Marc De Brabander

DNA Synthesis and Mitoses in Coronary Collateral Vessels of the Dog

DNA synthesis and mitotic activity in coronary collateral arterioles was assessed in dogs at different time intervals after gradual ameroid constriction of the left circumflex coronary artery. Labeling of nuclei and the mitotic index were highest at 3 weeks after implantation of the constrictor and gradually declined thereafter. Labeling persisted for at least 8 weeks but radioactive DNA was not found 12 months after coronary artery constriction nor in controls or animals with sham operations. Proliferative activity was, at any time interval, highest at the level of the smallest diameters of the collateral vessels. Labeled nuclei and mitoses were found in endothelial, medial, and adventitial cells. Myocardial mesenchymal cells also incorporated tritiated thymidine. The data provide evidence that after constriction of a major coronary artery, the coronary collateral vessels enlarge by an active growth process that follows the basic laws of cell kinetics.

Virological and immunological analysis of a triple combination pilot study with loviride, lamivudine and zidovudine in HIV-1-infected patients

Objective:To compare two antiretroviral regimens, loviride plus lamivudine (3TC) plus zidovudine (ZDV) (triple combination) and loviride plus ZDV (double combination) in terms of pharmacokinetic interactions, tolerability, safety, and immunological and virological efficacy. Study design:An open, case-controlled, pharmacokinetic and 24-week continuous treatment pilot study. Patients:Twenty p24 antigen-positive patients, 10 per treatment group, were matched according to p24 antigenaemia less or more than l00 pg, CD4 count less or more than 150×106/l, and gender. Eight out of 10 cases and seven out of 10 controls had received previous antiretroviral therapy. Results:No clinically relevant pharmacokinetic interactions were observed. Both treatment combinations were well tolerated. Median absolute and percentage CD4 count increases above baseline were more pronounced in the triple combination arm than in the double combination arm. Median p24-antigen and plasma viraemia level decreases below baseline were more pronounced in the triple combination arm. The M184I/V mutation was detected in all plasma samples of triple combination patients examined at week 12. Mutations conferring resistance to loviride and ZDV were found in a significant subset of patients in both treatment arms. Conclusions:Both combination regimens have an excellent safety/tolerability profile, but a higher level of in vivo efficacy is achieved by the triple combination, despite genotypic changes conferring resistance to one or all of these agents. The conclusions drawn are limited by small population size and the heterogenous pretreatment history. However, they support the validity of and strongly encourage a rationally designed multidrug combination approach to HIV therapy.

The mechanism of action of the new antimycotic ketoconazole

Ketoconazole is one of the new members of the imidazole series with a broad-spectrum antifungal profile. Although sharing its basic active principles with the other imidazoles, ketoconazole obtains its superior in vivo activity mainly from its good oral absorption and its lower degree of inactivation once absorbed. Its selective toxicity for yeasts and fungi is found to be primarily linked to the inhibition of ergosterol biosynthesis and to interference with other membrane lipids. In vitro growth studies revealed that ketoconazoles activity was more pronounced against the invasive morphogenetic form than against the saprophytic form of Candida albicans, which at least partly explains its prominent in vivo potency. At extremely low concentrations (10 ng/ml-1) ketoconazole prevents the development of the very form that is responsible for the expression of clinical symptoms. In contrast to other imidazoles, ketoconazoles action on the morphogenesis of the organism is not influenced by serum. The synergistic action with host defense cells, as demonstrated in culture systems, is another inherent property of this drug and may have a great impact on the eradication of systemic fungal infections. These effects of ketoconazole have been studied in a variety of fungal organisms with the aid of phase-contrast, scanning, and transmission electron microscopy in order to characterize ketoconazoles profile in comparison to the other imidazole derivatives.

Direct evidence for the contractile capacity of endothelial cells.

Abstract Dog aorta-derived endothelial cells, grown in culture and resuspended in normal human or canine citrated platelet-free plasma, invariably retracted the fibrin clot when stimulated with exogenously added or endogenously formed thrombin. The onset, rate and extent of this retraction depended upon the cell density, the concentration of thrombin, the cellular integrity, the homogeneous cell distribution and the availability of free extracellular Ca 2+ . Endothelial cell-induced clot retraction was completely inhibited by EGTA, by VK 774-induced phosphodiesterase inhibition and by combined treatment with PGE 1 and papaverine. It was partly reduced by nocodazole-induced microtubule disruption, by cytochalasin B, by verapamil- and flunarizine-induced blockade of Ca 2+ -fluxes but was not affected by cyclo-oxygenase inhibitor suprofen. Incorporation of additional Ca 2+ into non-retracting, Reptilase®-formed clots resulted in a retraction which was not blocked by heparin. In addition of 5-hydroxytryptamine, ADP, histamine or epinephrine slightly enhanced this retraction.

The Activity of Ketoconazole in Mixed Cultures of Fungi and Human Fibroblasts: Die Wirkung von Ketoconazol in Mischkulturen von Pilzen und menschlichen Fibroblasten

Summary: A system is described consisting of mixed cultures of human fibroblasts and fungi that can be used on a large scale for the semiquantitative determination of the fungistatic and cytotoxic activity of chemotherapeutic compounds.

Levamisole plus 5-fluorouracil inhibits the growth of human colorectal xenografts in nude mice

Fragments of human colorectal adenocarcinomas were inserted under the renal capsule of nude mice. The growth of these tumour grafts was significantly inhibited by the combination of 5-fluorouracil (5-FU) and levamisole. An alternating regimen of levamisole 2.5 mg/kg and 5-FU 20 mg/kg decreased the size of tumour implants by 33-59% and/or increased the number of macroscopically disappeared fragments in the combined group compared with ineffective monotherapy with saline, levamisole or 5-FU. This model could be valuable for investigating the mechanism of action of levamisole and to evaluate the effects of this adjuvant therapy in other oncological settings.

Serum and serotonin induce retraction of calf aortic smooth muscle (CASM) cells in vitro: Inhibition by ketanserin, a 5-HT2 receptor antagonist☆

Calf aortic smooth muscle (CASM) cells cultured in vitro at high cell density (4 x 10(4) cells/cm2) on bacteriological petri dishes in the presence of serum pile up in clusters and create open spaces in the monolayer. This phenomenon is clearly visible 6 days after plating and is markedly enhanced by the addition of fetal calf serum. Serotonin is essential for the serum-induced retraction since (1) dialyzed serum has no effect, (2) of all the vasoactive agents we tested, only serotonin induced a similar degree of retraction, and (3) the serum-induced retraction was completely blocked by preincubating the cells with serotonin 5-HT2 receptor blockers such as ketanserin and ritanserin but not by preincubation with adrenergic-alpha 1 blockers or histamine antagonists. Serotonin caused CASM cell retraction in a dose-dependent way, with a maximum effect at 10(-6) M. The serotonin-induced retraction was reversible in time and was effectively blocked by ketanserin (IC50 = 1.2 x 10(-9) M). It is therefore concluded that serotonin induces retraction of CASM cells, mediated by the serotonin 5-HT2 receptor.

Interaction between the microtubule inhibitor tubulozole and gamma-irradiation in murine tumors in vivo.

The combined effect of the microtubule inhibitor tubulozole and gamma-irradiation has been investigated in vivo in subcutaneous MO4 fibrosarcomas and Lewis Lung carcinomas. A marked interactive effect on tumor growth was observed when 160 mg/kg tubulozole was orally administered before the tumors were treated with 10 Gy radiation. Dose dependency and optimal effect were obtained on tumor growth of MO4 tumor bearing animals when the drug treatment was given 6 hr prior to the irradiation. The optimal pretreatment time coincided with the time at which a peak mitotic index in the tumor tissue was observed. An enhancing effect is also noticed at other doses of radiation in MO4 tumors pretreated 6 hr before with 160 mg/kg tubulozole. The interactive effect is maintained in a clinically relevant dose fractionation schedule whereby 8 fractions of 2 Gy each were pretreated 6 hr before with 80 mg/kg tubulozole. Tubulozole-T, the stereo-isomer of tubulozole, neither exhibits any antimicrotubular action nor exerts an antitumoral effect on its own or in combination with gamma-irradiation. The possible mechanisms of interaction between tubulozole and gamma-irradiation in tumor tissue are discussed.

Microtubule‐Dependent Intracellular Motility Investigated with Nanometer Particle Video Ultramicroscopy (Nanovid Ultramicroscopy)

The movement of intracellular organelles in vertebrate tissue-cultured cells, resulting in dynamic subcellular organization and polarity by seemingly guided and purposeful transport and mutual encounters, has been the subject of relatively few thorough studies. The motility of most optically detectable organelles is saltatory. It is fundamentally different from Brownian motion and displays both similarities and essential differences with other types of intracellular organelle transport such as axonal transport, pigment granule movement, and cytoplasmic streaming in plant cells. Saltations are dependent on the presence of, and occur along, microtubules. The molecular motor is unknown and may be located in the organelle membrane, or in the microtubule wall, or in both. We developed a new approach of general applicability to probe the role of various proteins in subcellular transport. Colloidal gold particles of 20 or 40 nm diameter microinjected into living cells are invisible to the eye in the light microscope. Individual particles, can, however, easily be discerned using transmitted light a t high numerical aperture and video contrast enhancement (FIGURE 1). They appear as black dots on a white background. In epipolarization microscopy they appear as brightly staining dots on a background consisting of the interference reflection picture of the cells (FIGURE 1). Ultrastructural observation of the same cells prepared as whole mounts or in thin sections, confirmed that individual 20 nm particles could be resolved in the light microscope. The injected particles were not within membrane-lined vesicles, but free in the cytoplasmic matrix often in the vicinity of microtubules. Uncoated particles stabilized with polyethylene glycol, or particles coated with albumin were transported along microtubules in a saltatory, ATP-dependent fashion a t the same velocity and with the same frequency as endogenous organelles (FIGURE 1). Because subunit flux within microtubules is a possible mechanism, we injected gold particles coupled to a monoclonal antitubulin antibody.’ Many of these particles assumed fixed positions for several hours, often forming linear arrays. The observations show that gold particles having the same size as the smallest cellular membrane-lined organelles are transported by a microtubule-associated mechanism, as has been described for larger (250-500 nm) polystyrene and other beads microinjected in axons’ or cultured cells.’ Microtubule treadmilling does not appear to be involved. The possibility of following 2 0 4 0 nm particles, and probably even smaller ones that can be coupled to many proteins within living cells, provides a tool of wide

Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells

Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cells long axis. The effect of a beta-agonist and of a calcium antagonist is described.