Molekulargenetische Analyse der minor Histokompatibilitäts-Antigene (mHag) und deren Relevanz für die allogene Blutstammzell-Transplantation

Klinische und experimentelle Daten weisen daraufhin, dass bei Patienten nach allogener Blutstammzell-Transplantation (HSCT) trotz genotypischer HLA-Identität des Spenders in 30-40% der Fälle eine Graft-versus-Host (GvH) Reaktion auftreten kann, die durch zytotoxische alloreaktive T-Lymphozyten des Spenders ausgelöst wird. Bei genotypischer HLA-Identität zwischen Spender und Empfänger verbleibt somit als allogene Differenz die Aminosäuresequenz-Variation in den dem T-Zell Rezeptor präsentierten Peptiden. Dieser Peptid-Polymorphismus ist die Grundlage für Auslösung einer GvH-Reaktion. Deshalb werden die allelischen Ausprägungen der präsentierten Peptide als minor Histokompatibilitäts-Antigene (mHag) bezeichnet. In der vorliegenden Arbeit wurden die mHag HA-1, CD31 und CD49b molekulargenetisch charakterisiert und deren allelischen Ausprägungen mit dem Auftreten einer akuten GvH Reaktion in einer gut definierten Patientenkohorte von 163 CML-Patienten und deren HLA-identischen Geschwisterspendern (n=94) sowie HLA-kompatiblen unverwandten Spendern (n=69) korreliert. Nach univariater Analyse ergab sich eine signifikante Assoziation von mHag-Mismatch und einer erhöhten aGvHD-Inzidenz in HLA-B44 Supertyp-positiven Patienten. Nach multivariater Untersuchung konnte jedoch einzig das Alter der Patienten zum Zeitpunkt der Transplantation als statistisch signifikanter aGvHD-Risikofaktor etabliert werden. Zur mHag-Testung wurde eine einheitliche PCR-SSP Methodik eingeführt und in einem populationsgenetischen Ansatz durch die Untersuchung von Normalpersonen (n=201) sowie eine Segregationsanalyse in 20 Familien mit 80 parentalen Haplotypen validiert. Die Verteilung aller im Rahmen der vorliegenden Arbeit untersuchten mHag-Allele befand sich dabei im Hardy-Weinberg Equilibrium. Innerhalb der Segregationsanalyse konnte eine bisher unbekannte Allelkombination im CD31 Gen beschrieben werden, die durch eine entsprechende Sequenzierungsstrategie weiter aufgeklärt wurde. Weiterführende Untersuchungen des für das mHag HA-1 kodierenden Gens KIAA0223 ergaben schließlich einen bisher unbeschriebenen T ® A Polymorphismus im an das Exon 22 anschließenden Intronbereich. Aufgrund der gegenüber den etablierten aGvHD-Risikofaktoren untergeordneten Relevanz der mHag und der widersprüchlichen Datenlage in der Literatur besteht weiterhin die Notwendigkeit nach zusätzlichen Studien mit großen Fallzahlen, um einen klinisch relevanten Effekt von multiplen mHag-Inkompatibilitäten im Rahmen der HSCT sicher nachzuweisen

Molekulargenetische Analyse der minor Histokompatibilitäts-Antigene (mHag) und deren Relevanz für die allogene Blutstammzell-Transplantation

Zugriff auf den Volltext ist aus datenschutzrechtlichen Gründen gesperrt, neue Version unter DuEPublico-ID 35212 Klinische und experimentelle Daten weisen daraufhin, dass bei Patienten nach allogener Blutstammzell-Transplantation (HSCT) trotz genotypischer HLA-Identität des Spenders in 30-40% der Fälle eine Graft-versus-Host (GvH) Reaktion auftreten kann, die durch zytotoxische alloreaktive T-Lymphozyten des Spenders ausgelöst wird. Bei genotypischer HLA-Identität zwischen Spender und Empfänger verbleibt somit als allogene Differenz die Aminosäuresequenz-Variation in den dem T-Zell Rezeptor präsentierten Peptiden. Dieser Peptid-Polymorphismus ist die Grundlage für Auslösung einer GvH-Reaktion. Deshalb werden die allelischen Ausprägungen der präsentierten Peptide als minor Histokompatibilitäts-Antigene (mHag) bezeichnet. In der vorliegenden Arbeit wurden die mHag HA-1, CD31 und CD49b molekulargenetisch charakterisiert und deren allelischen Ausprägungen mit dem Auftreten einer akuten GvH Reaktion in einer gut definierten Patientenkohorte von 163 CML-Patienten und deren HLA-identischen Geschwisterspendern (n=94) sowie HLA-kompatiblen unverwandten Spendern (n=69) korreliert. Nach univariater Analyse ergab sich eine signifikante Assoziation von mHag-Mismatch und einer erhöhten aGvHD-Inzidenz in HLA-B44 Supertyp-positiven Patienten. Nach multivariater Untersuchung konnte jedoch einzig das Alter der Patienten zum Zeitpunkt der Transplantation als statistisch signifikanter aGvHD-Risikofaktor etabliert werden. Zur mHag-Testung wurde eine einheitliche PCR-SSP Methodik eingeführt und in einem populationsgenetischen Ansatz durch die Untersuchung von Normalpersonen (n=201) sowie eine Segregationsanalyse in 20 Familien mit 80 parentalen Haplotypen validiert. Die Verteilung aller im Rahmen der vorliegenden Arbeit untersuchten mHag-Allele befand sich dabei im Hardy-Weinberg Equilibrium. Innerhalb der Segregationsanalyse konnte eine bisher unbekannte Allelkombination im CD31 Gen beschrieben werden, die durch eine entsprechende Sequenzierungsstrategie weiter aufgeklärt wurde. Weiterführende Untersuchungen des für das mHag HA-1 kodierenden Gens KIAA0223 ergaben schließlich einen bisher unbeschriebenen T ® A Polymorphismus im an das Exon 22 anschließenden Intronbereich. Aufgrund der gegenüber den etablierten aGvHD-Risikofaktoren untergeordneten Relevanz der mHag und der widersprüchlichen Datenlage in der Literatur besteht weiterhin die Notwendigkeit nach zusätzlichen Studien mit großen Fallzahlen, um einen klinisch relevanten Effekt von multiplen mHag-Inkompatibilitäten im Rahmen der HSCT sicher nachzuweisen

Mid-term results of a modified arterial switch operation using the direct reconstruction technique of the pulmonary artery

Background: There is ongoing discussion as to whether it is beneficial to avoid pulmonary sinus augmentation in the arterial switch operation. We report a single-surgeon series of mid-term results for direct pulmonary artery anastomosis during switch operation for transposition of the great arteries (TGA). Methods: This retrospective study includes 17 patients with TGA, combined with an atrial septal defect, patent foramen ovale or ventricular septal defect. Patient data was analyzed from hospital charts, including operative reports, post-operative course, and regular follow-up investigations. The protocol included cardiological examination by a single pediatric cardiologist. Echocardiographic examinations were performed immediately after arrival on the intensive unit, before discharge, and then after three, six, and 12 months, followed by yearly intervals. Pulmonary artery stenosis (PAS) was categorized into three groups according to the Doppler-measured pulmonary gradient: grade I (trivial stenosis) = increased pulmonary flow with a gradient below 25 mm Hg; grade II (moderate stenosis) = a gradient ranging from 25 to 49 mm Hg; and grade III (severe stenosis) = a gradient above 50 mm Hg. Follow-up data was available for all patients. The length of follow-up ranged from 1.2 to 9.7 years, median: 7.5 years (mean 6.1 years ± 14 months). Results: During follow-up, 12 patients (70.6%) had no (or only trivial) PAS, five patients (29.4%) had moderate stenosis without progress, and no patient had severe PAS. Cardiac catheterization after arterial switch operation was performed in 11 patients (64.7%) and showed a good correlation with echocardiographic findings. During follow-up there was no reintervention for PAS. Conclusions: Direct reconstruction of the neo-pulmonary artery is a good option in TGA with antero-posterior position of the great vessels, with very satisfactory mid-term results. (Cardiol J 2010; 17, 6: 574-579

Plasmonics on nanostructures for cell manipulation

A plasmon resonance based method for introducing foreign material into living cells is presented. By illuminating gold-coated or structured surfaces, near field enhancement is employed to selectively open the cell membrane using ultrashort laser pulses

Surgical treatment of aortic coarctation in adults: Beneficial effect on arterial hypertension

Background: The aim of this study was to determine the outcome after surgical repair of aortic coarctation in adults, analysing its effect on arterial blood pressure. Methods: Twenty-five adults (9 women, 16 men), mean age 43.4 years (19 to 70 years), underwent aortic coarctation surgical repair. All patients suffered from preoperative hypertension. Mean blood pressure was 182/97 mm Hg. Sixteen (64%) patients demonstrated reduced load capacity. Operative technique was resection and end-to-end anastomosis for 5 patients (20%), interposition of a Dacron-tube graft for 3 patients (12%), Dacron-patch dilatation was performed in 7 (28%) patients, and in 10 (40%) patients we performed an extra-anatomical bypass graft. Results: Early mortality occurred in 1 patient (4%). The mean blood pressure was reduced [systolic 182 mm Hg vs. 139 mm Hg (p < 0.001), diastolic 97 mm Hg vs. 83 mm Hg (p < 0.001)] in all patients. In 12 patients, blood pressure normalized immediately after surgery, in 7 patients it remained slightly elevated (systolic blood pressure between 140-160 mm Hg), and 1 patient suffered from prolonged arterial hypertension. Preoperatively, all patients were treated with antihypertensive drugs. Eleven of 20 patients received long-term medication during follow- up. In the remaining 4 patients, medication lists were unobtainable in retrospect. The mean follow-up was 7.1 years (min. 1.0 years; max. 16.6 years). One patient (5%) died from cardiac failure 12.4 years after the operation. On average, the New York Heart Association (NYHA) class was improved by 0.92. Conclusions: The surgical repair of aortic coarctation in adults can be performed with low surgical risk. Surgery reduces hypertension and permits more effective medical treatment

Cardiac myxomas: Short- and long-term follow-up

Background: Cardiac myxomas are the most frequently encountered benign intracardiac tumors, that, if left untreated, are inexorably progressive and potentially fatal. Patients with cardiac myxoma can be treated only by surgical removal. This study summarizes our experience over 22 years with these tumors. Methods: Fifty seven patients (M/F: 14/43, age: 57.9 &#177; 14.6 years) with cardiac myxomas underwent surgical resection at our institution. There were 82.4% left atrial myxomas, 14.0% right atrial myxomas, 3.6% biatrial myxomas. The duration of symptoms prior to surgery ranged from 6 to 1,373 days (median 96 days). The surgical approach comprised complete wide excision. The diagnostic methods, incidence of thromboembolic complications, valve degeneration, surgical repair techniques, recurrence and re-operation were reviewed and the Kaplan-Meier survival curve was calculated. Results: There were no in-hospital deaths. Hospital stay amounted to a mean of 13.7 &#177; 6.9 days. Late follow-up was available for 54 (94.7%) patients for a median 7.5 years after surgery (23 days to 21.4 years). Fifty two patients are alive, while five patients had died after a mean interval of 6.3 years. Cause of death was cardiac in 40% of the patients (n = 2) and non-cardiac in the other 60% (n = 3). Conclusions: Surgical excision of cardiac myxoma carries a low operative risk and gives excellent short-term and long-term results. Surgical excision of the tumor appears to be curative, with few recurrences at long-term follow-up. After diagnosis, surgery should be performed urgently, in order to prevent complications such as embolic events or obstruction of the mitral orifice. Follow-up examination, including echocardiography, should be performed regularly

Regional IFNγ expression is insufficient for efficacious control of food-borne bacterial pathogens at the gut epithelial barrier

IFNγ is critical for host defence against various food-borne pathogens including Salmonella enterica and Listeria monocytogenes, the causative agents of salmonellosis and listeriosis, respectively. We investigated the impact of regional IFNγ expression at the intestinal epithelial barrier on host invasion by salmonellae and listeriae following oral challenge. Transgenic mice (IFNγ-gut), generated on an IFNγ knock-out (KO) background, selectively expressed IFNγ in the gut driven by the modified liver fatty acid-binding protein (Fabpl4× at −132) promoter. Infections with attenuated S. enterica Typhimurium or with L. monocytogenes did not differ significantly in IFNγ-KO, IFNγ-gut and wild-type mice. Further, Listeria-specific CD4+ and CD8+ T cells were not altered in IFNγ-gut mice. Thus, this model indicates that local IFNγ expression by non-immunological cells in the distal part of the small intestine, caecum and colon is insufficient for prevention of gut penetration by S. enterica Typhimurium and L. monocytogene

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5′ exonuclease-mediated nucleic acid degradation

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.DFG/SFB/Transregio 37DFG/EXC/REBIRTHDFG/Transregio 37DFG/EXC/REBIRT