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In JoVE (1)
Other Publications (19)
- The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation
- Journal of Molecular Medicine (Berlin, Germany)
- Journal of Endovascular Therapy : an Official Journal of the International Society of Endovascular Specialists
- Endothelium : Journal of Endothelial Cell Research
- Journal of Endovascular Therapy : an Official Journal of the International Society of Endovascular Specialists
- Biochemistry
- Arteriosclerosis, Thrombosis, and Vascular Biology
- Acta Cardiologica
- Journal of Endovascular Therapy : an Official Journal of the International Society of Endovascular Specialists
- Artificial Organs
- Lab on a Chip
- The Journal of Gene Medicine
- Pediatric Transplantation
- Clinical Research in Cardiology : Official Journal of the German Cardiac Society
- Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
- Endothelium : Journal of Endothelial Cell Research
- Endothelium : Journal of Endothelial Cell Research
- Transplant International : Official Journal of the European Society for Organ Transplantation
- Pediatric Transplantation
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Articles by Alexandra Fuchs in JoVE
JoVE Bioengineering
İlaç Etkisi Micropatterned Hücreler Geliştirilmiş Görselleştirme ve Kantitatif Analiz
1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France
Hücresel mimarili normale Yapıştırıcı micropatterns, ilaç etkileri algılama duyarlılığını artırmak, tekrarlanabilirlik geliştirmek ve otomatik görüntü elde edilmesi ve analizi basitleştirmek için kullanılabilir. Bu teknoloji, geleneksel hücre kültürleri destekleyen üzerinde gerçekleştirilen ve dolayısıyla aşırı hücre-hücre değişkenlik muzdarip ilaç / siRNA tarama testleri, fayda sağlayacaktır.
Other articles by Alexandra Fuchs on PubMed
Heart Transplantation: an Approach to Treating Primary Cardiac Sarcoma?
Treating rare, primary cardiac soft-tissue sarcomas (C-STS) with heart transplantation (HTx) is controversial. Conventional tumor resection only partially alleviates the disease, and patients die of local recurrence of the tumor or of distant metastases. Heart transplantation offers the opportunity to eradicate the primary malignancy completely. In our experience of 4 patients with C-STS indicates, HTx followed by post-operative chemotherapy does not affect the long-term outcome. However, pre-operative chemotherapy can regress tumors in chemosensitive C-STS and potentially eradicate early micrometastases. Consecutive HTx for responders may then offer a chance of long-term survival.
Optimization of Nonviral Transfection: Variables Influencing Liposome-mediated Gene Transfer in Proliferating Vs. Quiescent Cells in Culture and in Vivo Using a Porcine Restenosis Model
Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.
Vascular Endothelial Growth Factor (VEGF165) and Its Influence on Angiogenesis Versus Arteriogenesis in Different Vascular Beds
To use local gene delivery to determine any district-specific influence of vascular endothelial growth factor (VEGF(165)) on angiogenesis and arteriogenesis in arteries of distinct developmental origin.
Vascular Endothelial Growth Factor Response in Porcine Coronary and Peripheral Arteries Using Nonsurgical Occlusion Model, Local Delivery, and Liposome-mediated Gene Transfer
Angiogenesis and arteriogenesis play an important role in advanced vascular occlusive diseases. Whether angiogenesis or arteriogenesis predominate depends on the preexisting collateral vessel network, the type and location of occlusion, and different developmental origin of the arteries. Angiogenesis and arteriogenesis were investigated following vascular endothelial growth factor (VEGF) treatment in different arteries important in occlusive arterial diseases using a newly developed porcine arterial occlusion model. Porcine coronary and peripheral arteries were occluded interventionally using blinded stent grafts. Gene transfer was performed using a needle injection catheter and cationic lipid DOCSPER as gene carrier. DNA and gene expression in arterial tissue was examined using polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR. Vessel development was determined by angiography, immunohistochemistry, and measurement of capillary density. The transfected gene and its expression were found 3 months following application. In tissue adjacent to coronary arteries, there was significantly enhanced capillary density but no increase in angiographic score. In contrast, tissue surrounding peripheral arteries demonstrated no enhancement of capillary density but an enhancement in angiographic score. These results demonstrate differential responses to VEGF treatment in coronary and peripheral arteries resulting predominantly in either angiogenesis or arteriogenesis. Further investigation of VEGF signaling pathway is necessary for better understanding of the processes of vascular development, which may have potential impact on the design of cardiovascular therapeutics.
Comparison of Surgical Versus Endovascular Occlusion Models in Pig Femoral Arteries
To compare an endovascular technique with a well established surgical approach to achieve long-term occlusions of large porcine arteries while preserving the integrity of periarterial tissue.
Phosphorylated P40PHOX As a Negative Regulator of NADPH Oxidase
The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.
Functional Analysis of Genomic DNA, CDNA, and Nucleotide Sequence of the Mature C-type Natriuretic Peptide Gene in Vascular Cells
The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells.
Interleukin-6 and Procalcitonin in Serum of Children Undergoing Cardiac Surgery with Cardiopulmonary Bypass
The aim of our study was to investigate the systemic inflammatory response in children with congenital heart disease undergoing surgical correction with cardiopulmonary bypass. We wanted to discuss interleukin 6 and procalcitonin as components of the systemic inflammatory response syndrome to cardiopulmonary bypass and evaluate postoperative kinetics of these parameters in case of an uncomplicated course.
C-type Natriuretic Peptide Inhibits Constrictive Remodeling Without Compromising Re-endothelialization in Balloon-dilated Renal Arteries
To investigate the long-term effect of local, liposome-mediated gene transfer of C-type natriuretic peptide (CNP) plasmid versus CNP protein on restenosis in porcine renal arteries following balloon angioplasty.
Mechanical Circulatory Support in Infants and Adults with the MEDOS/HIA Assist Device
Mechanical circulatory support is successfully applied to patients with low cardiac output. The MEDOS/HIA-System provides pulsatile ventricular assistance for pediatric and adult patients. Our experience with 13 consecutive patients with the MEDOS is reported. Perioperative survival was 84.6%, complications occurred in 61% (31% thrombembolism, 23% rethoracotomy, 7% infections). Mean duration of support was 17.6 +/- 14.6 days (1-45 days). Bilirubin decreased from 3.9 +/- 2.3 to 2.7 +/- 1.6 mg/dL; creatinine from 1.6 +/- 1 to 1.4 +/- 0.8 mg/dL; lactate from 5.8 +/- 4.2 to 1.7 +/- 1.5 (P = 0.027; Wilcoxon). All patients who underwent subsequent heart transplantation (6 of 13; 46%) were discharged from hospital. For 38.5% of the patients no organ offer was received. Mechanical circulatory support with the MEDOS/HIA-System can be performed successfully for bridging to transplantation. Secondary organ functions improve under this pulsatile circulatory assistance. Hemorrhage and thromboembolic events are the most frequent complications.
Electronic Sorting and Recovery of Single Live Cells from Microlitre Sized Samples
Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.
C-type Natriuretic Peptide for Reduction of Restenosis: Gene Transfer is Superior over Single Peptide Administration
Restenosis is still a significant clinical problem limiting the long-term therapeutic success following balloon dilation or stent implantation. New approaches are necessary inhibiting neointima formation and simultaneously promoting re-endothelialization. Therefore, long-term therapeutic effects of adventitial liposome-mediated C-type natriuretic protein (CNP) gene and CNP peptide applications in a porcine model for restenosis post-angioplasty were investigated.
Reinnervation After Heart Transplantation in Children: Results of Short-time Heart Rate Variability Testing
To detect impairment in short-term heart rate variability (HRV) in children after heart and heart-lung transplantation (TX) as reported in adults. To assess vagal and sympathetic influence on the donor heart rate using frequency domain analysis of HRV.
Efficacy of Extracorporeal Membrane Oxygenation in a Congenital Heart Surgery Program
To report our experience with extracorporeal membrane oxygenation (ECMO) in a congenital heart surgery program.
CNS or Bone Marrow Involvement As Risk Factors for Poor Survival in Post-transplantation Lymphoproliferative Disorders in Children After Solid Organ Transplantation
To identify prognostic factors of survival in pediatric post-transplantation lymphoproliferative disorder (PTLD) after solid organ transplantation.
Inhibition of Restenosis Formation Without Compromising Reendothelialization As a Potential Solution to Thrombosis Following Angioplasty?
Stent thrombosis remains an important problem after the implantation of different stent types. A potential solution to this problem may be vasoactive agents with dual effects on different cell types like C-type natriuretic peptide (CNP). Therefore, in vitro and in vivo effects of CNP were investigated in a porcine restenotic model. Gene transfer of CNP in cultures of porcine vascular cells revealed up to 30% reduction of growth of smooth muscle cells (p<.05), but no suppression of endothelial growth using CNP. Applied in vivo, angiography revealed a trend of reduced restenosis formation in balloon-injured porcine arteries treated with CNP gene or beta-galactosidase (beta-Gal) control gene after three months (2.59 +/- 2.04-fold reduction, p = n.s.). Histologically, morphometry revealed significantly reduced neointima formation after treatment with CNP plasmid (7.26 +/- 1.44-fold reduction, p < .05). Evans blue staining demonstrated complete endothelial repair already 3 weeks after intervention using CNP. Transfer of CNP gene resulted in a significant inhibition of neointima formation without compromising endothelial repair. Therefore, use of the CNP gene may offer a solution to suppress restenosis formation while preventing subacute or late thrombosis.
Meta-analysis Shows Similar Risk of Thrombosis After Drug-eluting Stent, Bare-metal Stent, or Angioplasty
Coronary stent thrombosis remains an important problem after the implantation of different stent types. This study investigates the risk of stent thrombosis associated with the use of drug-eluting stents (DESs), bare-metal stents (BMSs) compared to balloon angioplasty. A meta-analysis of 28 randomized trials involving 5612 versus 7639 versus 2994 patients with coronary heart disease treated with DES, BMS, or balloon angioplasty was therefore performed. Comparing the implantation of DES versus BMS, DES was not found to increase the hazard for thrombosis up to 15 months (odds ratio [OR] = 0.86, 95% confidence interval [CI] 0.58 to 1.3, p < .48). There was also no significant difference in the hazard for subacute thrombosis (SAT) or late stent thrombosis (LST) in the DES versus BMSs group (OR = 0.86, 95% CI 0.50 to 1.5, p < .6 and OR = 0.92, 95% CI 0.50 to 1.68, p < .78, respectively). Comparing incidences of stent thromboses in patients receiving balloon angioplasty or implantation of BMS, the rate of SAT in the balloon angioplasty group (1.7% SAT) versus BMS group (1.8% SAT) was also similar (OR = 0.93, 95% CI 0.61 to 1.4, p < .71). Finally, there was no significant difference in the occurrence of stent thrombosis for the different coatings of DESs. In conclusion, the use of DES was not observed to have a significant effect on stent thrombosis events, compared with the implantation of BMS or balloon angioplasty.
Lack of Serologic Immunity Against Vaccine-preventable Diseases in Children After Thoracic Transplantation
We investigated whether children after heart- (HTx) or heart-lung transplantation (HLTx) show protective antibody levels against recommended vaccinations, whether vaccination schedules are completed and which factors influence serologic immunity. We performed a cross sectional ELISA - quantification of specific antibodies in 46 patients after pediatric thoracic Tx. Findings were correlated to vaccination history, age at Tx, clinical course and immunosuppressive regimen. We found protective antibody levels against diphtheria in 74% of patients, against tetanus in 22%, against Haemophilus influenzae type b in 30% and against Streptococcus pneumoniae in 59%. Antibody concentrations against live attenuated vaccines were significantly lower in children transplanted in the first 2 years of life. Antibodies were absent for measles in 55% of late - and 81% of early transplanted children, for mumps in 66%/94%, for rubella in 30%/56% and for Varicella in 34%/63%. We found significant correlation of low antibody concentrations and age at Tx. Patients without protective antibody concentrations had significantly longer use of steroids. Vaccination schedules were incomplete or delayed in the majority of patients associated with more days in hospital pre-Tx. Our study shows that closer adherence to pretransplantation vaccination schedules and also post-transplantation monitoring of antibody levels are required in transplant patients.
Impaired Cellular Immune Response to Diphtheria and Tetanus Vaccines in Children After Thoracic Transplantation
Safety and immunogenicity of diphtheria and tetanus booster vaccination were evaluated in 28 children after thoracic transplantation. Adverse events were documented in a patient diary. Blood was collected prior to and four wk after vaccination. Specific antibody concentrations were measured by ELISA. Lymphocytes were investigated for expression of activation markers (CD25, HLA-DR) by flow cytometry and proliferation assays with and without stimulation. Post-vaccination antibody titers were higher than prevaccination (p < 0.001), with more patients having protective antibody levels against diphtheria (p < 0.02) and tetanus (p < 0.001). There was no increased proliferation in non-stimulated or stimulated cultures after vaccination. The number of T-lymphocytes activated by the vaccination antigens was similar pre- and post-vaccination, whereas HLA-DR-expression on stimulated and non-stimulated CD4(+) T-cells increased significantly. Increase in antibodies was negatively correlated with tacrolimus dose, and impaired cellular immunity was associated with higher tacrolimus dose and steroid use. Adverse events were similar to the general population; serious adverse events and rejection did not occur. Vaccination with inactivated vaccines can be performed safely in immunosuppressed children after thoracic transplantation and induces protective antibody levels in the majority of patients. Impaired induction of specific cellular immunity is correlated with intensity of immunosuppression and may explain reduced sustainability of antibodies.
