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Biomedical subjects

I Vermes

Publications and source records attributed to I Vermes.

At least 19 recordsLinked to original sources
PubMed2006

Differential effects of bisphosphonates on breast cancer cell lines.

Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.

Alendronate↗
PubMed2006

First steps towards tissue engineering of small-diameter blood vessels: preparation of flat scaffolds of collagen and elastin by means of freeze drying.

Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.

Biomechanical Phenomena↗
PubMed2005

Electrospinning of collagen and elastin for tissue engineering applications.

Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (M(w)=8 x 10(6)) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability (T(d)=79 degrees C) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.

Animals↗
PubMed2005

A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells.

BACKGROUND: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness). METHODS: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor-alpha/cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation. RESULTS: TNF-alpha treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed. CONCLUSIONS: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.

Annexin A5↗
PubMed2005

Apoptosis and proliferation in breast cancer cells, cultured in vitro: effects of SERMs.

OBJECTIVE: Selective estrogen receptor modulators (SERMs) decrease the risk of developing breast cancer. As an antagonistic effect, SERMs may aggravate or induce climacteric symptoms. Hormone therapy (HT) would be able to alleviate these symptoms. The present in vitro study tries to elucidate the effects of several HT preparations combined with SERMs on estrogen receptor-positive (ER +) (i.e. MCF-7 and T-47D) and -negative (ER-) (i.e. MDA-MB-231) human breast cancer cells in vitro. METHODS: We performed experiments with various HT preparations (estradiol (E2)/E2 + progesterone/E2 + dihydrodydrogesterone /E2 + norethisterone acetate/E2 + medroxyprogesterone acetate/tibolone) in the concentration of 10(-6) mol/l together with SERMs (raloxifene or tamoxifen) added to different breast cancer cell lines in vitro. After an incubation period of 144 h, proliferation and apoptosis were measured. The first was measured by quantification of the expression of cyclin D1 mRNA, the latter by the Nicoletti method. RESULTS: This in vitro study clearly demonstrates differences in results if various HT preparations, combined with SERMs, are added to ER + and ER- breast cancer cell lines. CONCLUSIONS: Adding estradiol/progestogens in combination with a SERM to estrogen receptor-positive breast cancer cell lines does not obligatorily lead to proliferation of tumor cells. Not all progestogens act equally.

Apoptosis↗
PubMed2004

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used.

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.

Apoptosis↗
PubMed2004

Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip.

INTRODUCTION: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. METHODS: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. RESULTS: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. CONCLUSIONS: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.

Apoptosis↗
PubMed2003

[Calprotectin: a fecal marker for diagnosis and follow-up in patients with chronic inflammatory bowel disease].

Chronic inflammatory bowel disease (IBD) is characterised clinically by periods of well being interspersed by exacerbations of disease activity. Differentiation between IBD and less severe disorders such as irritable bowel syndrome requires invasive and expensive diagnostic procedures. Diagnostic differentiation between active disease, symptoms due to residual constriction of the fibrotic lumen and functional symptoms is a well-known problem. There are not yet any laboratory parameters with sufficient discrimination in terms of sensitivity and specificity. Colonoscopy and histopathological examination remain the gold standards: in Crohn's disease this may be complex due to the variable localisation of the inflammatory process. Abdominal scintigraphic procedures, although informative, are complex and expensive. The recent assessment of faecal calprotectin, a calcium- and zinc-binding anti-inflammatory protein found in neutrophilic granulocytes and monocytes, offers an attractive alternative as an index of intestinal inflammation. We measured this stable marker in random stool samples from 187 patients including healthy volunteers, patients with endoscopically classified active IBD or IBD in remission, and patients with other gastrointestinal disorders. Disease activity was monitored by clinical symptoms, blood tests and endoscopy. Our results confirm previous literature findings that faecal calprotectin is a promising and useful non-invasive tool in the screening of patients presenting with abdominal pain and diarrhoea. Moreover, calprotectin seems helpful in differentiating between active and non-active IBD and possibly also in the monitoring of disease activity.

Biomarkers↗
PubMed2003

In vitro effects of estradiol, dydrogesterone, tamoxifen and cyclophosphamide on proliferation vs. death in human breast cancer cells.

The effects of 17 beta-estradiol, dihydrodydrogesterone, tamoxifen and cyclophosphamide upon parameters of cell maturation (Mucine1 expression), cell proliferation (Cyclin D1 expression) and apoptosis (loss of nuclear DNA) were studied in estrogen receptor positive (ER+) and negative (ER-) human breast cancer cells. Tamoxifen was the most potent inducer of apoptosis in ER+ and ER- breast cancer cells. 17 beta-estradiol in a concentration of 10(-6) M induced proliferation in ER+ cells after 144 h. incubation, while equimolar co-incubation with dihydrodydrogesterone prevented this effect and even induced a significant increase of cell death. It is speculated that the continuous use of combined 17 beta-estradiol plus dihydrodydrogesterone might be given as hormone replacement therapy without increased risk of breast cancer and even may reduce the relapse rate in breast cancer patients.

Antineoplastic Agents, Alkylating↗
PubMed2003

Tissue transglutaminase mRNA expression in apoptotic cell death.

INTRODUCTION: Tissue transglutaminase (t.TG) is an enzyme that catalyzes the cross-linking of intracellular proteins, thus assembling a protein scaffold that prevents leakage of intracellular components. t.TG is activated during the apoptotic cell death cascade and plays a key role in the formation of apoptotic bodies. The aim of this study was to determine to what amount t.TG-mRNA becomes expressed during apoptosis and whether the t.TG-mRNA expression level could be used as trace marker of recent apoptosis and in individual cases for quantification of apoptosis. METHODS: Expression of t.TG-mRNA was determined using TaqMan based, real-time RT-PCR, a semi-quantitative RT-PCR technique. The t.TG-mRNA expression was measured in cultured cells (MCF-7, human endothelial cells) and in peripheral blood mononuclear cells (PBMCs) before and after induction of apoptosis in vitro. RESULTS: The TaqMan RT-PCR of t.TG proved to be reliable, reproducible (CV's inter and intraassay precisions of 0.8-2.8%, measured at two levels), and specific for apoptotic cell death. t.TG-mRNA expression increases in response to apoptosis induction and is not expressed during the process of necrotic cell death. The expression during apoptotic cell death changes in the dose dependent manner in cultured cells as well as in the PBMCs, treated in vitro. The increase t.TG-mRNA expression level was up to 20 times, depending on the intensity of the apoptosis induction treatment and incubation time afterwards. PBMCs of patients with myelodysplasia showed spontaneous expression of t.TG-mRNA in agreement with their increased apoptotic cell death in vivo. CONCLUSION: t.TG-mRNA expression increases significantly in response to apoptosis inducing treatment. The observed changes are dose and time dependent. This leads to the conclusion that t.TG expression can be used as a trace marker for detection and quantification of apoptosis.

Apoptosis↗
PubMed2001

Patterns of corticosteroid-binding globulin and the free cortisol index during septic shock and multitrauma.

OBJECTIVE: To study the time course of corticosteroid binding-globulin (CBG) level and the free cortisol index (FCI) in comparison with total cortisol and ACTH concentrations during acute and prolonged critical illness. DESIGN: Prospective observational clinical study. SETTING: Twenty-bed medical/surgical intensive care unit. PATIENTS AND PARTICIPANTS: Thirty patients with septic shock, eight patients with multitrauma, and forty healthy control subjects. MEASUREMENTS AND RESULTS: During 14 days or until discharge/death, we serially measured serum concentrations of CBG, cortisol, TNF-alpha, IL-6, plasma ACTH immunoreactivity, and the FCI (=cortisol/CBG x 100). We also recorded haemodynamic parameters, APACHE II, ISS, SOFA scores, shock duration, inotrope use, and ICU mortality. In both groups we found markedly decreased CBG levels in the early phase (septic shock: 17.5+/-5.9, and trauma: 16.1+/-2.3 mg/l) in comparison with controls (37.3+/-5.3 mg/l). The FCI was high in this early phase (septic shock: 7.2+/-2.7; trauma: 6.5+/-1.3; controls: 1.25+/-0.76). During follow-up, CBG levels significantly increased, reaching normal levels from day 7 on. The FCI showed an opposite biphasic pattern, with near-normalising FCI values during the second phase. Regression analysis showed a negative correlation between CBG and IL-6 levels (rs=-0.63; P<0.05), but no relation between CBG concentrations and disease severity, shock duration or death was found. CONCLUSIONS: We found extremely low CBG levels in early stage septic shock and multitrauma. These dramatic changes are reflected in a concomitant higher FCI, indicating a higher free cortisol level. A second phase displays increasing and normalising CBG levels, independent from clinical parameters. We believe that CBG plays an active role in the glucocorticoid response to severe stress and in the regulation of cortisol availability to target tissues.

APACHE↗
PubMed2001

Beta 3-adrenergic receptor gene polymorphism and type 2 diabetes in a Caucasian population.

AIM: The beta 3-adrenergic receptor (beta 3-AR) is suspected to play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. A mutation in the beta 3-AR gene (Trp64Arg) has been associated with the capacity of weight gain and with early onset of noninsulin dependent diabetes mellitus (type 2 diabetes). In this study we investigated the prevalence of the two beta 3-AR alleles in a Caucasian population and studied the association between the beta 3-AR genotype and metabolic disorders (obesity and type 2 diabetes). METHODS: Genomic DNA extracted from peripheral blood leucocytes of 200 Caucasian subjects (137 subjects with and 63 subjects without type 2 diabetes). The MvaI polymorphism of beta 3-AR, which detects the Trp64Arg mutation, was determined by polymerase chain reaction (PCR). We studied the correlation between the Trp64Arg mutation and the body mass index (b.m.i. kg/m2). RESULTS: There was no significant difference between the patients with type 2 diabetes and control subjects in the frequency of the Arg64 allele (5.5% and 4.8%, respectively). Within the group of type 2 diabetes patients were 14 subjects with the Trp64Arg mutation (b.m.i., mean +/- s.d.: 31 +/- 8.5 kg/m2) and 123 without the mutation (b.m.i. 29 +/- 4.8). There was no association between the beta 3-AR gene polymorphism and sex, obesity, blood pressure, glycohaemoglobin concentration, proteinuria. CONCLUSION: Our results suggest that the Trp64Arg mutation is not a major determinant of metabolic disorders (type 2 diabetes, obesity) and chronic complications of type 2 diabetes in a Dutch population.

Adult↗
PubMed2001

The hypothalamic-pituitary-adrenal response to critical illness.

The maintenance of life depends on the capacity of the organism to sustain its equilibrium via allostasis'-the ability to achieve stability through change. Life-threatening disease induces acute adaptive responses specific to the stimulus and generalized responses when the disturbances are prolonged. These changes are associated with increased activity of the hypothalamic-pituitary-adrenal axis and may have survival value in preparing the body for fight or flight'. There is a shift towards an increase in glucocorticoid production and away from mineralocorticoid and androgen production, as well as an increase in the biological effects of glucocorticoids through an increased cortisol free fraction and an increased glucocorticoid receptor sensitivity. During the prolonged phase, there is a dissociation between high plasma cortisol and low adrenocorticotropin hormone levels, suggesting non-adrenocorticotropin hormone-mediated mechanisms for the regulation of the adrenal cortex. This hypercortisolism is in contrast to the very low dehydroepiandrosterone sulphate level, indicating an imbalance between the immunostimulatory and immunosuppressive adrenocortical hormones. The question is whether the total serum cortisol concentration represents sufficient glucocorticoid biological activity during the prolonged phase of critical illness.

Animals↗