Molecular identification of foreign inclusions in inflammatory tissue surrounding metal implants by Fourier transform laser microprobe mass spectrometry.

Fourier transform laser microprobe mass spectrometry (FT LMMS) is a novel technique for micro-analysis of solids with a lateral resolution in the 5 microns range. One of the major advantages of the technique is the capability to perform characterisation of the molecular composition of both organic and inorganic compounds. The information is directly deduced from the signals without the aid of reference spectra. FT LMMS was applied to the characterisation of black tissue fragments in a biopsy from a patient, in which a constrained condylar nodular knee system was implanted ten years ago. The tissue contained numerous foreign giant cells with a black non-birefringent pigment in their cytoplasm. FT LMMS analysis allowed us to detect directly by means of molecular signals, that the debris consisted primarily of titanium oxide and not metallic titanium, while the implant itself only contained titanium.

Laser microprobe mass spectrometry: principle and applications in biology and medicine.

Laser microprobe mass spectrometry (LMMS) is an interesting technique for micro- and surface analysis. It employs local ionization by a focused laser under high power density conditions and subsequent mass analysis of the generated ions. This paper surveys the main LMMS instruments and their operational principles. Sample preparation is discussed in the context of biological materials. The problem of quantification is addressed. Selected examples show the way that precise information on the molecular composition can be deduced from the detected signals. Both inorganic and organic substances can be identified, even without reference spectra, from in-situ analysis with a lateral resolution in the order of 1 to 5 micrograms.

Lidocaine does not prevent the calcium paradox in rat hearts: a laser microprobe mass analysis (LAMMA) study.

The calcium paradox stands for the cell damage that occurs when isolated hearts are perfused with a Ca(2+)-free solution followed by perfusion with a Ca(2+)-containing solution. Although it is generally accepted that a massive Ca2+ influx during the Ca(2+)-repletion phase is responsible for the cell damage, there is no consensus about what makes the heart susceptible to the calcium paradox during the Ca(2+)-depletion phase. It has been suggested that the extent of the calcium paradox is primarily determined by accumulation of Na+ during Ca2+ depletion and a subsequent accumulation of Ca2+ via reverse Na(+)-Ca2+ exchange during Ca2+ repletion. According to another theory, weakening of intercalated disc junctions during Ca2+ depletion and contracture-mediated disruption of the cell membrane during Ca2+ repletion are responsible for the calcium paradox. In the present study we further investigated the possible role of Na+ in the development of the calcium paradox. During Ca2+ depletion, lidocaine was used to inhibit Na+ entry through the Na+ channels. Isolated rat hearts were perfused with Krebs Henseleit buffer (KH) containing 1.4 mM Ca2+ for 15 min, followed by 10 min of Ca(2+)-free perfusion and 10 min of reperfusion with Ca2+. In the treated group 0.1 mM lidocaine was present throughout the experiment. At the end of each experiment, Ca2+ cytochemistry was performed and the intracellular Ca2+ content was analyzed by laser microprobe mass analysis (LAMMA). The results show that during Ca2+ depletion, the intracellular Ca2+ content did not change significantly. Ca2+ repletion, however, gave rise to a full calcium paradox irrespective of the presence of lidocaine: massive cell damage and Ca2+ accumulation in the mitochondria. The results provide further evidence that intracellular Na+ accumulation during Ca2+ depletion is not involved in the occurrence of the calcium paradox.

Distribution of calcium in a subset of chronic hibernating myocardium in man.

The structural correlates of 'chronic hibernating myocardium' in man consist of myocardial cells which transformed from a functional state (rich in contractile material) to a surviving state (poor in contractile material, rich in glycogen). Since the calcium-handling organelles such as SR, sarcolemma and mitochondria underwent structural changes in cells so affected, the distribution of calcium was investigated in biopsies obtained from 'hibernating' areas. The material was processed for microscopic localization of total calcium (laser microprobe mass analysis, LAMMA) and of exchangeable calcium (phosphate-pyroantimonate precipitation method, PPA). Subcellular distribution of total calcium as assessed by LAMMA revealed that in the structurally affected cells the areas in which sarcomeres were replaced by glycogen contained significantly more calcium than all other areas probed such as mitochondria, remaining sarcomeres at the cell periphery and subcellular areas of normally structured cells. Calcium precipitate, obtained after PPA assessment, was localized at the sarcolemma but was virtually absent in the mitochondria of affected cells. The high calcium content in the myolytic areas of affected cells most probably belongs to a pool of bound calcium. The observations that calcium is retained at the sarcolemma and that mitochondria are devoid of precipitate favour the hypothesis that cells structurally affected as such are not ischaemic and are still able to regulate their calcium homeostasis.

The prevention of Ca2+ overload in vascular smooth muscle by flunarizine as detected by Laser Microprobe Mass Analysis (LAMMA).

In vitro exposure of depolarized caudal artery preparations of the rat to a high calcium concentration resulted in a strong contraction of the smooth muscle cells. This muscle contraction was suppressed by flunarizine. It was shown cytochemically, using the combined oxalate-pyroantimonate method for the localization of calcium, that a considerable amount of electron-dense precipitate was seen over the depolarized muscle cells after incubation in a calcium containing medium. On the other hand this precipitate was not present on the smooth muscle cells when flunarizine was added to this incubation medium. The reaction product was only present in the extracellular space. These results were controlled by Laser Microprobe Mass Analysis. By evaporating and ionizing small parts of the smooth muscle cells (+/- 1 micron), it was confirmed that the cytochemical method indeed demonstrated calcium, with negligible interference of other cations.

Identification of calcium in the retina by the combined use of ultrastructural cytochemistry and laser microprobe mass analysis.

The specificity of the combined oxalate-pyroantimonate (OPA) technique for subcellular localization of calcium was examined by laser microprobe mass analysis (LAMMA) of the same tissue sections. The recorded spectra strongly suggest that the cytochemically detected precipitates contained calcium. This could be confirmed by LAMMA analysis of ethylene glycol tetraacetic acid-treated sections. The detected calcium was localized mainly in the rod outer segments, more particularly in the middle part. The validity of the OPA and LAMMA methods is discussed. A combination of both techniques is found to be a valuable tool to elucidate the role of calcium in physiological mechanisms.

Influence of econazole and 5-fluorocytosine on the ultrastructure of Aspergillus fumigatus and the cytochemical localization of calcium ions as measured by laser microprobe mass analysis.

The ultrastructural changes and the localization of Ca++ ions have been investigated in Aspergillus fumigatus after exposure in vitro to econazole (50 micrograms ml-1) and to 5-fluorocytosine (100 micrograms ml-1) for 24 h. The changes obtained with econazole concerned the cell periphery, necrotization of mitochondria and hyperactivity of the central vacuole. After cytochemical Ca++ localization a marked increase in precipitate was observed on mitochondria, vacuoles and collapsed membranes, compared to the control. Laser microprobe mass analysis confirmed that the measured amount of Ca++ ions corresponded to the degree of precipitate formation in the different cellular compartments. After exposure to 5-fluorocytosine, abnormal behaviour of the nuclei and internal lipidification of the mitochondria and of the cytoplasm were seen. No discernible Ca++ activity was present on the cellular structures by cytochemical localization. Assay by laser microprobe mass analysis, however, showed a slight increase in Ca++ which points to a structural bonding of the Ca++.

Protection of rats from compound 48/80-induced lethality. A simple test for inhibitors of mast cell-mediated shock.

The dose of 0.5 mg/kg i.v. of compound 48/80 was lethal in 97.2% of the injected rats. Observations before death and at autopsy were in accordance with the basic effect of compound 48/80 in rats i.e. the sustained release of mast cell mediators, whose action on the cardiovascular system leads to circulatory collapse. The administration of drugs with various pharmacological effects before the intravenous challenge with compound 48/80 allowed us to conclude that the following effects are not sufficient to prevent the lethal shock: inhibition of prostaglandin biosynthesis; H2-histamine antagonism; cholinergic, alpha- or beta-adrenergic blockade; beta-adrenergic stimulation; CNS-effects of antidepressants, hypnotics, sedatives, neuroleptics or narcotic analgesics; ganglion blockade; glucocorticoid or cromoglycate-like activity. Dose-dependent protection from the lethal reaction was obtained with compounds known to exert a single or several actions of the following types: oxatomide-like inhibition of mast cell mediator release; h1-histamine antagonism; serotonin antagonism. Quantitatively, however, when measured in in vitro systems these effects are poorly related to the protection from lethal compound 48/80 challenge. The new test offers the advantage of a simple, comprehensive measure of the potency of a compound to prevent mast cell-mediated shock.

[Toxocariasis in calves. Recent diagnosis and treatment (author's transl)]. / Toxocarose bij kalveren. Herontdekking en behandeling.

Toxocara vitulorum, a parasite of suckling calves, was again diagnosed in Belgium in imported cattle of French origin. The infectious larvae are present in the milk and colostrum; they do not undergo further migration in the tissues of calves. This form of transmammary migration is not specific of T. vitulorum, but was also reported in S. ransomi in pigs. Treatment with levamisole at a dosage of 5 mg/kg results in complete disappearance of the worms. In addition to the cycles, the elements of a specific parasitological diagnosis are also described.

Cytochemical localization of NADH oxidase in Candida albicans.

The application of a recently published technique to localize reduced nicotinamide adenine dinucleotide oxidase activity is described in glutaraldehyde-fixed Candida albicans. The reaction product appears as a finely granular precipitate on the mitochondrial cristae and on the central vacuolar membrane, and, if present, on the vacuolar contents. Fixation should be kept to a minimum and prolonged incubation times up to 2 hr are necessary to show these reactive sites. The reaction appears to be strongly substrate-dependent and not affected by cyanide. Exposure of C. albicans cells to the antimycotic miconazole resulted in a strong increase in reduced nicotinamide, adenine dinucleotide and oxidase activity. The hypothesis is put forward that this enzyme, together with peroxidative and catalatic enzymes, may be implicated in the mechanism by which miconazole exerts its lethal effect on C. albicans.

Cytochemical and biochemical studies of yeasts after in vitro exposure to miconazole.

Yeast cells exposed to different doses of the antimycotic agent miconazole revealed important cytochemical changes in the topographic distribution of the phosphatases. A strong effect was observed on the behavior of oxidative and peroxidative enzymes. Decreased cytochrome c oxidase and peroxidase activity and increased catalase activity were seen after treatment with a fungistatic dose of miconazole, whereas a complete disappearance of these enzymes was observed after treatment with a minimal fungicidal dose of miconazole. This was in complete agreement with the quantitative biochemical data. A hypothesis is advanced concerning the possible involvement of peroxidase and catalase in the mechanism of action of this drug.

The effects of miconazole on the ultrastructure of Candida albicans.

Ultrastructural changes in Candida albicans induced by increasing concentrations of miconazole in vitro are described. Fungistatic concentrations (10(-8) to 10(-7) M) induced minimal morphologic changes at the cell periphery. At 10(-6) M cell volume increased and peroxisomes became numerous in the cell interior. The minimal fungicidal dose of 10(-5) M caused severe damage to most of the cell population and a total fungicidal dose of 10(-4) M caused total internal cellular necrosis even when cell walls remained intact. It is suggested that miconazole inhibits the peroxidative enzymes cytochrome c-peroxidase and catalase. Cell necrosis then results from peroxide accumulation.

Morphological changes in cysticerci of Taenia taeniaeformis after mebendazole treatment.

The progressive micromorphological changes in Taenia taeniaeformis cysticerci, induced by a single parenteral treatment of the infected mice with mebendazole, are described. The time-related alterations concerned the tegument and tegumental cells and were successively: disappearance of microtubules, accumulation of secretory substances in the Golgi areas, decrease in number to complete loss of microtriches, "ballooning" of all tegumental cells with subsequent burst, vacuolization and degeneration of the tegument, and finally necrosis of the pseudoproglottids. Similar but less pronounced injuries were seen in the scolices, although microtubules disappeared as early as in the pseudoproglottids. Microtubules from the host tissues remained intact. The meaning of the apparent primary interference of mebendazole with the microtubular system in relation to the subsequently observed death of the cysticercoids is discussed.

Enzyme cytochemistry of Candida albicans.

The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed.

Influence of the anthelmintic mebendazole on microtubules and intracellular organelle movement in nematode intestinal cells.

The ultrastructural changes in the intestines of Ascaris suum and Syngamus trachea, induced by in vivo treatment of the hosts with the anthelmintic mebendazole, are reported. The primary site of drug action seemed to be the organelles involved in the secretory mechanism of the intestinal cells. The block in the transport of secretory granules and in the movement of other subcellular organelles coincided clearly with the disappearance of cytoplasmic microtubules. On the other hand, the microtubular system of the host cells was unaffected by the treatment. Degenerative changes in the intestinal cells of the parasites observed afterwards were correlated with the primary deteriorative effect of the drug on cytoplasmic microtubules.

The development of alkaline phosphatase in trichinous muscle.

The development of alkaline phosphatase during invasion and encystment of Trichinella spiralis in rat skeletal muscle fibres was studied at the ultrastructural level. On day 14 after infection, the enzymatic activity is found in proliferating parts of the T-tubular system and in parts of the plasmalemma. In cells, in which a strong hyperplasia of this system is noted. AlPase is present in the abundant network of stratified and concentric membranes from which a large number of pinocytic vesicles arise. From day 50 till 1 year after infection the enzyme activity was invariably present in the matrix surrounding the larvae and was confined to the enormous amounts of cytoplasmic membranes. The possible functional significance of this enzyme in the matrix, in view of its peculiar localization in the immediate vicinity of the parasite, is discussed. In the presence of 0.1 mM of the levamisole analogue, compound R 30402, which is a stereospecific inhibitor of AlPase, the activity is completely lost.

Scanning electron microscopy of Candida albicans after in vitro treatment with miconazole.

A study was made on the in vitro action of the antimycotic miconazole on Candida albicans yeast cells with scanning electron microscopy, and the effects were compared with those seen on the yeast cells by means of transmission electron microscopy. It was found that cells exposed to fungistatic and minimal fungicidal doses of miconazole (10(-7) M and 10(-6) M) presented rough surfaces and had multiple, desoriented buds and bud scars. Whereas in control cultures the cells were well separated, the treated ones formed small clusters of interconnected cells. After exposure to a fungicidal concentration (10(-4) M) of the drug, most of the remaining cells showed smooth surfaces and were covered with numerous vesicular structures probably representing cytoplasmic remnants derived from broken cells. This has been substantiated by the presence of abundant fragments of cell walls and confirmed by examination of similarly treated cultures in the transmission electron microscope. Moreover, the cells with an apparently intact surface when examined with scanning electron microscopy were shown with transmission electron microscopical examination to be completely necrotic inside.