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C Skarke

Publications and source records attributed to C Skarke.

11 recordsLinked to original sources
PubMed2005

[Fundamental principles of clinical trials].

Medical conduct relies on the efficacy and tolerability of a given pharmacotherapy. On the basis of an experimental comparison of different treatment options, a clinical trial provides the foundation for an evidence-based medicine that complies with state-of-the-art scientific practice. The quality of the data assessment determines the clinical relevance of the study results. Clinical trials are classified into Phases I-IV to ensure the development of an effective and safe pharmaceutical. The investigator is responsible for being experienced in the relevant area of expertise, for conducting the study according to the investigational plan, and for complying with ethical guidelines and drug safety.

Adverse Drug Reaction Reporting Systems↗
PubMed2005

Comparison of two screening methods for in-house genotyping in clinical pharmacology units.

Two genetic screening methods, the fluorescence resonance energy transfer (FRET) technique on the LightCycler and the real-time pyrophosphate detection technique on the Pyrosequencer have been compared with regard to their usefulness as screening methods for subject recruitment in clinical studies in pharmacology units. Two SNPs of possible clinical relevance were selected, namely the 118A>G SNP of the OPRM1 gene and the 3435C>T SNP of the ABCB1 gene. Genotypes diagnosed using conventional sequencing served as control. The allelic frequency of the mutated 118G allele of the OPRM1 gene was 12.7% and that of the mutated 3435T allele of the ABCB1 gene was 50.7%. All results obtained with the Pyrosequencer were in accord with those obtained using conventional sequencing. With the LightCycler, an incorrect genotype was assigned to 1 of the 130 DNA samples corresponding to an error rate of 0.8%. Although both methods were found suitable for rapid SNP detection, Pyrosequencing was the preferred method since it provides the nucleotide sequence directly thus facilitating interpretation.

ATP-Binding Cassette Transporters↗
PubMed2004

Single-step identification of all length polymorphisms in the UGT1A1 gene promoter.

AIM: To provide a sensitive genetic screening method for rapid identification of all known length polymorphisms in the promoter region of the uridine 5'-diphosphoglucose glucuronosyltransferase (UGT) 1A1 gene comprising (TA)5, (TA)7 and (TA)8 repeats as opposed to the non-mutated (TA)6 allele. METHODS: The UGT1A1 promoter genotype was assessed in 115 subjects by means of a newly developed pyrosequencing method. PCR-generated DNA templates of heterozygous (TA)5 and (TA)7 carriers were cloned into a TOPO TA vector and verified by sequencing. In addition, a (TA)8 segment was produced by cloning to demonstrate the ability of the method to detect this mutation. RESULTS: All length polymorphisms of the UGT1A1 promoter described in the literature were clearly identified. Fifteen subjects had Gilbert's syndrome with elevated serum bilirubin associated with a homozygous (TA)7TAA/(TA)7TAA genotype. Two subjects with the rare genotypes (TA)5TAA/(TA)6TAA and (TA)5TAA/(TA)7TAA were found, where only the latter one displayed elevated serum bilirubin levels. Allelic frequencies were 0.9%, 66.1% and 33% for the (TA)5TAA, (TA)6TAA and (TA)7TAA allele, respectively. CONCLUSION: Our method enables reliable genetic single-step screening for all known length polymorphisms in the UGT1A1 gene promoter that cause Gilbert's syndrome. This facilitates pharmacogenetic-guided dosing of drugs with known toxicity metabolized by UGT1A1.

Genetic Testing↗
PubMed2001

Effects of the opioid remifentanil on olfactory function in healthy volunteers.

The effects of opioids on human subjective olfactory function have rarely been investigated. This is despite the fact that opioid receptors are widely distributed throughout the olfactory systems. Using an established validated test of subjective olfactory function, olfactory threshold, odor discrimination and odor identification performance were tested in 16 healthy volunteers before opioid administration and at steady state after 3 hours remifentanil infusion. Each one man and one women were assigned randomly to one out of eight predefined remifentanil target plasma concentrations: 0, 1.2, 1.8, 2.4, 3, 3.6, 4.8, and 6 ng/ml. In the thirteen subjects that had completed the tests, olfactory thresholds were elevated with increasing remifentanil dose, and this correlated statistically significant with the remifentanil dose. Remifentanil plasma concentrations were linearly related to changes in olfactory thresholds. In contrast, effects of remifentanil on odor discrimination and identification were not statistically significant. However, remifentanil target plasma concentrations were also significantly correlated with the subjects' ratings of tiredness and drowsiness, although only drowsiness was significantly correlated with the differences in odor thresholds. We conclude that opioid administration leads to impaired olfactory function expressed in raised olfactory thresholds. This is compatible with previously reported opioidergic effects at the level of the olfactory bulb.

Adult↗
PubMed2001

A rapid screening method for a single nucleotide polymorphism (SNP) in the human MOR gene.

AIMS: Genetic association studies have suggested that the single nucleotide polymorphism (SNP) at position 118 of the human mu-opioid receptor (MOR) gene could be a potential risk factor for drug treatment variability in patients. Therefore, we wanted to develop a fast and reliable detection method for this SNP which is applicable in a clinical setting. METHODS: To detect the polymorphism at position A118-->G in the human MOR gene we used the fluorescence resonance energy transfer (FRET)-PCR technique with subsequent melting curve analysis. RESULTS: The polymorphism at position A118-->G in the human MOR gene could be clearly discriminated with melting peak temperatures of 69.8 degrees C and 63.8 degrees C, corresponding to the wild type and mutated MOR allele, respectively. The results from FRET-PCR were validated by sequencing and restriction-fragment length polymorphism (RFLP). Screening of blood samples from 100 subjects showed an allelic distribution for the human MOR alleles of 79% (homozygous wild type), 20% (heterozygous) and 0.9% (homozygous mutated). CONCLUSIONS: The FRET-PCR protocol for detection of the human MOR gene polymorphism at position 118 offers a rapid and reliable method which could be used for population screening of this and other genes.

DNA↗
PubMed2001

The transfer half-life of morphine-6-glucuronide from plasma to effect site assessed by pupil size measurement in healthy volunteers.

BACKGROUND: Clinical and experimental data suggested a long delay between the plasma concentration versus time course of morphine-6-glucuronide and the time course of its central opioid effects. This study was aimed at the quantification of the transfer half-life (t(1/2,ke0)) of this delay. METHODS: Pupil size was used as a measure of central opioid effect. Eight healthy volunteers (four men, four women) participated in that single-blind randomized crossover study. Median dosages administered intravenously were 0.5 mg morphine as loading dose followed by 10.7 mg given as infusion over a period of 4.7 h, and 10.2 mg M6G as loading dose followed by 39.1 mg M6G given over a period of 3.7 h. The duration of the infusion was tailored to achieve submaximum pupil constriction. The pupil diameter was assessed every 20 min for approximately 18 h. Values of t(1/2,ke0) were obtained by semiparametric pharmacokinetic-pharmacodynamic modeling. RESULTS: The estimated median t(1/2,ke0) of M6G was 6.4 h (range, 2.9-16.2 h), and that of morphine was 2.8 h (range, 1.8-4.4 h). The individual t(1/2,ke0) of M6G was always longer than that of morphine. Judged by the concentration at half-maximun effect (EC50) values of the sigmoid pupil size at maximum constriction (Emax) model describing concentration-response relation, M6G was apparently 22 times less potent than morphine (EC50 = 740.5 nm [range, 500-1,520 nm] for M6G and 36.2 nm [range, 19.7-43.3 nm] for morphine). The steepness of the sigmoid Emax model did not significantly differ between morphine and M6G (gamma = 1.9 and 2.6, respectively). To produce similar pupil effects, the M6G dose had to be 2.8 times greater than the morphine dose. CONCLUSIONS: The reported numerical value of the t(1/2,ke0) of M6G in humans obtained after direct administration of M6G is a step toward a complete modeling approach to the prediction of the clinical effects of morphine. The study raises questions about the high interindividual variability of the transfer half-life between plasma and effect site (ke0) values and the apparent low potency of M6G.

Adult↗
PubMed1999

Remodeling of peritoneal-like structures by mesothelial cells: its role in peritoneal healing.

BACKGROUND: Intraabdominal adhesions are a common complication following laparotomy. Since the exact mechanisms involved in this processes are unknown we have analyzed in vitro the role of mesothelial cells in peritoneal healing. MATERIAL AND METHODS: Human mesothelial cells from omental tissue were cultivated for 2 weeks in a three-dimensional culture either on or in a collagen type I matrix. The effects of blood and collagen matrix were analyzed by exposing mesothelial cells to an overlying blood clot, simulating intraperitoneal bleeding, or a second collagen layer. The production of collagen types III and IV, fibronectin, and laminin was analyzed with immunohistochemical methods. RESULTS: Mesothelial cells grown on a collagen matrix formed a monolayer of flat or cobblestone-like cells whereas those cultivated in a collagen matrix exhibited spindle-like morphology. Mesothelial cells failed to grow into an overlying collagen matrix, but did grow into a blood clot, emphasizing a potential role of blood clots in peritoneal adhesion formation. Independent of the culture systems mesothelial cells produced collagen type III, fibronectin, and laminin but not collagen type IV. CONCLUSIONS: Our experiments demonstrate remodeling of peritoneal-like structures by mesothelial cells in a three-dimensional culture reflecting their putative role in the reepithelialization after serosal defects, and also in the formation of peritoneal adhesions.

Cells, Cultured↗
PubMed1998

Differential expression of cell adhesion molecules in inflamed appendix: correlation with clinical stage.

The diagnosis of 'early inflamed', 'recurrent' or 'sub-acute' appendicitis is often difficult and accompanied by controversies between clinical data, histological findings, and their interpretation. The expression of the intercellular cell adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1), and E-selectin has been studied in 61 appendicectomy specimens for possible use as a diagnostic tool. This study demonstrates a different expression of CAM by endothelial (EC) and mesothelial cells (MC) in the various stages of appendicitis, with early E-selectin and ICAM-1 expression in EC, followed by VCAM-1 in EC and MC. Appendices from patients with prolonged clinical symptoms defined by clinicians as 'chronic' appendicitis showed VCAM-1 expression and occasionally weak expression of E-selectin in EC. In several cases, discrepancies were found between the pre-operative 'clinical' diagnosis, the histomorphological findings, and the immunohistological results. In this context, the expression of E-selectin and VCAM-1 in comparison with the histological features has potential significance in the diagnosis of 'early acute', 'sub-acute' or 'recurrent' appendicitis. In addition, a correlation was demonstrated between the histological stages of appendicitis and the kinetics of CAM expression. The study also indicates that the time course of E-selectin expression in vivo is longer than is suggested from in vitro data.

Acute Disease↗
PubMed1997

Reconstruction of peritoneal-like structure in three-dimensional collagen gel matrix culture.

The peritoneum is a serous membrane consisting of different kinds of cells and extracellular matrix components (ECM). The aim of the present study was to develop a three-dimensional (3D) in vitro culture system for possible investigation of pathological conditions of the peritoneum. Human omental mesothelial cells (MC) and endothelial cells from the umbilical vein (EC) were cultivated either on (MC) or in (EC) a preformed type I collagen matrix. In 3D culture mesothelial cells showed their phenotypical in vivo characteristics and the synthesis of a new basal membrane (BM). Endothelial cells developed vessel-like structures, produce a BM and express E-selectin after TNF-alpha stimulation. This 3D culture system presents extended possibilities for analyzing mesothelial and endothelial cell behavior as well as the cell-cell and cell-matrix interactions involved in several pathological processes in the peritoneum.

Adipose Tissue↗
PubMed1996

PECAM-1 expression in human mesothelial cells: an in vitro study.

Mesothelial cells are actively involved in inflammatory processes by expressing a set of cell adhesion molecules (CAMs). Transmigration of leukocytes into inflamed tissues requires a chemotactic stimulus and engagement of platelet-endothelial cell adhesion molecule-1 (PECAM-1). To investigate the kinetics involved in peritonitis, pure cultures of mesothelial cells are necessary. In previous studies, we have found that human mesothelial cells (HOMES) show a weak constitutive expression of PECAM-1, which cannot be further stimulated by cytokines. It is known that all serous cavities and body fluids contain numerous macrophages which strongly express this adhesion molecule. To identify the cells responsible for the expression of PECAM-1, mesothelial cells freshly obtained from omental tissue were isolated using PECAM-1-conjugated magnetic beads by cell sorting. For these studies, the negative as well as the positive fraction of isolated cells were used. As a control, freshly isolated monocytes were studied. Cell cultures were characterized by light and electron microscopy, as well as immunocytochemistry. The negative cell fraction was cultivated and stimulated for different times with tumor necrosis factor-alpha (30 and 300 U/ml), interleukin-1 beta (10 and 100 U/ml) and interferon-gamma (500 U/ml) and PECAM-1 expression was analyzed by a comparative quantitative cell enzyme immunoassay (EIA). The positive cell fraction was treated in the same manner. Both fractions of isolated cells showed strong positivity for cytokeratins 8, 18, 7 and 19, as well as vimentin. CD68, a monocyte marker, was not detected on mesothelial cells. In addition, EIA analysis confirmed the constitutive expression of PECAM-1 obtained from previous studies. This expression on HOMES was not inducible, irrespective of the type and concentration of cytokine studied. These data confirm PECAM-1 expression on mesothelial cells obtained from human omental tissue and suggest a critical role in transmigration of leukocytes during peritoneal inflammation.

Cell Separation↗