Sirolimus inhibits key events of restenosis in vitro/ex vivo: evaluation of the clinical relevance of the data by SI/MPL- and SI/DES-ratio's

<p>Abstract</p> <p>Background</p> <p>Sirolimus (SRL, Rapamycin) has been used successfully to inhibit restenosis both in drug eluting stents (DES) and after systemic application. The current study reports on the effects of SRL in various human in vitro/ex vivo models and evaluates the theoretical clinical relevance of the data by SI/MPL- and SI/DES-ratio's.</p> <p>Methods</p> <p>Definition of the SI/MPL-ratio: relation between <b>s</b>ignificant <b>i</b>nhibitory effects in vitro/ex vivo and the <b>m</b>aximal <b>p</b>lasma <b>l</b>evel after systemic administration in vivo (6.4 ng/ml for SRL). Definition of the SI/DES-ratio: relation between <b>s</b>ignificant <b>i</b>nhibitory effects in vitro/ex vivo and the drug concentration in <b>DES </b>(7.5 mg/ml in the ISAR drug-eluting stent platform). Part I of the study investigated in cytoflow studies the effect of SRL (0.01–1000 ng/ml) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary smooth muscle cells (HCMSMC). Part II of the study analysed the effect of SRL (0.01–1000 ng/ml) on cell migration of HCMSMC. In part III, IV, and V of the study ex vivo angioplasty (9 bar) was carried out in a human organ culture model (HOC-model). SRL (50 ng/ml) was added for a period of 21 days, after 21 and 56 days cell proliferation, apoptosis, and neointimal hyperplasia was studied.</p> <p>Results</p> <p>Expression of ICAM-1 was significantly inhibited both in HCAEC (SRL ≥ 0.01 ng/ml) and HCMSMC (SRL ≥ 10 ng/ml). SRL in concentrations ≥ 0.1 ng/ml significantly inhibited migration of HCMSMC. Cell proliferation and neointimal hyperplasia was inhibited at day 21 and day 56, significance (p < 0.01) was achieved for the inhibitory effect on cell proliferation in the media at day 21. The number of apoptotic cells was always below 1%.</p> <p>Conclusion</p> <p>SI/MPL-ratio's ≤ 1 (ICAM-1 expression, cell migration) characterize inhibitory effects of SRL that can be theoretically expected both after systemic and local high dose administration, a SI/MPL-ratio of 7.81 (cell proliferation) represents an effect that was achieved with drug concentrations 7.81-times the MPL. SI/DES-ratio's between 10^-6and 10^-8indicate that the described inhibitory effects of SRL have been detected with micro to nano parts of the SRL concentration in the ISAR drug-eluting stent platform. Drug concentrations in DES will be a central issue in the future.</p

Simultaneous intra/extravascular administration of antiproliferative agents as a new strategy to inhibit restenosis: The peak of reactive cell proliferation as a hallmark for the duration of the treatment

BACKGROUND: Strictly intravascular approaches for the treatment of postangioplasty restenosis are effective in the intima and the inner parts of the media but may be insufficient to control redundant pathways in the more outer parts of the media and the adventitia. An inverse situation may occur subsequently to a strictly extravascular approach, like the recently suggested pericardial approach in pigs. We hypothesized that simultaneous intra/extravascular administration of anti-restenotic agents inhibits restenosis by blocking all stimulatory pathways in the entire arterial wall. METHODS: Fresh hearts of 25 domestic pigs were obtained from a local slaughterhouse. Left anterior descending coronary arteries (LAD) were harvested, cut into cylindric 5 mm segments, and cultured as ex vivo porcine organ cultures (POCs). After 9 bar ballooning simultaneous intra/extravascular administration of high dose diltiazem (50 μg/mL) was carried out for a period of 1, 2, 3, 4, 5, 6, and 7 days. At day 7 and 28 proliferative activity (BrdU), neointimal thickening, and staining against smooth muscle α-actin and vWF was analysed. RESULTS: 7 days after ballooning administration of diltiazem for 4, 5, 6, and 7 days inhibited reactive cell proliferation by more than 50% (n.s.) as compared to control, 28 days after ballooning administration for 6 and 7 days inhibited neointimal thickening by more than 75% (p < 0.05). Simultaneous intra/extravascular administration of high dose diltiazem did not affect the expression of vWF in endothelial cells or smooth muscle α-actin in smooth muscle cells. CONCLUSIONS: Simultaneous intra/extravascular administration of high dose diltiazem (50 μg/mL) has to be maintained for at least 6 days to achieve a significant inhibition of neointimal thickening. The data demonstrate the importance of the maximal reactive cell proliferation (= day 7 in the POC-model) for the calculation of the duration of the treatment period

Effects of mycophenolate mofetil on key pattern of coronary restenosis: a cascade of in vitro and ex vivo models

BACKGROUND: Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is a rationally designed immunosuppressive drug. The current study investigates the effect of MMF on key pattern of restenosis in a cascade of in vitro and ex vivo models. METHODS: Part I of the study investigated in northern blot and cytoflow studies the effect of MMF (50, 100, 150, 200, 250, and 300 μg/mL) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC). Part II of the study applied a human coronary 3D model of leukocyte attack, the 3DLA-model. HCAEC and HCMSMC were cultured on both sides of a polycarbonate filters, mimicking the internal elastic membrane. Leukocyte attack (LA) was carried out by adding human monocytes (MC) on the endothelial side. The effect of MMF (50 μg/mL) on adhesion and chemotaxis (0.5, 1, 2, 3, 4, 6, and 24 h after LA) and the effect on proliferation of co-cultured HCMSMC (24 h after LA) was studied. In part III of the study a porcine coronary organ culture model of restenosis (POC-model) was used. After ex vivo ballooning MMF (50 μg/mL) was added to the cultures for a period of 1, 2, 3, 4, 5, 6, and 7 days. The effect on reactive cell proliferation and neointimal thickening was studied at day 7 and day 28 after ballooning. RESULTS: Expression of ICAM-1 in northern blot and cytoflow studies was neither clearly inhibited nor stimulated after administration of MMF in the clinical relevant concentration of 50 μg/mL. In the 3DLA-model 50 μg/mL of MMF caused a significant antiproliferative effect (p < 0.001) in co-cultured HCMSMC but had no effect on MC-adhesion and MC-chemotaxis. In the ex vivo POC-model neighter reactive cell proliferation at day 7 nor neointimal hyperplasia at day 28 were significantly inhibited by MMF (50 μg/mL). CONCLUSION: Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 μg/mL) in the 3DLA-model. The antiproliferative effect was a direct antiproliferative effect that was not triggered via reduced expression of ICAM-1 or via an inhibition of MC-adhesion and chemotaxis. Probably due to technical limitations (as e.g. the missing of perfusion) the antiproliferative effect of MMF (50 μg/mL) could not be reproduced in the coronary organ culture model. A cascade of focused in vitro and ex vivo models may help to gather informations on drug effects before large experimental studies are initiated

Effects of abciximab on key pattern of human coronary restenosis in vitro: impact of the SI/MPL-ratio

BACKGROUND: The significant reduction of angiographic restenosis rates in the ISAR-SWEET study (intracoronary stenting and antithrombotic regimen: is abciximab a superior way to eliminate elevated thrombotic risk in diabetes) raises the question of whether abciximab acts on clopidogrel-independent mechanisms in suppressing neointimal hyperplasia. The current study investigates the direct effect of abciximab on ICAM-1 expression, migration and proliferation. METHODS: ICAM-1: Part I of the study investigates in cytoflow studies the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1). Migration: Part II of the study explored the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on migration of HCMSMC over a period of 24 h. Proliferation: Part III of the study investigated the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on proliferation of HUVEC, HCAEC, and HCMSMC after an incubation period of 5 days. RESULTS: ICAM-1: In human venous endothelial cells (HUVEC), human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC) no inhibitory or stimulatory effect on expression of ICAM-1 was detected. Migration: After incubation of HCMSMC with abciximab in concentrations of 0.0002 – 2 μg/ml a stimulatory effect on cell migration was detected, statistical significance was achieved after incubation with 0.002 μg/ml (p < 0.05), 0.002 μg/ml (p < 0.001), and 0.2 μg/ml (p < 0.05). Proliferation: Small but statistically significant antiproliferative effects of abciximab were detected after incubation of HUVEC (0.02 and 2.0 μg/ml; p = 0.01 and p < 0.01), HCAEC (2.0 and 20.0 μg/ml; p < 0.05 and p < 0,01), and HCMSMC (2.0 and 20.0 μg/ml; p < 0.05 and p < 0.05). The significant inhibition (SI) of cell proliferation found in HCAEC and HCMSMC was achieved with drug concentrations more than 10 times beyond the maximal plasma level (MPL), resulting in a SI/MPL-ratio > 1. CONCLUSION: Thus, the anti-restenotic effects of systemically administered abciximab reported in the ISAR-SWEET-study were not caused by a direct inhibitory effect on ICAM-1 expression, migration or proliferation

How to turn the Fast-Track into a Fast-Track: Process integration for evaluation of the quality of Digital Health Applications (DiGAs) on the example of the German Fast-Track Process

In this paper, we address the research question of which integration points in the \textit{German Fast-Track process} are particularly well suited for the integration of evaluation platforms for digital health applications. For this purpose, possible integration points are first identified and then analyzed with the help of a utility analysis with regard to the posed research question. Finally, a recommendation for action is made based on the results of the conducted utility analysis

Micro-Scale Restraint Methodology for Humidity Induced Swelling Investigated by Phase Contrast X-Ray Tomography

A new methodology for restraining the swelling of spruce wood samples in the micrometre range is developed and presented. We show that the restraining device successfully prevents the free swelling of wood during moisture adsorption, thus modifying significantly the anisotropy of swelling and provoking the intended collapse and large deformations of the wood cells at the edges of the sample in contact with the restraining device. The device consists in a slotted cube designed to restrain swelling and is made of PMMA manufactured by laser ablation. The sample undergoing the restraining experiment is imaged with high-resolution synchrotron radiation phase contrast X-Ray Tomographic Microscopy. The deformation of the restraining device itself is only approximately 2μm with respect to a 500μm width in cubes containing latewood samples and half of that in the case of cubes containing earlywood

Edge restenosis: impact of low dose irradiation on cell proliferation and ICAM-1 expression

BACKGROUND: Low dose irradiation (LDI) of uninjured segments is the consequence of the suggestion of many authors to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Atherosclerosis is a general disease and the uninjured segment close to the intervention area is often atherosclerotic as well, consisting of neointimal smooth muscle cells (SMC) and quiescent monocytes (MC). The current study imitates this complex situation in vitro and investigates the effect of LDI on proliferation of SMC and expression of intercellular adhesion molecule-1 (ICAM-1) in MC. METHODS: Plaque tissue from advanced primary stenosing lesions of human coronary arteries (9 patients, age: 61 ± 7 years) was extracted by local or extensive thrombendarterectomy. SMC were isolated and identified by positive reaction with smooth muscle α-actin. MC were isolated from buffy coat leukocytes using the MACS cell isolation kit. For identification of MC flow-cytometry analysis of FITC-conjugated CD68 and CD14 (FACScan) was applied. SMC and MC were irradiated using megavoltage photon irradiation (CLINAC2300 C/D, VARIAN, USA) of 6 mV at a focus-surface distance of 100 cm and a dose rate of 6 Gy min(-1 )with single doses of 1 Gy, 4 Gy, and 10 Gy. The effect on proliferation of SMC was analysed at day 10, 15, and 20. Secondly, total RNA of MC was isolated 1 h, 2 h, 3 h, and 4 h after irradiation and 5 μg of RNA was used in standard Northern blot analysis with ICAM-1 cDNA-probes. RESULTS: Both inhibitory and stimulatory effects were detected after irradiation of SMC with a dose of 1 Gy. At day 10 and 15 a significant antiproliferative effect was found; at day 20 after irradiation cell proliferation was significantly stimulated. Irradiation with 4 Gy and 10 Gy caused dose dependent inhibitory effects at day 10, 15, and 20. Expression of ICAM-1 in human MC was neihter inhibited nor stimulated by LDI. CONCLUSION: Thus, the stimulatory effect of LDI on SMC proliferation at day 20 days after irradiation may be the in vitro equivalent of a beginning edge effect. Extending the irradiation area in vascular brachytherapy in vivo may therefore merely postpone and not inhibit the edge effect. The data do not indicate that expression of ICAM-1 in quiescent MC is involved in the process

The present status of childhood cancer therapy in Korea.

We have studied the incidence pattern of childhood cancers in Korea. Although the incidence of many tumors in Korea is similar to that in other countries, the incidence of acute myelogenous leukemia, non-Hodgkin's lymphoma and hepatoma is greater in Korean children. Yonsei Cancer Center commenced a study of multi-modality treatment of childhood cancers in July 1974. The most striking improvement of survival rate was seen in patients with acute lymphocytic leukemia (50% at 5 years), Wilms' tumor (65% at 5 years), neuroblastoma (45% at 2 years), osteogenic sarcoma (55% at 2 years) and malignant histiocytosis (20% at 5 years). This study is an attempt to create a basic framework providing the best possible treatment of childhood cancer in Korea. The data obtained in Korea are briefly compared with those in Japan and the United States.</p

The effect of autonomy, training opportunities, age and salaries on job satisfaction in the South East Asian retail petroleum industry

South East Asian petroleum retailers are under considerable pressure to improve service quality by reducing turnover. An empirical methodology from this industry determined the extent to which job characteristics, training opportunities, age and salary influenced the level of job satisfaction, an indicator of turnover. Responses are reported on a random sample of 165 site employees (a 68% response rate) of a Singaporean retail petroleum firm. A restricted multivariate regression model of autonomy and training opportunities explained the majority (35.4%) of the variability of job satisfaction. Age did not moderate these relationships, except for employees >21 years of age, who reported enhanced job satisfaction with additional salary. Human Capital theory, Life Cycle theory and Job Enrichment theory are invoked and explored in the context of these findings in the South East Asian retail petroleum industry. In the South East Asian retail petroleum industry, jobs providing employees with the opportunity to undertake a variety of tasks that enhanced the experienced meaningfulness of work are likely to promote job satisfaction, reduce turnover and increase the quality of service

SREB, a GATA Transcription Factor That Directs Disparate Fates in Blastomyces dermatitidis Including Morphogenesis and Siderophore Biosynthesis

Blastomyces dermatitidis belongs to a group of human pathogenic fungi that exhibit thermal dimorphism. At 22°C, these fungi grow as mold that produce conidia or infectious particles, whereas at 37°C they convert to budding yeast. The ability to switch between these forms is essential for virulence in mammals and may enable these organisms to survive in the soil. To identify genes that regulate this phase transition, we used Agrobacterium tumefaciens to mutagenize B. dermatitidis conidia and screened transformants for defects in morphogenesis. We found that the GATA transcription factor SREB governs multiple fates in B. dermatitidis: phase transition from yeast to mold, cell growth at 22°C, and biosynthesis of siderophores under iron-replete conditions. Insertional and null mutants fail to convert to mold, do not accumulate significant biomass at 22°C, and are unable to suppress siderophore biosynthesis under iron-replete conditions. The defect in morphogenesis in the SREB mutant was independent of exogenous iron concentration, suggesting that SREB promotes the phase transition by altering the expression of genes that are unrelated to siderophore biosynthesis. Using bioinformatic and gene expression analyses, we identified candidate genes with upstream GATA sites whose expression is altered in the null mutant that may be direct or indirect targets of SREB and promote the phase transition. We conclude that SREB functions as a transcription factor that promotes morphogenesis and regulates siderophore biosynthesis. To our knowledge, this is the first gene identified that promotes the conversion from yeast to mold in the dimorphic fungi, and may shed light on environmental persistence of these pathogens