Biomedical subjects

L De Ridder

Publications and source records attributed to L De Ridder.

At least 37 records · Page 2Linked to original sources

PubMed1999

Apoptosis induced by gamma irradiation in peripheral blood mononuclear cells is not mediated by cytochrome-c release and only partially involves caspase-3-like proteases.

Caspase 3 has been shown to be actively involved in the apoptotic process in thymocytes after gamma-irradiation. We examined caspase 3 activation in mature peripheral blood lymphocytes (PBL) after gamma irradiation. Since the activation of caspase 3 is generally prceded by a decrease in mitochondrial membrane potential (delta psi m) and cytochrome c release, these two parameters were also examined. Apoptosis in PBL after a 5-Gy gamma irradiation, is characterized by a decrease in delta psi m, but surprisingly no release of cytochrome-c and only a weak caspase 3 activation was noticed. In contrast, staurosporin treated PBL showed a decrease in delta psi m with cytochrome-c release and a clear caspase 3 activation. We were unable to block the decrease in delta psi m with the caspase-inhibitors zVAD-fmk or zDEVD-fmk after gamma irradiation, but DNA fragmentation as measured by the TUNEL assay was partially inhibited. Therefore, in gamma irradiated mature PBL, caspase-dependent and -independent pathways, but not cytochrome c, seem to be involved in the apoptotic process.

Apoptosis↗

PubMed1999

A multidisciplinary approach to the healing of cranial and residual maxillary cleft defects by means of allogenous demineralized osseous implants and polylactic acid casts in dogs.

OBJECTIVE: The aim of this study was to evaluate the healing of artificially induced bony defects in dogs by means of demineralized allogenous bone powder (DBP) implants covered with polyhydroxy lactic acid (PLA) casts compared with DBP implants without the casts. DESIGN: Prospective animal study. SETTING: Research university. SAMPLE: Following a pilot study in which two dogs were used, four mongrel dogs between the ages of 18 and 24 months and weighing approximately 20 kg were used as subjects. INTERVENTIONS: Each experimental animal had bilateral maxillary alveolar clefts created. In a later procedure, each defect was repaired with a DBP implant, half of which were covered with a PLA96 matrix. Each animal also had a circular defect created in each parietal bone that was immediately covered with DBP implants, half of which were similarly covered with a PLA96 disk. MAIN OUTCOME MEASURE: Repeated technetium-99m methylene-diphosphate (99mTC MDP) uptake measurements were performed to evaluate bone metabolism during the healing period, while at relevant intervals, radiographs were taken of the healing alveolar cleft defects to register bone repair. After 1 year, the animals were euthanized for macroscopic and histologic evaluation. RESULTS: Histologically, the grafts covered with PLA96 were at a more advanced stage of healing than those without, and the cranial defects similarly were more advanced in the healing process than the alveolar defects. Uptake of 99mTC MDP into the cranial implants was at its maximal level after 1 week and then gradually decreased until, after 7 weeks, it was not significantly different from zero. Cranial defects covered with a PLA95-enhanced implant showed a mean maximum count rate of 275, while the plain DBP side showed a mean maximal count rate of 150. Alveolar defects with the plain DBP implant showed a maximum count rate in the first week; those with the PLA95 enhanced implant showed maximum uptake during the second week. On both sides, there was a gradual decrease to the base value in the seventh week. The mean maximum count on the PLA96-enhanced side was 285, while on the plain DBP side, the corresponding value was 320. CONCLUSION: Although an advantage of the combination was established for parietal cranial defects, no advantage was seen for alveolar cleft defects in this experimental setup.

Alveoloplasty↗

PubMed1999

Influence of polysulfated polysaccharides on aggrecans synthesized by differentiated human articular chondrocytes.

OBJECTIVE: Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates have been used as variables reflecting the capability of these cells to restore the extracellular matrix of articular cartilage in vitro. The effects of heparin, chondroitin polysulfate, and xylosan polysulfate on these variables were investigated. METHODS: Human articular chondrocytes were maintained in culture in gelled agarose. After 2 weeks of culture, the media were supplemented for one week with 10 microg/ml each of heparin, chondroitin polysulfate, and xylosan polysulfate. Synthesis of aggrecans was investigated using Na2(35SO4) as a radioactive precursor during the last 24 h of exposure to polysaccharides. Size exclusion chromatography was used to assay the proportions of aggrecans immobilized in aggregates. Native aggrecan aggregates accumulated during culture were liberated in associating conditions by agarase digestion of the artificial agarose matrix and studied by electron microscopy. Hyaluronan synthesis was studied in monolayer cultured chondrocyte derived fibroblasts. The cells were exposed to 3H glucosamine for 24 h after the nutrient media were supplemented with 3 polysaccharides for one week. Size exclusion chromatography was used to assess the length of the hyaluronan filaments. RESULTS: Xylosan polysulfate and chondroitin polysulfate but not heparin significantly increased total 35S incorporation rates in aggrecan. Electron microscopy study of aggrecan aggregates showed that xylosan and chondroitin polysulfate but not heparin significantly increased aggrecan aggregate sizes. The same polysulfated polysaccharides increased the synthesis of high molecular weight hyaluronan by chondrocyte derived fibroblast-like cells. CONCLUSION: Polysulfated polysaccharides significantly increased the synthesis rates and the accumulation of aggrecan in aggregates in the extracellular environment in this in vitro system.

Adult↗

PubMed1998

Cathepsin B-mediated activation of the proinflammatory caspase-11.

Members of the caspase (CASP) family of cysteine proteases can be subdivided in proapoptotic caspases and proinflammatory caspases. Whereas the apical activation pathways for the caspases that are involved in the execution of the apoptotic process are beginning to be understood, the pathways that lead to the activation of proinflammatory caspases are still largely unknown. Analysis of subcellular fractions for their ability to process and activate several caspases in vitro led to the identification of lysosomes as the source for a protease that could proteolytically activate the proinflammatory CASP-11. Although this lysosomal activity was sensitive to caspase inhibitors, affinity purification with the biotinylated broad spectrum caspase inhibitor z-VAD.fmk revealed the CASP-11 activating protease as cathepsin B. Activation of CASP-11 by cathepsin B as well as its sensitivity to several caspase inhibitors was further confirmed with purified proteases. Similar to the role of mitochondrial factors in the activation of proapoptotic caspases, our results suggest a potential role for lysosomes and cathepsin B as activators of specific proinflammatory caspases. In addition, the aspecific inhibition of cathepsin B by so-called specific caspase inhibitors implicates that results obtained with these inhibitors should be interpreted with care.

Animals↗

PubMed1998

Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes.

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in gamma irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5 Gy and 20 Gy gamma irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5 Gy gamma irradiation of PBMCs mainly causes apoptosis, whereas a 20 Gy gamma irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.

Adult↗

PubMed1998

Real-life radiation burden to relatives of patients treated with iodine-131: a study in eight centres in Flanders (Belgium).

In view of the EURATOM 96/29 [1] regulations, a prospective multicentre study was performed to evaluate the present guidelines given to relatives of patients treated with iodine-131 for both thyroid carcinoma and thyrotoxicosis, based on the real-life radiation burden. This study comprised 166 measurements carried out on a group of 94 relatives of 65 patients. All relatives wore a thermoluminescent dosemeter (TLD) on the wrist for 7 days. Sixty-one relatives agreed to wear another TLD for an additional 7 days. TLD were placed on nine patients' bedside tables. The eight participating centres were arbitrarily divided into three groups according to the period of time they advised their patients to sleep separately. Groups I, II and III respectively advised their patients to sleep separately for 0, 7-10 and 14-21 days. The median dose received by in-living relatives of thyroid carcinoma patients during the 14 days following hospital discharge was 281 microSv (doses to infinity not calculated); the median dose to infinity received by in-living relatives of ambulatory treated thyrotoxicosis patients was 596 microSv, as compared with 802 microSv for in-living relatives of hospitalised thyrotoxicosis patients. In general the children of patients received a significantly (P < 0.1) lower mean dose than their partners. For thyroid carcinoma patients, only two relatives out of 19 (10%) exceeded the EURATOM 96/29 limit of 1 mSv/year. For thyrotoxic patients, 28% of relatives exceeded the EURATOM 96/29 limit, but none of them were relatives of patients who followed guidelines for 21 days. The results of this study indicate that sleeping separately for 7 days, after a period of hospitalisation of 2-3 days, will usually be sufficient for thyroid carcinoma patients. For thyrotoxicosis patients, up to 21 days of sleeping separately could be necessary in order to strictly abide by EURATOM 96/29. Therefore, the authors propose the implementation of a non-rigid dose constraint for people who "knowingly and willingly" help patients treated with 131I, while still following the ALARA principle.

Adolescent↗

PubMed1998

Effect of testosterone replacement after neonatal castration on craniofacial growth in rats.

Neonatal castration precludes the pubertal increase in serum testosterone and reduces general and craniofacial growth in the male Wistar rat. This study aimed to determine whether exogenous testosterone given at an age beyond the normal pubertal peak restores general and craniofacial growth in male, neonatally castrated rats. The design was a randomized, double-controlled, cross-sectional trial. Male Wistar rats were assigned by weighted randomization to be either castrated early after birth (n = 35) or not (n = 15). On day 57, a 1.5-cm Silastic tube with testosterone was implanted in 18 of the castrated rats. On day 70 and day 110, body length, weight, and craniofacial growth were measured together with the weight of the prostate, and blood samples taken. The exogenous testosterone resulted in a significant increase in serum testosterone and prostate weight. All measures of general and craniofacial growth had higher mean values in the non-castrated control group than in the castrated group, while in the testosterone-implant group the mean values lay between these of the castrated animals and the non-castrated controls. Two-way ANOVA indicated a significant effect of the testosterone administration on lower-incisor growth and the size of the total skull vault.

Analysis of Variance↗

PubMed1998

Comparative effects of neonatal and prepubertal castration on craniofacial growth in rats.

The role of endogenous testosterone in the craniofacial growth of the young male rat was investigated. First, the effect of neonatal surgical castration was examined in a randomized, cross-sectional study in which male Wistar rats were allocated to be either castrated or sham-operated 4 h after birth. Then, the effect of prepubertal chemical castration was analysed in a second, randomized longitudinal study in which male Wistar rats were randomly allocated either to a control group or to two experimental groups, one injected with triptorelin at day 25 and the other injected on day 25 and on day 45. Every tenth day between 20 and 70 days of age for the first study, and between 30 and 110 days of age for the second, body length and weight were measured, cephalometric X-rays taken, and blood samples obtained. Neonatal and prepubertal castration resulted in decreased plasma concentrations of testosterone and in delayed growth of somatic and craniofacial components. The initiation, duration and magnitude of the effect was dependent on individual bones (cranial base, skull roof) and on the lower incisor, and related to the testosterone concentrations. These results suggest that testosterone effects participate in the process of normal craniofacial growth, particularly during puberty.

Aging↗

PubMed1998

Apoptosis induced by fast neutrons versus 60Co gamma-rays in human peripheral blood lymphocytes.

PURPOSE: To investigate the effectiveness of high LET fast neutrons compared with low LET 60Co gamma-rays to induce apoptosis in resting lymphocytes. MATERIALS AND METHODS: Apoptosis induction was quantified by light microscopic analysis of irradiated lymphocyte samples from healthy donors after 24 h culture. For the dose-response analyses doses ranging from 0.05 to 5 Gy were applied at 1.5 Gy/min (gamma-rays) or 0.2 Gy/min (fast neutrons). To investigate the role of DNA repair in apoptosis induction a dose of 2 Gy was also given at low dose-rate (0.006 Gy/min). RESULTS: Dose-response curves obtained with both radiation qualities were characterized by an initial steep increase in the number of apoptotic cells below 1 Gy, with a flattening of the curves at higher doses towards 5 Gy. The calculated relative biological effectiveness (RBE) values for fast neutrons were close to unity. When a 2 Gy dose was administered at low rather than high dose-rate no decrease in apoptotic cell yield was observed. CONCLUSION: The dose-response data confirm the high radiosensitivity of lymphocytes and demonstrate that their response to undergo early interphase cell death by apoptosis is largely independent of LET. The observations that apoptosis induction is independent of LET and dose-rate may suggest that initial DNA damage, as opposed to DNA repair, dominate the induction of apoptosis in resting lymphocytes.

Adult↗

PubMed1998

Induction of micronuclei and apoptosis in natural killer cells compared to T lymphocytes after gamma-irradiation.

PURPOSE: To investigate the chromosomal damage caused by gamma-irradiation in T lymphocytes and natural killer (NK) cells and compare this with apoptosis induction in both lymphocyte subsets. MATERIALS AND METHODS: Apoptosis induction by gamma-irradiation in T lymphocytes and NK cells was quantified using the annexin V flow cytometric assay. The cytokinesis-block micronucleus (MN) assay was used to evaluate the induced cytogenetic damage. For the MN assays on NK cells, gamma-irradiated peripheral blood mononuclear cells were cultured and stimulated with interleukin 15 (IL-15). Afterwards the NK cells (characterized by the CD3-/CD56+ phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binuclear cells was scored. Doses of 1 and 2 Gy gamma-irradiation were applied. RESULTS: Higher numbers of MN in NK cells were found compared with the MN yield in T lymphocytes. In contrast, NK cells were less than T lymphocytes prone to apoptosis after gamma-irradiation. CONCLUSION: The results support the view that cytogenetic damage and apoptosis after gamma-irradiation are not necessarily correlated.

Apoptosis↗

PubMed1997

Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens.

Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose gamma-irradiation prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change.

Animals↗

PubMed1997

Semi-automated micronucleus scoring in cytokinesis-blocked lymphocytes after irradiation.

A semi-automated scoring procedure for the cytokinesis-block micronucleus assay based on computerized image analysis was validated by comparing results of the automated analysis with those from manual analysis by those experienced scorers of micronuclei. Each object selected by the computer system was also relocated and visually inspected. The validation was based on slides of blood samples after in vitro irradiation with X-rays in the low-dose range up to 2 Gy. Up to 1 Gy a very satisfactory agreement between manual and semi-automated scoring was obtained. At 2 Gy the micronucleus yields obtained by the semi-automated scoring were systematically below the manual data due to a small percentage of false negatives (3%). Using the appropriate dose-response relationship the validation study indicates that the results obtained by computerized image analysis are comparable to manual scoring with respect to accuracy. The computerized assay is superior regarding reproducibility. At present, semi-automated scoring of micronuclei is practically feasible for a limited number of slides for large-scale applications of the semi-automated micronucleus is assay in the field of radiation protection an increase of the speed of the system is necessary.

Adult↗

PubMed1997

Flow cytometry as a quantitative and sensitive method to evaluate low dose radiation induced apoptosis in vitro in human peripheral blood lymphocytes.

Human peripheral blood lymphocytes, irradiated in vitro, die by an apoptotic process. The number of apoptotic cells after in vitro gamma-irradiation (0, 0.1, 0.2, 1, 2 and 5 Gy) was measured by flow cytometry using Annexin V and DiOC6 (a cationic dye) after 24 and 48 h incubation. The mean dose-response curves for apoptosis of six healthy volunteers obtained with both methods were steep below 1 Gy and flatter at higher doses. A slightly higher number of apoptotic cells was observed with DiOC6, compared to Annexin V. This can be assigned to a minor DiOC6-int/PI- population. Forty-eight hour cultures contained higher numbers of apoptotic cells compared with 24 h cultures. For both culture times, DiOC6 and Annexin V detected a statistically significant difference between a control sample and a 0.1 Gy irradiated one, illustrating the high sensitivity of the methods.

Annexin A5↗

PubMed1997

In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytes.

One of the major drawbacks of the in vitro micronucleus (MN) assay for human lymphocytes is its reduced sensitivity for the detection of damage induced by low radiation doses, due to the high variability among the spontaneous MN frequencies. In this paper we investigated the enhancement of the sensitivity of the MN assay by analysing spontaneous and radiation-induced MN for the presence of centromeres. For this, in situ hybridization (FISH) with the human pancentromeric DNA probe, p82H, was performed. Our results revealed that a high percentage (73%) of the spontaneous MN contain a centromere. These centromere-positive MN indicate the presence of a whole chromosome/chromatid. After in vitro irradiation with low doses (0.1-2 Gy) 60Co gamma-rays mainly centromere-negative MN were induced while only a very small number of additional centromere-positive MN were formed. This demonstrates that radiation-induced MN mainly contain acentric fragments pointing to the clastogenic action of ionizing radiation. Furthermore, our data show that the sensitivity of the MN assay for low dose detection is increased by scoring only centromere-negative MN.

Adult↗

PubMed1997

Generation of a monoclonal antibody directed against a human cell substrate adhesion molecule and the expression of the antigen in human tissues.

Cell substrate adhesion is a prerequisite for invasion and the subsequent formation of metastases. Therefore, we designed monoclonal antibodies (MAbs) against epitopes on the extracellular cell membrane domain of SK-BR-3 cells. One of the antibodies, called MAb 14C5, binds to an extracellular epitope of a plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells. This MAb 14C5 is able to inhibit cell substrate adhesion, not only on culture-treated plastic but also on host tissue, and therefore prevents invasion and metastases. We evaluated the tissue distribution of the 14C5 antigen by immunohistochemistry. The antigen is specifically overexpressed in 64% of invasive ductal adenocarcinomas of the breast (n = 33), in all investigated cases of invasive squamous cell carcinoma (n = 7) and in 40% of basocellular carcinomas of the skin (n = 5). The 14C5 molecule is located on the cell membrane of the carcinoma cells. However, when the tumor is characterized by a highly invasive phenotype, 65% of the cases also show an extensive stromal expression on the fibroblasts between the tumor cells (n = 71). This stromal expression is caused by the presence of the 14C5 antigen on the membrane of the adjacent fibroblasts. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast (n = 15), no expression of the 14C5 antigen occurred. A 90-kDa protein was purified from lysates of human breast cancer cells using a 14C5 MAb Sepharose column and is considered as the antigen recognized by the MAb 14C5.

Antibodies, Monoclonal↗

PubMed1996

Mechanism of age-dependent involution in embryonic chick notochords.

To study the possible mechanism of the age-dependent involution of the notochord, isolated mesenchyme-free notochords of chick embryos were cultured in vitro and compared with their counterparts in vivo. Two different aspects were evaluated: (1) DNA synthesis measured by [3H]thymidine incorporation and visualized by autoradiography and (2) cell death quantified by counting the number of pyknotic nuclei. The results demonstrate that [3H]thymidine uptake by notochords shows an age-dependent decrease in vitro as well as in vivo. The number of [3H]thymidine-labelled notochord cells, however, is higher in vitro than in vivo. At the same time, there is an age-dependent increase in pyknosis in the notochord in vivo and in vitro. So, during the aging process, the number of both pyknotic nuclei and of [3H]thymidine-labelled nuclei suggest a high turnover of notochord cells in vitro. From these results, we can conclude that the process of involution in aging notochord seems to be controlled by a programmed intrinsic process, which might be influenced partially by the microenvironment in vivo.

Animals↗

PubMed1996

A cytogenetic study of radiological workers: effect of age, smoking and radiation burden on the micronucleus frequency.

A large scale cytogenetic study of the radiation damage in nuclear power plant workers and medical workers handling X-ray machines (269 individuals) was undertaken using the micronucleus assay for peripheral blood lymphocytes. The micronucleus frequency was found to increase systematically with donor age. After correction for the age-dependence, no correlation of the micronucleus frequency with smoking habits, expressed as cigarette-years and cigarette consumption per day, could be observed. Compared to the group of administrative workers receiving doses below 1 mSv/year, limit recommended by the ICRP for public exposure, the micronucleus frequency was slightly increased in the group of radiation workers, exposed occupationally. However, applying the Mann-Whitney test, the observed differences are not statistically significant. After correction of the dose accumulation pattern for the turn-over of the lymphocyte pool, a weak correlation between the micronucleus frequency and the equivalent dose accumulated over the 10 years preceding the study was obtained. For clear-cut conclusions on the radiation damage of low-dose worker cohorts, an increase in the sensitivity of the assay, e.g., by analysis of the micronuclei for the presence of centromeres is necessary.

Adult↗

PubMed1996

Micronucleus induction by 60Co gamma-rays and fast neutrons in ataxia telangiectasia lymphocytes.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by a progressive neuronal degeneration, immunodeficiency, cancer proneness and an extreme sensitivity to ionizing radiation. In this work, micronucleus dose-response curves for lymphocytes of normal and AT individuals, exposed in G(zero) to low LET gamma-rays and high LET fast neutrons, are compared. After gamma-irradiation, the micronucleus yields for AT lymphocytes are strongly increased compared with controls. The micronucleus dose-response curve for AT cells shows a linear dependence instead of a linear-quadratic one which is found for normal cells. After neutron irradiation, the increase in micronucleus yield above controls is less pronounced than with gamma-rays and the micronucleus dose-response curves are linear, as expected. The high increase in micronucleus yield compared with controls after gamma-irradiation further suggests the application of the micronucleus assay as a diagnostic tool for ataxia telangiectasia.

Ataxia Telangiectasia↗