Bettina Kulle

NAD(P)H Oxidase and Multidrug Resistance Protein Genetic Polymorphisms Are Associated With Doxorubicin-Induced Cardiotoxicity

Background— A significant number of patients treated with anthracyclines develop cardiotoxicity (anthracycline-induced cardiotoxicity [ACT]), mainly presenting as arrhythmias (acute ACT) or congestive heart failure (chronic ACT). There are no data on pharmacogenomic predictors of ACT. Methods and Results— We genotyped participants of the German non-Hodgkin lymphoma study (NHL-B) who were followed up for the development of heart failure for a median of >3 years. Single-nucleotide polymorphisms (SNPs) were selected from 82 genes with conceivable relevance to ACT. Of 1697 patients, 55 developed acute and 54 developed chronic ACT (cumulative incidence of either form, 3.2%). We detected 5 significant associations with polymorphisms of the NAD(P)H oxidase and doxorubicin efflux transporters. Chronic ACT was associated with a variant of the NAD(P)H oxidase subunit NCF4 (rs1883112, −212A→G; symbols with right-pointing arrows, as edited?‘ odds ratio [OR], 2.5; 95% CI, 1.3 to 5.0). Acute ACT was associated with the His72Tyr polymorphism in the p22phox subunit (rs4673; OR, 2.0; 95% CI, 1.0 to 3.9) and with the variant 7508T→A (rs13058338; OR, 2.6; 95% CI, 1.3 to 5.1) of the RAC2 subunit of the same enzyme. In agreement with these results, mice deficient in NAD(P)H oxidase activity, unlike wild-type mice, were resistant to chronic doxorubicin treatment. In addition, acute ACT was associated with the Gly671Val variant of the doxorubicin efflux transporter multidrug resistance protein 1 (MRP1) (OR, 3.6; 95% CI, 1.6 to 8.4) and with the Val1188Glu-Cys1515Tyr (rs8187694-rs8187710) haplotype of the functionally similar MRP2 (OR, 2.3; 95% CI, 1.0 to 5.4). Polymorphisms in adrenergic receptors previously demonstrated to be predictive of heart failure were not associated with ACT. Conclusions— Genetic variants in doxorubicin transport and free radical metabolism may modulate the individual risk to develop ACT.

Sex-dependent genetic markers of CYP3A4 expression and activity in human liver microsomes.

OBJECTIVEnTo find genetic markers of the individual cytochrome P450 (CYP)3A expression.nnnMETHODSnA large collection of liver samples phenotyped for CYP3A expression and activity was genotyped for CYP3A variants. Data were analyzed for associations between CYP3A phenotypes and genotypes, and for evidence of recent selection.nnnRESULTSnWe report associations between the hepatic CYP3A4 protein expression level, as well as its enzymatic activity, measured as verapamil N-dealkylation, and genetic polymorphisms from two regions within the CYP3A gene cluster. One region is defined by several variants, mostly located within CYP3A7, the other by a single nucleotide polymorphism in intron 7 of CYP3A4. The effects of these single nucleotide polymorphisms are sex-dependent. For example, female carriers of T alleles of the single nucleotide polymorphism rs4646437C>T in CYP3A4 intron 7 have, respectively, 5.1-fold and 2.7-fold higher expression and activity compared with male T-carriers, but only 2.2-fold and 1.4-fold higher expression and activity compared with males of genotype CC. A regression analysis indicates that the impact of these single nucleotide polymorphisms in men goes beyond the previously reported sex effect. The rs4646437C undergoes positive selection in Caucasians, as evidenced by its relative extended haplotype homozygosity value located within the uppermost percentile of a genome-wide test set of haplotypes in the same 5% frequency bin.nnnCONCLUSIONSnOur findings reconcile the apparent contradiction between the evidence for the influence of the individual genetic makeup on CYP3A4 expression and activity suggested by clinical studies, and the failure to identify the responsible gene variants.

Survival probabilities and hazard functions of malignant melanoma in Germany 1972–1996, an analysis of 10 433 patients. Evolution of gender differences and malignancy

The evaluation of the impact of prevention activities on the course of survival in conjunction with the individual hazard rate of dying is described using data from a follow-up study of 10433 melanoma patients during three observation periods (1972-1980, 1981-1988, 1989-1996). Kaplan-Meier survival curves combined with hazard functions were calculated. At all observation periods, survival of men was lower compared with women and their maximum dying risk was earlier (70 versus 100 months after removal of the primary tumour). In 1989-1996, differences in the survival rates were approximately halved compared with those for 1972-1980 or 1981-1988, respectively. This improvement was predominantly seen in young men. There was a lower survival rate of men compared with women with identical thickness categories. The maximum dying risk for those men with tumours >4 mm peaked at approximately 60 months, the other thickness categories showing a lower and later maximum; in women, the maximum dying risk for tumours >4 mm was also seen at approximately 60 months, but less pronounced. Over time, the influence of Breslow thickness on the survival rates remained constant in women; in men, with the exception of thick tumours, there was a trend towards a better survival. Melanoma awareness campaigns conducted in Germany since the late 1980s have resulted in a trend towards a remarkable increase of thin tumours in recent years, whereas the number of new cases with thick tumours has remained constant.

[In-vitro study of the cellular response of human fibroblasts cultured on alloplastic hernia meshes. Influence of mesh material and structure].

ZusammenfassungHintergrundZur Therapie von Bauchwandhernien steht eine Vielzahl unterschiedlicher Kunststoffnetze zur Verfügung. Neben dem Material selbst scheint für die Gewebeverträglichkeit dieser Netze deren Oberflächenstruktur eine wesentliche Rolle zu spielen. Fibroblasten sind im Zusammenhang mit der Netzeinheilung von besonderer Bedeutung. Mit dieser In-vitro-Studie sollte das Wachstum und die Zellmorphologie humaner Fibroblasten auf unterschiedlich strukturierten handelsüblichen Kunststoffnetzen untersucht werden.MethodenDrei Polypropylennetze wurden verglichen: ein leichtgewichtiges, kleinporiges (NK1®), ein schwergewichtiges, mittelporiges (BiomeshP1®) und ein leichtgewichtiges, großporiges Netz (Vypro®), letzteres mit einem Zusatzanteil resorbierbaren Polyglactins. Humane Fibroblasten (1,5·105·Zellen) wurden auf den drei Netzen (je 12xa0mm2) über einen Zeitraum von 3xa0Monaten kultiviert. Mithilfe der Rasterelektronenmikroskopie (REM) erfolgte eine morphometrische Analyse des Fibroblastenwachstums zu den Zeitpunkten 6xa0Stunden, 5xa0Tage sowie 2, 4, 6 und 12xa0Wochen.ErgebnisseDas Vypro®-Netz zeigte über die Zeit die signifikant höchste Zelldichte unter den drei Netzen (p <0,001). Allerdings wuchsen die Fibroblasten auf diesem Netz in den ersten 6xa0Wochen nahezu ausschließlich auf dem Polyglactin, nicht auf dem Polypropylen (p <0,006). Auf dem BiomeshP1® konzentrierte sich das Zellwachstum eindeutig auf die Netzknoten, verglichen mit den geraden Netzfasern (p <0,001). Zudem ließen sich auf diesem Netz bereits ab der 2. Woche erhebliche regressive Zellveränderungen nachweisen, die auf den anderen Netzen nicht zu beobachten waren. Auf dem NK1®-Netz fand sich ab der 2. Woche ein konfluierendes Fibroblastenwachstum, allerdings ausschließlich auf den Netzfasern, nicht auf den gepressten, porenfreien Netzflächen (p <0,001).SchlussfolgerungNeben dem Material beeinflusst die Oberflächenstruktur der Kunststoffnetze eindeutig das Wachstumsverhalten humaner Fibroblasten. Diese wachsen bevorzugt auf dünnen Netzfasern (<200xa0µm), multifilen Fasern, Netzknoten und Netzen mit einer Mindestporengröße von 130xa0µm. Auf schwergewichtigen Netzen zeigen sich frühzeitig regressive Zellveränderungen; auf porenfreien Netzflächen findet kein Zellwachstum statt. Polyglactin fördert das Fibroblastenwachstum in besonderer Weise.AbstractBackgroundThe biocompatibility of meshes in hernia surgery seems to be influenced markedly by the amount of the selected material and its structure. Fibroblasts play a major key role during the process of mesh incorporation. This study was performed to investigate differences in cell morphology and proliferation of human fibroblasts cultured on different polypropylene meshes.MethodsIn the present in vitro study the cellular response of human fibroblasts was investigated by scanning electron microscopy (SEM), comparing three different polypropylene meshes: a newly constructed low-weight and microporous mesh (NK1), a low-weight and macroporous mesh with absorbable polyglactin filaments (Vypro), and a heavy-weight and microporous mesh (BiomeshP1). Human fibroblasts (1,5·105 cells) were incubated with the meshes (each 12xa0mm2) for 6xa0hours, 5xa0days, 2, 4, 6, and 12xa0weeks. Computer-assisted morphometry of the fibroblast/mesh surface ratio served to reflect the biological cell response.ResultsThe Vypro mesh showed the significantly highest fibroblast density during the first 6xa0weeks, but cell growth was nearly exclusively limited to the polyglactin filaments. At 3xa0months, after reabsorption of the polyglactin, the fibroblast-coated polypropylene mesh surface was only 50% compared to NK1 and BiomeshP1. The morphologic aspect of the fibroblasts on the BiomeshP1 mesh was much more degenerative and unphysiological, compared to NK1 and Vypro, with isolated, single cells instead of a broad, connective growth. The BiomeshP1 showed a significantly higher fibroblast proliferation around the nodes of the mesh compared to the straight filaments. On the NK1 mesh fibroblasts exclusively proliferated on the filaments but not on the pressed mesh surface.ConclusionsThe polymer surface and structure appears to be of major importance for the biocompatibility of meshes: human fibroblasts preferably grow on low-weight meshes, thin filaments, and mesh nodes. Heavy-weight meshes induce degenerative cell reactions. Polyglactin seems to further improve cell proliferation whereas a pressed mesh surface without pores hinders fibroblast growth.

Hernienchirurgie: Wachstumsverhalten humaner Fibroblasten auf alloplastischen Kunststoffnetzen

ZusammenfassungHintergrundZur Therapie von Bauchwandhernien steht eine Vielzahl unterschiedlicher Kunststoffnetze zur Verfügung. Neben dem Material selbst scheint für die Gewebeverträglichkeit dieser Netze deren Oberflächenstruktur eine wesentliche Rolle zu spielen. Fibroblasten sind im Zusammenhang mit der Netzeinheilung von besonderer Bedeutung. Mit dieser In-vitro-Studie sollte das Wachstum und die Zellmorphologie humaner Fibroblasten auf unterschiedlich strukturierten handelsüblichen Kunststoffnetzen untersucht werden.MethodenDrei Polypropylennetze wurden verglichen: ein leichtgewichtiges, kleinporiges (NK1®), ein schwergewichtiges, mittelporiges (BiomeshP1®) und ein leichtgewichtiges, großporiges Netz (Vypro®), letzteres mit einem Zusatzanteil resorbierbaren Polyglactins. Humane Fibroblasten (1,5·105·Zellen) wurden auf den drei Netzen (je 12xa0mm2) über einen Zeitraum von 3xa0Monaten kultiviert. Mithilfe der Rasterelektronenmikroskopie (REM) erfolgte eine morphometrische Analyse des Fibroblastenwachstums zu den Zeitpunkten 6xa0Stunden, 5xa0Tage sowie 2, 4, 6 und 12xa0Wochen.ErgebnisseDas Vypro®-Netz zeigte über die Zeit die signifikant höchste Zelldichte unter den drei Netzen (p <0,001). Allerdings wuchsen die Fibroblasten auf diesem Netz in den ersten 6xa0Wochen nahezu ausschließlich auf dem Polyglactin, nicht auf dem Polypropylen (p <0,006). Auf dem BiomeshP1® konzentrierte sich das Zellwachstum eindeutig auf die Netzknoten, verglichen mit den geraden Netzfasern (p <0,001). Zudem ließen sich auf diesem Netz bereits ab der 2. Woche erhebliche regressive Zellveränderungen nachweisen, die auf den anderen Netzen nicht zu beobachten waren. Auf dem NK1®-Netz fand sich ab der 2. Woche ein konfluierendes Fibroblastenwachstum, allerdings ausschließlich auf den Netzfasern, nicht auf den gepressten, porenfreien Netzflächen (p <0,001).SchlussfolgerungNeben dem Material beeinflusst die Oberflächenstruktur der Kunststoffnetze eindeutig das Wachstumsverhalten humaner Fibroblasten. Diese wachsen bevorzugt auf dünnen Netzfasern (<200xa0µm), multifilen Fasern, Netzknoten und Netzen mit einer Mindestporengröße von 130xa0µm. Auf schwergewichtigen Netzen zeigen sich frühzeitig regressive Zellveränderungen; auf porenfreien Netzflächen findet kein Zellwachstum statt. Polyglactin fördert das Fibroblastenwachstum in besonderer Weise.AbstractBackgroundThe biocompatibility of meshes in hernia surgery seems to be influenced markedly by the amount of the selected material and its structure. Fibroblasts play a major key role during the process of mesh incorporation. This study was performed to investigate differences in cell morphology and proliferation of human fibroblasts cultured on different polypropylene meshes.MethodsIn the present in vitro study the cellular response of human fibroblasts was investigated by scanning electron microscopy (SEM), comparing three different polypropylene meshes: a newly constructed low-weight and microporous mesh (NK1), a low-weight and macroporous mesh with absorbable polyglactin filaments (Vypro), and a heavy-weight and microporous mesh (BiomeshP1). Human fibroblasts (1,5·105 cells) were incubated with the meshes (each 12xa0mm2) for 6xa0hours, 5xa0days, 2, 4, 6, and 12xa0weeks. Computer-assisted morphometry of the fibroblast/mesh surface ratio served to reflect the biological cell response.ResultsThe Vypro mesh showed the significantly highest fibroblast density during the first 6xa0weeks, but cell growth was nearly exclusively limited to the polyglactin filaments. At 3xa0months, after reabsorption of the polyglactin, the fibroblast-coated polypropylene mesh surface was only 50% compared to NK1 and BiomeshP1. The morphologic aspect of the fibroblasts on the BiomeshP1 mesh was much more degenerative and unphysiological, compared to NK1 and Vypro, with isolated, single cells instead of a broad, connective growth. The BiomeshP1 showed a significantly higher fibroblast proliferation around the nodes of the mesh compared to the straight filaments. On the NK1 mesh fibroblasts exclusively proliferated on the filaments but not on the pressed mesh surface.ConclusionsThe polymer surface and structure appears to be of major importance for the biocompatibility of meshes: human fibroblasts preferably grow on low-weight meshes, thin filaments, and mesh nodes. Heavy-weight meshes induce degenerative cell reactions. Polyglactin seems to further improve cell proliferation whereas a pressed mesh surface without pores hinders fibroblast growth.

Topoisomerase II beta expression level correlates with doxorubicin-induced apoptosis in peripheral blood cells

Anthracyclines are widely used in oncology. Both the response and side-effects of anthracyclines are individually variable, but determinants or predictive markers of this variability are not available. We investigated the variability in the expression of the anthracycline targets topoisomerases II (topo II) alpha and beta and its significance for the apoptotic response following exposure to the anthracycline doxorubicin. Only topo II beta protein expression was detected in peripheral blood cells. Usually considered a constitutively expressed protein, topo II beta varied 3-, 18-, and 16-fold on the mRNA, protein and activity levels, respectively, among the volunteers tested. In addition, the expression of topo II beta was modified by several mitogens, suggesting a role in the regulation of cell cycle. Strikingly, topo II beta activity correlated statistically significantly with the apoptotic response in peripheral blood leukocytes exposed to 1xa0μM doxorubicin. A longitudinal study in a subset of study subjects demonstrated that 30% of the topo II expression variability may be inherited. However, resequencing of the TOP2B gene in 48 unrelated individuals revealed only 8 gene variants, none of them with obvious effects on the expression or protein sequence of topo II beta. Taken together, the apoptotic response to doxorubicin in peripheral blood cells may be mediated by topo II beta. The expression level of topo II beta is intra- and inter-individually variable, and may in part determine the apoptotic response to doxorubicin and other anthracyclines.

Distinct chromosomal profiles in metastasizing and non-metastasizing colorectal carcinomas

Background: The prognosis of colorectal cancer patients is to a considerable extent determined by the metastatic potency of the primary tumor. However, despite the fact that liver metastases are the leading cause of death for cancer patients, the molecular basis still remains poorly understood and independent prognostic markers have not been established. Materials and methods: Comparative genomic hybridization (CGH) was used to screen colorectal carcinomas without distant metastases (n=18) and carcinomas synchronously metastatic to the liver (n=18). We aimed to detect distinct chromosomal aberrations indicating a metastatic phenotype. Results and discussion: Metastatic tumors exhibited a significantly (P=0.03) higher ANCA value (13.8) if compared with non-metastatic cancers (10.0). Furthermore, we observed that losses of chromosomal regions 1p32-ter and 9q33-ter were present at much higher frequencies in metastatic than in non-metastatic cancers, respectively (P=0.02 and 0.04). Conclusion: These data indicate that metastatic tumors may be separated from non-metastatic colorectal cancers based on their genomic profile.

Association of ADAMDEC1 haplotype with high factor VIII levels in venous thromboembolism

A suggestive locus on chromosome 8 could be shown to be associated with familial high factor VIII (FVIII) levels in venous thromboembolism. The ADAMDEC 1 gene is a candidate expressing an ectodomain sheddase. However, the ectodomain of the clearance receptor for FVIII, the low-density lipoprotein receptor-related protein (LRP), is subject to proteolysis by metalloproteases like ADAMDEC1. Other LRP-interacting proteins are lipoprotein lipase (LPL) and t-PA. For an association study, 165 thrombotic patients with high FVIII levels (from the MAISTHRO, i.e. Main-Isar-thrombosis register) were included. All patients with known causes for high FVIII levels had been previously excluded. The patients were compared with 214 healthy blood donors. Polymorphisms with usually a minor allele frequency >5%, i.e. 24 SNPs and two insertion/deletion polymorphisms of LPL gene, eight SNPs of the t-PA gene, and five SNPs of the ADAMDEC1 gene, were analyzed. Haplotype differences were calculated using PHASE. A new polymorphism in intron 7 of the t-PA gene with a minor allele frequency of 2.2% was identified. Analysis of each SNP by the Cochrane-Armitage trend test did not show any significant association between genotype and disease status. Interestingly, the ADAMDEC1 haplotype (rs12674766, rs10087305, rs2291577, rs2291578, rs3765124) differed between cases and controls (p = 0.04). In particular, the TGTGG haplotype showed a difference. In conclusion, the ADAMDEC 1 haplotype may indicate an underlying mechanism for high FVIII levels. The only moderate linkage disequilibrium may be due to a possible causal polymorphism in distant introns or the promoter region against a polygenic background.

Zur Sterblichkeit am kutanen invasiven malignen Melanom Übersicht unter besonderer Berücksichtigung der Geschlechterverteilung

ZusammenfassungHintergrund und Fragestellung. Anhand des Parameters Sterblichkeit wird bei Männern und Frauen versucht, die Ausgangssituation einer differenzierten Ansprache der Geschlechter für die zukünftige Melanomprävention zu beschreiben. Die Zeiträume 1972–1980, 1981–1988, 1989–1996 werden verglichen, und der Einfluss des Lebensalters sowie der Tumordicke nach Breslow auf die Sterblichkeit wird bestimmt.nn Patienten/Methodik. Insgesamt wurden 10.433 Patienten nachbeobachtet. Überlebenswahrscheinlichkeitskurven wurden nach Kaplan-Meier berechnet und Unterschiede mit dem Logrank-Test bestimmt.nn Ergebnisse. Die Sterblichkeit der Frauen war niedriger als die der Männer, jedoch nur mit einer geringen Abnahme über die Zeit. Diese Entwicklung betraf besonders jüngere Männer. Prognosebestimmender intraindividueller Faktor war die Tumordicke nach Breslow. In allen Zeiträumen war deren Medianwert bei Männern höher als bei Frauen. Er zeigte über die Zeit bei beiden Geschlechtern einen Abwärtstrend mit stärker reduzierender Wirkung auf die Sterblichkeit der Männer.nn Schlussfolgerung. Die Befunde rechtfertigen an Männer gerichtete Präventionsmaßnahmen. Deren Evaluation hat jedoch mit dem Umstand zu rechnen, dass bei ihnen eine Besserung der Überlebensrate zu beobachten ist, die die der Frauen übertrifft.AbstractBackground and Objective. The final goals of malignant melanoma prevention are lowering incidence and mortality. We assessed the parameter “survival” for both men and women as the beginning point for future gender-directed prevention campaigns. We compared the periods 1972–1980, 1981–1988, 1989–1996, and determined the influence of age and of Breslow tumor thickness on survival.nn Patients/Methods. We had sufficient follow-up on 10.433 patients. We calculated survival curves according to Kaplan-Meier and defined differences by the logrank test.nn Results. At all periods of time, survival of women was higher compared with men, but with no impressive changes over time. This was especially true for younger men. The most important prognostic factor was the Breslow tumor thickness. Within all periods of time, its median was higher in men. A trend downwards for both genders could be observed with higher influence on survival in men.nn Conclusions. Our findings justify melanoma prevention campaigns addressed to men. Evaluation of such campaigns has to take into account an already existing upwards trend for male survival, which exceeds that of female survival.

Surrogate phenotype definition for alcohol use disorders: a genome-wide search for linkage and association

For the identification of susceptibility loci in complex diseases the choice of the target phenotype is very important. We compared results of genome-wide searches for linkage or for association related to three phenotypes for alcohol use disorder. These are a behavioral score BQ, based on a 12-item questionnaire about drinking behavior and the subjects report of drinking-related health problems, and ERP pattern and ERP magnitude, both derived from the eyes closed resting ERP measures to quantify brain activity. Overall, we were able to identify 11 candidate regions for linkage. Only two regions were found to be related to both BQ and one of the ERP phenotypes. The genome-wide search for association using single-nucleotide polymorphisms did not yield interesting leads.