term deliveries and no known pregnancy losses. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292, rs8176592 and rs10153820) SNPs were analysed by Real Time PCR. Genotype frequencies for PROC 2418A > G, PROC 2405C > T, THBD 1418C > T, TFPI (T-33C and TFPI C-399T) SNPs were similar in cases and controls. The carriers of SERPINC1 786A allele (GA + AA genotypes) had an increased risk for RPL (odds ratio [OR]: 1.77, 95% confidence interval [CI]: 1.05-3.00, p=0.034) while women carrying the TFPI-287C allele (TC + CC genotypes) had a protection effect on having RPL (OR: 0.46, 95% CI: 0.26 - 0.83, p=0.009). The TCC haplotype for TFPI T-33C/TFPI T-287C/TFPI C-399T SNPs was less frequent in cases (5.7%) than in controls (11.6%) (OR: 0.45, 95% CI: 0.23 - 0.90, p=0.025). In conclusion, our data indicate that SERPINC1 786G > A variant increases the risk for RPL, while TFPI T-287C variant is protective; however, further studies are required to confirm our findings.

Surgical approach to medullary thyroid carcinoma associated with multiple endocrine neoplasia type 2

We briefly review the surgical approaches to medullary thyroid carcinoma associated with multiple endocrine neoplasia type 2 (medullary thyroid carcinoma/multiple endocrine neoplasia type 2). The recommended surgical approaches are usually based on the age of the affected carrier/patient, tumor staging and the specific rearranged during transfection codon mutation. We have focused mainly on young children with no apparent disease who are carrying a germline rearranged during transfection mutation. Successful management of medullary thyroid carcinoma in these cases depends on early diagnosis and treatment. Total thyroidectomy should be performed before 6 months of age in infants carrying the rearranged during transfection 918 codon mutation, by the age of 3 years in rearranged during transfection 634 mutation carriers, at 5 years of age in carriers with level 3 risk rearranged during transfection mutations, and by the age of 10 years in level 4 risk rearranged during transfection mutations. Patients with thyroid tumor >5 mm detected by ultrasound, and basal calcitonin levels >40 pg/ml, frequently have cervical and upper mediastinal lymph node metastasis. In the latter patients, total thyroidectomy should be complemented by extensive lymph node dissection. Also, we briefly review our data from a large familial medullary thyroid carcinoma genealogy harboring a germline rearranged during transfection Cys620Arg mutation. All 14 screened carriers of the rearranged during transfection Cys620Arg mutation who underwent total thyroidectomy before the age of 12 years presented persistently undetectable serum levels of calcitonin (<2 pg/ml) during the follow-up period of 2-6 years. Although it is recommended that preventive total thyroidectomy in rearranged during transfection codon 620 mutation carriers is performed before the age of 5 years, in this particular family the surgical intervention performed before the age of 12 years led to an apparent biochemical cure.

Fra il nome e la storia. Trasformazioni del discorso politico e concetto di classe al principio della monarchia di Luglio (1831-1832)

La ricerca indaga tensioni e trasformazioni che investono le principali correnti di pensiero politico in Francia nei primi anni della monarchia di Luglio, e vi osserva l’emergere del concetto di classe. Assumendo la dimensione dell’avvenimento come punto di intersezione fra storia e teoria, l’elaborato si concentra sul periodo novembre 1831-giugno 1832 per analizzare il modo in cui, nell’ordine del discorso politico repubblicano, liberale e socialista, le vicende di questi mesi vengono interpretate cercando di dar nome alle figure sociali che esse fanno irrompere nel dibattito pubblico. Il titolo Fra il nome e la storia fa dunque riferimento allo sforzo di indagare il campo di tensione che si apre fra concreto divenire storico e grandi operazioni di nominazione che segnano l’affiorare di strutture concettuali della lunga durata. L’emergere della nozione di classe operaia e delle categorie che intorno a essa si organizzano viene interpretata come una «formazione discorsiva» che pone in questione significato e confini del politico. La frattura del 1848 è assunta come orizzonte e margine esterno della ricerca nella misura in cui si ipotizza che essa segni una prima affermazione del regime di verità di tale formazione discorsiva: lo statuto politico del lavoro. L’elaborato consta di quattro capitoli. I primi tre indagano la riflessione sul politico e la funzione che in essa svolge il concetto di classe a partire dall’interpretazione di alcuni avvenimenti del tornante 1831-32 proposta nel discorso repubblicano del quotidiano «Le National» e della Société des Amis du Peuple, in quello del liberalismo dottrinario di François Guizot e in quello socialista nascente, prima del movimento sansimoniano, e poi muovendo fino al 1848 francese con l’analisi propostane da Karl Marx. Il quarto capitolo indaga infine la dimensione del «sociale», la sua elaborazione e articolazione attraverso il lavoro di studio e oggettivazione delle figure del mondo del lavoro.

Nanoparticles for efficient uptake by human dendritic cells and T lymphocytes to be used in adoptive immunotherapy

Dendritic cells (DCs) are the most potent cell type for capture, processing, and presentation of antigens. They are able to activate naïve T cells as well as to initiate memory T-cell immune responses. T lymphocytes are key elements in eliciting cellular immunity against bacteria and viruses as well as in the generation of anti-tumor and anti-leukemia immune responses. Because of their central position in the immunological network, specific manipulations of these cell types provide promising possibilities for novel immunotherapies. Nanoparticles (NP) that have just recently been investigated for use as carriers of drugs or imaging agents, are well suited for therapeutic applications in vitro and also in vivo since they can be addressed to cells with a high target specificity upon surface functionalization. As a first prerequisite, an efficient in vitro labeling of cells with NP has to be established. In this work we developed protocols allowing an effective loading of human monocyte-derived DCs and primary antigen-specific T cells with newly designed NP without affecting biological cell functions. Polystyrene NP that have been synthesized by the miniemulsion technique contained perylenmonoimide (PMI) as a fluorochrome, allowing the rapid determination of intracellular uptake by flow cytometry. To confirm intracellular localization, NP-loaded cells were analyzed by confocal laser scanning microscopy (cLSM) and transmission electron microscopy (TEM). Functional analyses of NP-loaded cells were performed by IFN-γ ELISPOT, 51Chromium-release, and 3H-thymidine proliferation assays. In the first part of this study, we observed strong labeling of DCs with amino-functionalized NP. Even after 8 days 95% of DCs had retained nanoparticles with a median fluorescence intensity of 67% compared to day 1. NP loading did not influence expression of cell surface molecules that are specific for mature DCs (mDCs) nor did it influence the immunostimulatory capacity of mDCs. This procedure did also not impair the capability of DCs for uptake, processing and presentation of viral antigens that has not been shown before for NP in DCs. In the second part of this work, the protocol was adapted to the very different conditions with T lymphocytes. We used leukemia-, tumor-, and allo-human leukocyte antigen (HLA) reactive CD8+ or CD4+ T cells as model systems. Our data showed that amino-functionalized NP were taken up very efficiently also by T lymphocytes, which usually had a lower capacity for NP incorporation compared to other cell types. In contrast to DCs, T cells released 70-90% of incorporated NP during the first 24 h, which points to the need to escape from intracellular uptake pathways before export to the outside can occur. Preliminary data with biodegradable nanocapsules (NC) revealed that encapsulated cargo molecules could, in principle, escape from the endolysosomal compartment after loading into T lymphocytes. T cell function was not influenced by NP load at low to intermediate concentrations of 25 to 150 μg/mL. Overall, our data suggest that NP and NC are promising tools for the delivery of drugs, antigens, and other molecules into DCs and T lymphocytes.

Caratterizzazione dei sintomi neurovegetativi e neuropsicologici nella malattia di Parkinson associata a mutazioni del gene glucocerebrosidasi

Pochi studi hanno indagato il profilo dei sintomi non-motori nella malattia di Parkinson associata al gene glucocerebrosidasi (GBA). Questo studio è mirato alla caratterizzazione dei sintomi non-motori, con particolare attenzione alla valutazione delle funzioni neurovegetativa, cognitiva e comportamentale, nel parkinsonismo associato a mutazione del gene GBA con la finalità di verificare se tali sintomi non-motori siano parte dello spettro clinico di questi pazienti. E’ stato condotto su una coorte di pazienti affetti da malattia di Parkinson che erano stati tutti sottoposti ad una analisi genetica per la ricerca di mutazioni in uno dei geni finora associati alla malattia di Parkinson. All’interno di questa coorte omogenea sono stati identificati due gruppi diversi in relazione al genotipo (pazienti portatori della mutazione GBA e pazienti non portatori di nessuna mutazione) e le caratteristiche non-motorie sono state confrontate nei due gruppi. Sono state pertanto indagati il sistema nervoso autonomo, mediante studio dei riflessi cardiovascolari e analisi dei sintomi disautonomici, e le funzioni cognitivo-comportamentali in pazienti affetti da malattia di Parkinson associata a mutazione del gene GBA. I risultati sono stati messi a confronto con il gruppo di controllo. Lo studio ha mostrato che i pazienti affetti da malattia di Parkinson associata a mutazione del gene GBA presentavano maggiore frequenza di disfunzioni ortosimpatiche, depressione, ansia, apatia, impulsività, oltre che di disturbi del controllo degli impulsi rispetto ai pazienti non portatori. In conclusione, i pazienti GBA positivi possono esprimere una sintomatologia non-motoria multidominio con sintomi autonomici, cognitivi e comportamentali in primo piano. Pertanto l’impostazione terapeutica in questi pazienti dovrebbe includere una accurata valutazione dei sintomi non-motori e un loro monitoraggio nel follow up clinico, allo scopo di ottimizzare i risultati e ridurre i rischi di complicazioni.

Raccontare la storia al tempo delle crisi

La ricerca analizza il tema della relazione tra storia e narrazione nella letteratura degli ultimi quindici anni in tre contesti nazionali: Italia, Spagna e Portogalo. Per indagare un campo così vasto si sono identificate tre direttrici principali connesse tra loro, coincidenti con tre "crisi": la crisi del rapporto tra letteratura e mercato, la crisi del concetto di verità e la crisi dello stato nazione. Attraverso le riflessioni sul postmoderno (Lyotard, Jameson Hutcheon) e l’analisi di Bourdieu si indaga il rapporto tra mercato e autore letterario, facendo particolare riferimento ai percorsi letterari di Rafael Chirbes, Mia Couto e Wu Ming. Il tema della forma letteraria è invece letto atttraverso le analisi di Hutcheon e analizzando i testi di Helder Macedo (Pedro e Paula), Isaac Rosa (¡Otra maldita novela sobre la guerra civil!) e Tommaso De Lorenzis-Guido Favale (L’aspra stagione). La crisi del concetto di verità viene analizzata alla luce del dibattito sulla storiografia nella seconda metà del Novecento. In particolare si evidenzia la tensione tra Hayden White e Carlo Ginzuburg. Per evidenziare come le relazioni di potere influenzino la narrazione della storia si fa inoltre riferimento alle analisi di Michel Foucault, Michel de Certeau, Stephen Greenbaltt e Gayatri Spivak. Si analizzano quindi Anatomía de un instante, di Javier Cercas, Romanzo criminale, di Giancarlo de Cataldo e As três vidas, di João Tordo. Infine ci si riferisce alla crisi dello stato-nazione individuando una tensione tra le analisi di György Lukács e Franco Moretti, e allargando la riflessione agli studi sociologici di Immanuel Wallerstein e Saskia Sassen. Inoltre, attraverso i testi di Benedict Anderson, Homi B. Bhabha, José Saramao e Eduardo Lourenço si articola una riflessione sull’immaginario politico nazionale. I testi analizzati sono Victus, di Albert Sánchez Piñol, Pro Patria, di Ascanio Celestini e A voz da terra di Miguel Real.

La strage di Ustica nell'opinione pubblica italiana (1980-1992): analisi di un caso politico e mediatico

Questa ricerca indaga come il “caso Ustica” si è articolato nell’opinione pubblica italiana negli anni compresi tra il 1980 e il 1992. Con l'espressione “caso Ustica” ci si riferisce al problema politico determinato dalle vicende legate all’abbattimento dell’aereo civile DC-9 dell’Itavia, avvenuto il 27 giugno 1980 in circostanze che, come noto, furono chiarite solamente a distanza di molti anni dal fatto. L’analisi intende cogliere le specificità del processo che ha portato la vicenda di Ustica ad acquisire rilevanza politica nell’ambito della sfera pubblica italiana, in particolare prendendo in considerazione il ruolo svolto dall’opinione pubblica in un decennio, quale quello degli anni ’80 e dei primi anni ’90 italiani, caratterizzato da una nuova centralità dei media rispetto alla sfera politica. Attraverso l’analisi di un’ampia selezione di fonti a stampa (circa 1500 articoli dei principali quotidiani italiani e circa 700 articoli tratti dagli organi dei partiti politici italiani) si sono pertanto messe in luce le dinamiche mediatiche e politiche che hanno portato alla tematizzazione di una vicenda che era rimasta fino al 1986 totalmente assente dall’agenda politica nazionale. L’analisi delle fonti giudiziarie ha permesso inoltre di verificare come la politicizzazione del caso Ustica, costruita intorno alla tensione opacità/trasparenza del potere politico e all’efficace quanto banalizzante paradigma delle “stragi di Stato”, sia risultata funzionale al raggiungimento, dopo il 1990, dei primi elementi di verità sulla tragedia e all’ampiamento del caso a una dimensione internazionale.

Die Expression und Regulation der CYP3A in menschlicher Haut

In Leber und Dünndarm bauen CYP3A-Enzyme eine Vielzahl von Fremdstoffen ab, die in den Körper gelangt sind. Zudem aber sind diese Enzyme auch in anderen Organen, wie der Haut exprimiert. Doch weder die genaue Zusammensetzung der CYP3A-Isozyme noch deren physiologische Rolle in der Haut sind bisher bekannt. Basierend auf begrenzten in vitro-Daten ist eine Rolle der CYP3A in der kutanen Vitamin D-Synthese denkbar. Auf der anderen Seite könnten die kutanen CYP3A auch lokal oder systemisch verabreichte Medikamente in der Haut verstoffwechseln und so zur Entstehung immunologischer und nicht-immunologischer unerwünschter Arzneimittelwirkungen beitragen, von denen sich bis zu 45 % in der Haut manifestieren.rnDie Arbeitshypothese dieses Projekts war, dass die CYP3A die kutane Synthese von Vitamin D regulieren. In dieser Funktion wurden sie zur Vermeidung von Vitamin D-Mangel-Erkrankungen wie Rachitis oder Osteomalazie in Europäern negativ selektiert. rnDie Expression und Regulation der CYP3A wurde in Hautbiopsien, einer Zelllinie epidermalen Ursprungs und primären Hautzellen wie auch in transgenen Mäusen untersucht. Die metabolische Aktivität der CYP3A gegenüber den kutanen Vitamin D-Vorstufen wurde mit Hilfe rekombinant exprimierter Enzyme untersucht. CYP3A5-mRNA war die häufigste der CYP3A in humanen Hautproben und überstieg die von CYP3A4 um das Dreifache, die von CYP3A7 um das 130-Fache. Damit entsprach diese 1,3 %, 0,01 % bzw. 0,01 % der jeweiligen hepatischen Genexpression. Die Expression von CYP3A43 war zu vernachlässigen. CYP3A5 zeigte eine bimodale Expression sowohl auf mRNA- als auch auf Proteinebene. So zeigten Träger der Wildtyp-Allels *1 eine 3,3-fach höhere mRNA- und 1,8-fach höhere Proteinmenge als homozygote Träger des Nullallels *3. CYP3A4/7- und CYP3A5-Protein wurde v. a. in den Keratinozyten der Epidermis und den Talgdrüsen, also den Bereichen der kutanen Vitamin D-Synthese lokalisiert. Die CYP3A5-Expression wurde ferner in der Haut transgener Mäusen gezeigt, die das Reportergen Luziferase unter Kontrolle des humanen CYP3A5-Promoters exprimieren. Verglichen mit der Leber war die kutane Expression des Vitamin D-Rezeptors (VDR) 100-fach höher, die der Xenosensoren CAR und PXR vergleichbar bzw. zu vernachlässigen. Dementsprechend erhöhte die Behandlung mit 1,25-Dihydroxyvitamin D, dem aktiven Vitamin D-Hormon, und dessen Vorstufen außer 7-Dehydrocholesterol, jedoch nicht der PXR-Ligand Rifampicin, die Expression der CYP3A. Wie in Zwei-Hybrid-Experimenten gezeigt, wurden die Effekte des 1,25-Dihydroxyvitamin D und dessen Vorstufen alleinig durch VDR vermittelt. Die Effektstärke hingegen war abhängig von Zellspender, Zellpassage und Zelltypus. Alle drei CYP3A-Isozyme metabolisieren Vitamin D zu einem oder mehreren unbekannten Metaboliten, jedoch nicht zu 25-Hydroxyvitamin D, dem direkten Vorläufer des aktiven Vitamin D. rnZusammengefasst legen die Daten nahe, dass die kutanen CYP3A, allen voran CYP3A5, die Vitamin D-Homöostase durch VDR-vermittelte Induktion des Abbaus von Vitamin D-Vorstufen regulieren. Dies zusammen mit Sequenzdaten liefert starke Indizien für Vitamin D als treibende Kraft der Selektion des CYP3A-Lokus in Europäern. Der Einfluss der CYP3A-Expression auf selektiv wirksame, klinisch relevante Knochenveränderungen wie Rachitis oder Osteomalazie müssen folgen.rn

Funktionelle HPMA-Copolymere zur möglichen Anwendung in der Tumor-Immuntherapie

Der Fokus dieser Arbeit lag in der Synthese von funktionellen HPMA-Copolymeren, sowohl für die Darstellung definierter Polymer-Antikörper Konjugate, als auch zum effizienten Transport von p-DNA in Polymer-DNA Komplexen (Polyplexe). Nach ausführlicher physikalischer und chemischer Charakterisierung wurden gezielt ihre Wechselwirkungen mit (Immun)-Zellen untersucht und so ihr Potential für die Verwendung in der Tumor-Immuntherapie aufgezeigt.rnFür das gezielte Ansprechen von bestimmten Immunzellen mit Schlüsselfunktionen besitzen monoklonale Antikörper ein großes Potential. Im Rahmen dieser Arbeit gelang die Darstellung definierter Polymer-Antikörper Konjugate über das gezielte Einführen von Thiol-Gruppen an Antikörper und die Synthese eng verteilter, Maleinimid funktionalisierter HPMA-Copolymere. Diese sehr gut definierten, funktionellen HPMA-Copolymere konnten über die Kombination der RAFT-Polymerisation und Reaktivester Polymeren gewonnen werden. Unterschiedliche Polymerstrukturen ermöglichten die Synthese verschiedener Arten von Polymer-Antikörper Konjugaten. Speziell die Untersuchung der verschiedenen Konjugate aus dem für dendritische Zellen spezifischen aDEC-205 Antikörper an Immunzellen aus dem Knochenmark von Mäusen lieferten wertvolle Erkenntnisse über Struktur-Wirkungsbeziehungen und zeigten die Möglichkeit der gezielten Adressierung von Immunzellen mit Schlüsselfunktionen bei der Aktivierung einer (Tumor)-Immunabwehr am Beispiel von dendritischen Zellen. Gleichzeitig erlaubt der Syntheseweg sowohl die gleichzeitige und kontrollierte Einführung auch komplexerer Stimuli am Polymerrückgrat als auch die Verwendung verschiedener Antikörper.rnÜber die Kombination der RAFT-Polymerisation und polymeren Reaktivestern wurde ebenso die Synthese von neuartigen kationisch-hydrophilen Polylysin-b-poly(HPMA) Blockcopolymeren als effiziente Transporter für den komplexen aber wirkungsvollen Wirkstoff p-DNA in Form von Polymer-DNA Komplexen (Polyplexe) realisiert. Da diese Polyplexe gleichzeitig eine Abschirmung der sensitiven p-DNA über eine poly(HPMA)-Korona vermitteln, stellen sie allgemein ein geeignetes Transportmittel für einen therapeutischen Transport von p-DNA dar. Diese Polyplexe sind in der Lage, humane Nierenkarzinomzellen (HEK-293T Zelllinie) zu transfizieren ohne signifikante Zytotoxizität zu zeigen. Darüber hinaus gelang eine große Steigerung der Transfektionseffizienz, ohne eine gleichzeitige Erhöhung der Zytotoxizität, durch die gezielte Einführung von Redox-stimuliresponsiven Disulfid-Gruppen zwischen den einzelnen Blöcken. Diese Polyplexe stellen einen polymeren Vektor zur transkriptionellen Regulierung von Zellen dar, zum Beispiel für die transkriptionelle Aktivierung von dendritischen Zellen, durch die Verwendung speziell dafür modifizierter p-DNA-Konstrukte. rnDurch die Verknüpfung einer ortsspezifischen enzymatischen Kopplung und kupferfreien Cyclooctin-Azid Kupplung gelang die kontrollierte und kovalente Modifizierung von polymeren Mizellen mit aDEC-205 Antikörpern an der hydrophilen poly(HPMA)-Korona. Diese Methode bietet die Möglichkeit der Anbindung der effektiven aber anspruchsvollen Erkennungsstruktur Antikörper an komplexere Polymerstrukturen und andere nano-partikulären Systeme, zum Beispiel an die zuvor genannten Polyplexe, um eine zellspezifische und verbesserte Aufnahme und Prozessierung zu erreichen.rnDiese Studien zeigen somit, sowohl die Möglichkeit der selektiven Addressierung von Immunzellen mit Schlüsselfunktionen wie dendritischer Zellen, als auch die Möglichkeit der transkriptionellen Regulation von Zellen durch Polyplexe. Sie stellen somit einen ersten Schritt zur Herstellung funktioneller, nanopartikulärer Systeme zur Verwendung in der Tumor-Immuntherapie dar. rn

Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in caucasians

A recent genome-wide study revealed an association between variation in the PNPLA3 gene and liver fat content. In addition, the PNPLA3 single-nucleotide polymorphism rs738409 (M148I) was reported to be associated with advanced alcoholic liver disease in alcohol-dependent individuals of Mestizo descent. We therefore evaluated the impact of rs738409 on the manifestation of alcoholic liver disease in two independent German cohorts. Genotype and allele frequencies of rs738409 (M148I) were determined in 1,043 alcoholic patients with or without alcoholic liver injury and in 376 at-risk drinkers from a population-based cohort. Relative to alcoholic patients without liver damage (n = 439), rs738409 genotype GG was strongly overrepresented in patients with alcoholic liver cirrhosis (n = 210; OR 2.79; P(genotype) = 1.2 × 10(-5) ; P(allelic) = 1.6 × 10(-6) ) and in alcoholic patients without cirrhosis but with elevated alanine aminotransferase levels (n = 219; OR 2.33; P(genotype) = 0.0085; P(allelic) = 0.0042). The latter, biochemically defined association was confirmed in an independent population-based cohort of at-risk drinkers with a median alcohol intake of 300 g/week (OR 4.75; P(genotype) = 0.040; P(allelic) = 0.022), and for aspartate aminotransferase (AST) levels. Frequencies of allele PNPLA3 rs738409(G) in individuals with steatosis and normal alanine aminotransferase (ALT) and AST levels were lower than in alcoholics without steatosis and normal ALT/AST (P(combined) = 0.03). The population attributable risk of cirrhosis in alcoholic carriers of allele PNPLA3 rs738409(G) was estimated at 26.6%. CONCLUSION: Genotype PNPLA3 rs738409(GG) is associated with alcoholic liver cirrhosis and elevated aminotransferase levels in alcoholic Caucasians.

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.

Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets

Background PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters. Methods We performed nutritional interventions in young healthy male volunteers and offspring of type 2 diabetic (OffT2D) patients that are more prone to develop insulin resistance, including: i) acute post-prandial hyperlipidemic challenge (n=10), ii) 4 days of high-fat (HF) or high-fat/high-protein (HFHP) (n=10), iii) 7 (HFruc1, n=16) or 6 (HFruc2, n=9) days of hypercaloric high-fructose diets. An acute oral fat load was also performed in two patients bearing the R104C-V114A loss-of-function (LOF) PCSK9 mutation. Plasma PCSK9 concentrations were measured by ELISA. For the HFruc1 study, intrahepatocellular (IHCL) and intramyocellular lipids were measured by 1H magnetic resonance spectroscopy. Hepatic and whole-body insulin sensitivity was assessed with a two-step hyperinsulinemic-euglycemic clamp (0.3 and 1.0 mU.kg-1.min-1). Findings HF and HFHP short-term diets, as well as an acute hyperlipidemic oral load, did not significantly change PCSK9 concentrations. In addition, post-prandial plasma triglyceride excursion was not altered in two carriers of PCSK9 LOF mutation compared with non carriers. In contrast, hypercaloric 7-day HFruc1 diet increased plasma PCSK9 concentrations by 28% (p=0.05) in healthy volunteers and by 34% (p=0.001) in OffT2D patients. In another independent study, 6-day HFruc2 diet increased plasma PCSK9 levels by 93% (p<0.0001) in young healthy male volunteers. Spearman’s correlations revealed that plasma PCSK9 concentrations upon 7-day HFruc1 diet were positively associated with plasma triglycerides (r=0.54, p=0.01) and IHCL (r=0.56, p=0.001), and inversely correlated with hepatic (r=0.54, p=0.014) and whole-body (r=−0.59, p=0.0065) insulin sensitivity. Conclusions Plasma PCSK9 concentrations vary minimally in response to a short term high-fat diet and they are not accompanied with changes in cholesterolemia upon high-fructose diet. Short-term high-fructose intake increased plasma PCSK9 levels, independent on cholesterol synthesis, suggesting a regulation independent of SREBP-2. Upon this diet, PCSK9 is associated with insulin resistance, hepatic steatosis and plasma triglycerides.