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Biomedical subjects

D Janssens

Publications and source records attributed to D Janssens.

At least 19 recordsLinked to original sources
PubMed2001

Expression, refolding and crystallization of the OpcA invasin from Neisseria meningitidis.

OpcA is an integral outer membrane from the Gram-negative pathogen Neisseria meningitidis that plays a role in adhesion of meningococci to host cells. The protein was overexpressed in Escherichia coli in an insoluble form and a procedure developed for refolding by rapid dilution from denaturant into detergent solution. The refolded material was identical to native OpcA isolated from meningococci, as judged by overall molecular weight, migration on SDS-PAGE and reaction against monoclonal antibodies. Both native and recombinant OpcA crystallized under similar conditions to give an orthorhombic crystal form (P2(1)2(1)2), with unit-cell parameters a = 96.9, b = 46.3, c = 74.0 A. Complete data sets of reflections were collected from native and refolded OpcA to 2.0 A resolution.

Bacterial Proteins↗
PubMed2001

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

Thirty-one heavy-metal-resistant bacteria isolated from industrial biotopes were subjected to polyphasic characterization, including 16S rDNA sequence analysis, DNA-DNA hybridizations, biochemical tests, whole-cell protein and fatty-acid analyses. All strains were shown to belong to the Ralstonia branch of the beta-Proteobacteria. Whole-cell protein profiles and DNA-DNA hybridizations revealed two clearly distinct groups, showing low similarity to known Ralstonia species. These two groups, of 8 and 17 isolates, were assigned to two new species, for which the names Ralstonia campinensis sp. nov. and Ralstonia metallidurans sp. nov. are proposed. The type strains are WS2T (= LMG 19282T = CCUG 44526T) and CH34T (= LMG 1195T = DSM 2839T), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain; an emendation of the latter species description is therefore proposed.

Bacterial Proteins↗
PubMed2000

Assessment of electroporation by flow cytometry.

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.

Antibiotics, Antineoplastic↗
PubMed2000

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells.

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.

Adenosine Triphosphate↗
PubMed1999

Protection of mitochondrial respiration activity by bilobalide.

Mitochondria alteration is an early event in ischemia-induced damage, and its prevention improves tissue survival upon reperfusion. Adenine translocase and complex I activities are rapidly affected by ischemia. Ginkgo biloba extract demonstrates anti-ischemic properties attributable to the terpenoid fraction, mainly due to the presence of bilobalide. The mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated. Mitochondria isolated from rats treated with bilobalide (2 to 8 mg/kg) showed a dose-dependent increase in the respiratory control ratio, due to a lower oxygen consumption during state 4. Bilobalide also decreased the sensitivity of oxygen consumption to inhibition of complex I by Amytal or to inhibition of complex III by antimycin A or myxothiazol. There was no protection of complexes IV and V. It also increased the activity of complex I but not of adenine translocase. Similar effects were also obtained in vitro when control mitochondria were preincubated for 1 hr with 0.8 microg/mL bilobalide. Treatment of the rats with 8 mg/kg bilobalide also prevented the ischemia-induced decrease in state 3 of the mitochondrial respiration and thus the decrease in RCR. The protective effect of bilobalide on cellular ATP content observed under ischemic conditions can be correlated with the above observations. By protecting complex I and III activities, bilobalide allows mitochondria to maintain their respiratory activity under ischemic conditions as long as some oxygen is present, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Animals↗
PubMed1999

Increase in circulating endothelial cells in patients with primary chronic venous insufficiency: protective effect of Ginkor Fort in a randomized double-blind, placebo-controlled clinical trial.

One possible mechanism that accounts for the alterations observed in varicose veins is the activation of endothelial cells by ischemia occurring in the leg veins during blood stasis and the cascade of reactions that follows. Because in vitro data suggest that endothelium alteration is a key event in the development of the pathology, it was important to confirm this hypothesis in patients. We used the number of circulating endothelial cells detached from the vascular wall as a criterion of the endothelium injury. We first compared the number of circulating endothelial cells (CECs) in patients with chronic venous insufficiency (CVI) with those of a control population. A twofold increase in the CEC count (1,001+/-127 CEC/ml of plasma compared with 514+/-82 CECs/ml) was observed in CVI patients, which indeed suggests an alteration of the endothelium in this disease. Second, the protective effect of a venotropic drug, Ginkgo biloba extract, troxerutine, and heptaminol (Ginkor Fort), was tested by a randomized double-blind, placebo-controlled clinical trial. In the active-treatment group, the mean values of the CEC count decreased by 14.5% after a 4-week treatment, whereas in the placebo group, the decrease was less (8.4%). The decrease from week 0 to the end of treatment was significantly higher in the active-treatment group than in the placebo group. These results confirm the important role of the endothelium alterations in the development of varicose veins and suggest a potential beneficial action of a venotropic drug on the venous wall.

Adult↗
PubMed1999

Assignment of Centers for Disease Control group IVc-2 to the genus Ralstonia as Ralstonia paucula sp. nov.

An integrated genotypic and phenotypic analysis of 12 Centers for Disease Control (CDC) group IVc-2 strains revealed that this taxon represents a novel species belonging to the genus Ralstonia. Comparative 16S rDNA sequence analysis allocated a representative CDC group IVc-2 strain to the Ralstonia branch of the beta subclass of the Proteobacteria. DNA-DNA hybridizations did not detect significant binding levels towards any presently known Ralstonia species, including Ralstonia pickettii. Its DNA base ratio is between 65 and 67 mol%. The name Ralstonia paucula sp. nov. is proposed, with strain LMG 3244 (= CDC E6793), isolated from a human respiratory tract, as the type strain. R. paucula can be differentiated from other Ralstonia species by whole-cell protein analysis, amplified rDNA restriction analysis and a variety of classical biochemical tests. Strains have been isolated from various human clinical and environmental sources.

Bacterial Proteins↗
PubMed1999

Effect of Ruscus extract and hesperidin methylchalcone on hypoxia-induced activation of endothelial cells.

BACKGROUND: Ruscus aculeatus extract and the flavonoid hesperidin methylchalcone (HMC) are drugs used in the treatment of chronic venous insufficiency. METHODS: In the present study, we investigated their effects on the activation of endothelial cells by hypoxia, a condition which mimics venous blood stasis. RESULTS: We observed that Ruscus extract was able to inhibit the activation of endothelial cells by hypoxia: the decrease in ATP content, the activation of phospholipase A2 as well as the subsequent increase in neutrophil adherence with a maximal protection obtained at 50 microg/ml. HMC was also able to inhibit the hypoxia-induced decrease in ATP content. Furthermore, the effects of Ruscus extract and of HMC on this decrease seem to be additive. CONCLUSIONS: The biochemical mechanism evidenced in this work might explain some of the beneficial therapeutic effects of these products in the treatment of chronic venous insufficiency patients.

Adenosine Triphosphate↗
PubMed1998

Effect of Ginkor Fort on hypoxia-induced neutrophil adherence to human saphenous vein endothelium.

This study was performed to evaluate the effects of Ginkor Fort, a venotropic drug composed of Ginkgo biloba extract, troxerutine, and heptaminol, on neutrophil adherence to the endothelium of saphenous veins. When saphenous veins were incubated 2 h in hypoxic conditions, they showed a five- to sixfold increase in neutrophil adherence to the endothelium. Ginkor Fort at 0.3 mg/ml was able to inhibit this increase by 69%. These results were confirmed by observations in scanning electron microscopy. Ginkor Fort also inhibited the subsequent activation of these neutrophils, as evidenced by the inhibition of superoxide anion release. The biochemical mechanism of this inhibition of neutrophil adherence was studied on endothelial cells in culture. We observed that Ginkor Fort was able to inhibit the different steps of the activation of endothelial cells by hypoxia: the activation of phospholipase A2 and the decrease in adenosine triphosphate (ATP) content. By preventing the first step of the activation cascade, the decrease in ATP content, Ginkor Fort blocks the subsequent increase in neutrophil adherence as well as neutrophil activation. The biochemical mechanism evidenced in this work might explain the beneficial effect of this drug in the treatment of patients with chronic venous insufficiency.

Cell Adhesion↗
PubMed1997

Perfused human saphenous veins for the study of the origin of varicose veins: role of the endothelium and of hypoxia.

If venous stasis due to blood stagnation has been recognized to be involved in the development of varicose veins, the mechanism linking this situation to the modifications of the venous wall observed in varicoses is still unclear. In order to study this mechanism, human saphenous veins were incubated in normoxic or hypoxic conditions and the interactions between the endothelium and neutrophils were investigated. We observed that many neutrophils adhered to the endothelium of veins incubated in hypoxic conditions rather than in normoxia and that these adherent neutrophils were activated: they released high amounts of superoxide anion and of leukotriene B4. Studies in scanning electron microscopy confirmed the increased neutrophil adherence to the endothelium as well as their activation. These results were then related to the histological observation of varicose veins. These veins show a thickening of the media with extracellular matrix deposit as well as an alteration of the elastic lamina with the presence of smooth muscle cells in the intima. These results are in agreement with in vitro studies on isolated endothelial cells. They all show that hypoxia is able to activate endothelial cells: they release inflammatory mediators and become adhesive for neutrophils which are then activated. These activated leukocytes release free radicals and proteases which are able to degrade the extracellular matrix. In addition, hypoxia-activated endothelial cells secrete growth factors which will trigger smooth muscle cell proliferation and the synthesis of extracellular matrix components. Altogether and because they are frequently repeated, these processes could eventually lead to alterations of the venous wall similar to those observed in varicose veins.

Cell Adhesion↗
PubMed1996

Effect of aescine on hypoxia-induced activation of human endothelial cells.

Phlebotonic drugs are very often old drugs which improve symptoms in chronic venous insufficiency but their precise mechanism remains unclear. One reason for this lack of information is our poor understanding of the aetiology of the varicose vein. One hypothesis which is being more and more substantiated is that the origin of the disease lies in the activation of the endothelium during blood stasis, leading to a cascade of reactions which, in the long term, alter the structure of the vein wall. In this work, we tested aescine (Reparil i.v. form), a phlebotonic drug, in an in vitro model which mimics this situation, i.e. human endothelial cells exposed to hypoxic conditions. Aescine was shown to inhibit 2 important steps of the activation of endothelial cells incubated 120 min under hypoxia the decrease in ATP content, which is the starting point of the activation cascade, and the increase in the activity of phospholipase A2, an enzyme responsible for the release of precursors of inflammatory mediators. Hypoxia-activated endothelial cells also increase their adhesiveness for neutrophils. This process could also be prevented in a dose-dependent manner if endothelial cells were incubated in the presence of aescine. This inhibition was confirmed by morphological observations in scanning electron microscopy. All 3 effects were already evidenced at 100 ng/ml and were maximal at 750 ng/ml. These effects obtained at very low concentrations probably represent one of the main molecular and cellular mechanisms that underlie, among others, protection of the vessel wall. Objective criteria for our understanding of the preventive action of this phlebotonic drug are, thus, provided.

Adenosine Triphosphate↗
PubMed1996

Effects of hydroxyethylrutosides on hypoxia-induced activation of human endothelial cells in vitro.

1. A clinically available mixture of hydroxyethylrutosides (HR) was examined as inhibitors of endothelial cell activation by hypoxia in vitro. Thus, the effects of HR on ATP depletion, phospholipase A2 activation and neutrophil adherence were investigated in hypoxia-activated human umbilical vein endothelial cells in primary cell culture. 2. Our results show that HR inhibited two important steps of the activation of endothelial cells by hypoxia: the decrease in ATP content, which is the starting point of the process, and the activation of phospholipase A2 one enzyme responsible for the release of inflammatory mediators. This inhibition was dose-dependent with 70 to 90% inhibition at 500 micrograms ml-1 of HR. 3. In addition, hypoxia-activated endothelial cells increased their adhesiveness for neutrophils. This process could also be prevented in a dose-dependent manner if endothelial cells were incubated in the presence of HR. This inhibition was confirmed by a morphological study. 4. In conclusion, the results of this study suggest that a possible explanation for the improvement in venous insufficiency by HR observed clinically could be their ability to inhibit the activation of endothelial cells during blood stasis.

Adenosine Triphosphate↗
PubMed1996

Interactions between endothelial cells and smooth muscle cells after their activation by hypoxia. A possible etiology for venous disease.

Because of their localization at the interface between blood and tissue, endothelial cells are responsible for the maintenance of vascular homeostasis. They fulfil a series of various functions and constantly interact with circulating leukocytes and with the smooth muscle cells (SMC) present in the media. Any disturbance of their metabolism can thus lead to alterations of the blood vessel functions. We have shown that hypoxia, for example resulting from venous stasis, induces the activation of endothelial cells which then release inflammatory mediators able to activate neutrophils and to induce their infiltration as well as growth factors for SMC. We propose that these processes are the beginning of a cascade of events eventually leading to structural and functional modifications of the venous wall similar to the ones observed in varicose vein wall. The endothelium alterations resulting from venous stasis would thus be the origin of the development of the venous disease. Pharmacological and clinical evidence reinforce this hypothesis.

Cell Hypoxia↗
PubMed1995

Protection of hypoxia-induced ATP decrease in endothelial cells by ginkgo biloba extract and bilobalide.

Due to their localization at the interface between blood and tissue, endothelial cells are the first target of any change occurring within the blood, and alterations of their functions can seriously impair organs. During hypoxia, which mimics in vivo ischemia, a cascade of events occurs in the endothelial cells, starting with a decrease in ATP content and leading to their activation and release of inflammatory mediators. EGb 761 and one of its constituents, bilobalide, were shown to inhibit the hypoxia-induced decrease in ATP content in endothelial cells in vitro. Under these conditions, glycolysis was activated, as evidenced by increased glucose transport, as well as increased lactate production. Bilobalide was found to increase glucose transport under normoxic but not hypoxic conditions. In addition, EGb and bilobalide prevented the increase in total lactate production observed after 60 min of hypoxia. However, after 120 min of hypoxia, the total lactate production was similar under normoxic and hypoxic conditions, and both compounds increased this production. These results indicate that glycolysis slowed down between the 60th and 120th minute of hypoxia, while EGb and bilobalide delayed the onset of glycolysis activation. In another experimental model, both compounds were shown to increase the respiratory control ratio of mitochondria isolated from liver of rats treated orally. Since ischemia is known to uncouple mitochondria, the protection of ATP content and the delay in glycolysis activation observed during hypoxia in the presence of EGb 761 or bilobalide is best explained by a protection of mitochondrial respiratory activity, at least during the first 60 min of hypoxia incubation. Both products retain the ability to form ATP, thereby reducing the cell's need to induce glycolysis, probably by preserving ATP regeneration by mitochondria as long as oxygen is available.

Adenosine Triphosphate↗
PubMed1995

Hypoxia induces PMN adherence to umbilical vein endothelium.

OBJECTIVE: In vitro incubation of cultured endothelial cells under hypoxia leads to the activation of these cells and results in an increase of their adhesiveness for neutrophils (PMN). Because of the possible relevance of these observations for pathological situations, we investigated whether adherence of PMN also occurs in an entire vein after its incubation in hypoxic conditions. METHODS: Human umbilical veins in complete cords were incubated for 2 h in normoxic or hypoxic conditions and the adherence of unstimulated human 51Cr-labelled-PMN was measured under flow conditions. Experiments with human umbilical vein endothelial cells (HUVEC) were performed in parallel for comparison. Morphological studies in scanning electron microscopy were carried out in both in vitro and ex vivo situations. RESULTS: Hypoxia induced an increase in the adherence of PMN either to HUVEC or to the umbilical vein endothelium up to 5- to 6-fold when compared to normoxic conditions (P < 0.001). In both cases, this hypoxia-induced adherence was inhibited by anti-ICAM-1 antibodies or when the PAF (platelet-activating factor) synthesis was blocked during hypoxia by oleic acid. Furthermore, the adherence of PMN was inhibited when PMN were pre-incubated with WEB 2086 (a selective PAF receptor antagonist). These results indicate a crucial role of PAF in this process. Morphological studies confirmed that the number of PMN adherent to hypoxic HUVEC or to the hypoxic umbilical vein endothelium was much greater than the number of PMN on normoxic endothelial cells. Both in vitro and ex vivo, PMN adherent to the hypoxic endothelial cells to the contrary of the ones adherent to normoxic endothelial cells demonstrated membrane foldings typical of an activated state. CONCLUSION: These results show that in a complete vein, hypoxia induced an increased adhesiveness of endothelial cells for PMN by a similar mechanism to the one observed for cultured endothelial cells. They suggest an active role of endothelial cells in the initiation of the inflammatory response often described in ischemic-reperfused organs.

Antibodies, Monoclonal↗
PubMed1993

Effects of naftidrofuryl on hypoxia-induced activation and mortality of human endothelial cells.

The present study was designed to elucidate the possible beneficial effects of naftidrofuryl on ischemia-induced endothelium damage. For this purpose, an in vitro model was developed wherein human endothelial cells isolated from umbilical vein were submitted to hypoxia. Long-term hypoxia incubation (6 h) induced cell mortality, and naftidrofuryl strongly protected endothelial cells against this mortality in a dose-dependent manner and at concentrations as low as 10(-9) M. 66% protection was still observed after 16 h of hypoxia. Naftidrofuryl had to be present during the hypoxia incubation to exert its action; preincubation up to 24 h in the presence of naftidrofuryl could not protect endothelial cells incubated under hypoxia without naftidrofuryl. Short-term hypoxia, which does not induce mortality, strongly activates the endothelial cells with an increase in the cytosolic calcium concentration, in the phospholipase A2 activity, and in the synthesis of prostaglandin and of platelet-activating factor. It also enhances the adherence of polymorphonuclear neutrophils. Naftidrofuryl was able to markedly inhibit this whole cascade of events in a dose-dependent manner. We also demonstrated that naftidrofuryl could block the decrease in ATP concentration that results from the hypoxic conditions. These results indicate that by preserving the energetic level of the cells, naftidrofuryl prevents the activation of endothelial cells and the cell mortality induced by hypoxia. By maintaining an intact endothelium in vivo during ischemia, naftidrofuryl could prevent the further damage induced by leukocyte recruitment and activation.

Adenosine Triphosphate↗