Ofloxacin alters expression of integrins on chondrocytes from mouse fetuses in vitro.
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Animals↗
Biomedical subjects
H J Merker
Publications and source records attributed to H J Merker.
Synovial and peritoneal macrophages in organoid culture.
Cultivation of macrophages and their progenitors has been very useful for elucidation of function, behaviour and morphology of these cells. The purpose of this contribution is to describe a new in vitro system (organoid, high density or micromass culture) which proved to be convenient for cultivation of macrophages derived from human synovial fluid and tissue and mouse peritoneal fluid. Using this method, highly differentiated and functionally active macrophages of marked purity and long maintenance (up to 2 weeks) could be obtained even after previous cultivation and subcultivation in monolayer culture. The macrophages were identified by electron microscopy and immunomorphology using HLA-DR-DP, CD-68 (markers for human macrophages), anti-human-polymorphonuclear leukocyte-gelatinase and F4/80 (a mouse macrophage surface marker). The significance of this method as a research tool in the study of cartilage degradation by macrophages in co-cultures is stressed.
Animals↗
The thyroid gland of Callithrix jacchus in organ culture.
Thyroid glands from 7 marmosets (Callithrix jacchus) of different age groups (newborn, 2 weeks, 2, 7, 8 and 11 months, and 8 years) were grown as organ culture according to Trowell at the medium/air interphase for 3 to 30 days. The morphology of thyroid tissue was well preserved until the end of the culture period. Necroses were only occasionally seen in connective tissue. In contrast to the in vivo situation, the number of lysosome-like inclusions changed considerably and cell-cell contacts became loose. After the addition of TSH the number of apical microvilli, apical granules and coated vesicles rose. The basal surface of follicle cells enlarged due to the formation of processes or ridges and invaginations. Mitosis could be observed. These findings show that, using the Trowell culture technique, thyroid tissue can be grown for up to 4 weeks without showing distinct changes in its morphology. Its responsiveness to the application of TSH is maintained in vitro. Hence, this technique is suitable also for long-term endocrinological, pharmacological and toxicological investigations.
Aging↗
Morphology of the basement membrane.
The aim of this contribution is to summarize our knowledge of the morphology of the basement membrane (BM). The first step in this direction is the attempt to define this term. The BM is composed of the Lamina lucida, densa, and fibroreticularis. Subsequently, the historical development of this term is discussed. Our main interest is, of course, focused on the description of the BM-structure up to the macromolecular level and the special forms of this structure. This is supplemented by discussing its chemical composition and establishing a relationship between morphology and biochemistry. The obtained findings yielded some indications as to the molecular composition of the BM which may serve for the construction of "models." The composition of the Lamina lucida (L.l.) and the Lamina or Pars fibroreticularis (L.f.) must be discussed separately, since, if present, they show a different and strongly varying structure (L.f.). An important aspect is the function of this extracellular layer which comprises mechanical tasks up to inductive effects. Finally, the concepts of the formation of the BM, especially of the Lamina densa (L.d.), are summarized. It obviously consists of a sequence of individual steps which starts with expression and secretion of the L.d.-components and is followed by an induction of integrin expression.
Animals↗
Embryotoxic effects of thalidomide derivatives in the non-human primate callithrix jacchus. IV. Teratogenicity of micrograms/kg doses of the EM12 enantiomers.
The dose-response of the teratogenic potency of the thalidomide (Thd) derivative EM12 was evaluated in the common marmoset (Callithrix jacchus). The smallest daily dose found to be effective was 30 micrograms EM12/kg body wt. This is the lowest dose of a Thd derivative ever reported to induce severe skeletal abnormalities. Ten micrograms EM12/kg body wt may be considered the no-observed-adverse-effect-level (NOAEL) under the experimental conditions chosen. The teratogenic potencies of the two EM12 enantiomers were tested at 100 micrograms/kg body wt, the dose which just induces an almost 100% effect in the case of the racemate. The S(-)-EM12 was found to induce typical severe limb abnormalities such as amelia, phocomelia, and radius aplasia, and none of the exposed fetuses were devoid of skeletal defects. In contrast, only few and minor skeletal defects were observed after application of the R(+) enantiomer. Although a pronounced teratogenic potency of the R(+)-EM12 can now largely be excluded, these low-dose studies are not sufficient to completely rule out any teratogenic potential of this enantiomer, since racemisation to small amounts of the S(-) form may occur in vivo. Further studies with Thd derivatives which are unable to racemise are necessary to prove the assumed complete ineffectiveness of the R(+) enantiomers.
Abnormalities, Drug-Induced↗
Toxicity of beta-blockers in a rat whole embryo culture: concentration-response relationships and tissue concentrations.
Beta-adrenoceptor blockers are widely used drugs for the treatment of cardiovascular diseases. Since beta-blockers cross the placenta, it is essential to consider possible adverse effects on the embryo. Six beta-adrenoceptor blockers were tested at various concentrations (10-5000 microM) in a rat whole embryo culture. Although inducing a very similar pattern of dysmorphogenetic effects (incomplete flexure, disturbed development of the neural tube, the head, the heart and the tail bud), the compounds exhibited a wide range of embryotoxic potency. Estimation of the EC50 (median-concentration producing dysmorphogenesis in 50% of the embryos) for the six compounds revealed differences of more than two orders of magnitude: propranolol 25 microM, alprenolol 30 microM, metoprolol 100 microM, pindolol 150 microM, acebutolol 500 microM, atenolol 4000 microM. Measurements of the concentrations of the various drugs in the cultured embryos at corresponding EC50 levels showed differing values: metoprolol 4.5 microM, propranolol 5.2 microM, alprenolol 8.4 microM, pindolol 9.0 microM, acebutolol 12.5 microM and atenolol 77.0 microM. With regard to the EC50 and the degree of substance transfer to the embryo it can be stated that propranolol and metoprolol show a much higher intrinsic potency to interfere with normal in vitro embryonic development than, e.g. atenolol.
Acebutolol↗
Changes associated with the basal lamina during metamorphosis of Xenopus laevis.
We examined the changes in the morphology of the basal lamina of the colon, abdominal wall and tail muscles of tadpoles during metamorphosis and compared our results to those previously obtained for the small intestine. At the early stage of metamorphic climax (stages 60-62), we observed a curving of the basal lamina along the processes of the epithelial cells. At a later stage of metamorphic climax (stages 63-66), more extensive curving and folding of the basal lamina were observed. The basal lamina of the mesothelial cells in the abdominal wall did not change extensively as compared to the colon. Folded basal lamina was observed among degenerated muscle cells. Our results suggest that the folding of the basal lamina depends on the degree of deformation of the particular organ, which occurs mainly by physical factors such as the shortening or shrinking of the organ due to cellular degeneration. Cells that are newly differentiated produce an additional basal lamina, which results in transient doubling or layering. The combination of physical factors and cell differentiation results in a basal lamina that is more complex and contains folds and layers.
Animals↗
Histochemical and immunoelectron microscopic localization of proteoglycans in calcified cartilage of rat epiphyseal growth plate.
Localization and ultrastructure of proteoglycans during endochondral calcification or mineralization in the rat epiphyseal growth plate cartilage were investigated histochemically and by immunoelectron microscopy, using ruthenium hexammine trichloride and/or mouse monoclonal antibody (2-B-6) which recognizes chondroitin-4-sulphated glycosaminoglycans. The gold particles of the second antibodies were distributed solitarily, lineally or in small clusters in calcified cartilage as well as in the surrounding non-calcified matrix. The labelled matrix glycosaminoglycans in the longitudinal septa increased from the proliferative to the upper hypertrophic zones and remained constant in calcified cartilage. After fixation with ruthenium hexammine trichloride, matrix proteoglycans precipitated and were observed as a reticular network of strands. Immunogold-labelled antibodies for the demonstration of the glycosaminoglycans were localized on these strands. Proteoglycans in calcified cartilage were observed as a network of fine-filamentous structures after ruthenium hexammine trichloride, showing their non-precipitated native state. Immunostaining for chondroitin-4-sulphate indicated that there was no change in the concentration of glycosaminoglycans on the small calcified nodules in the areas where calcification was just beginning.
Animals↗
Influence of electromagnetic fields on the enzyme activity of rheumatoid synovial fluid cells in vitro.
Since positive clinical effects have been observed in the treatment of rheumatoid arthritis with electromagnetic fields of weak strength and low frequency range (magnetic field strength: 70 microT; frequency: 1.36-14.44 Hz), an attempt was made to analyse the effects of these electromagnetic fields on enzyme activity in monolayer cultures of rheumatoid synovial fluid cells after single irradiation of the cultures for 24 hours. We only investigated the matrix metalloproteinases (collagenase, gelatinase, proteinase 24.11 and aminopeptidases). It was found that electromagnetic fields of such a weak strength and low frequency range do not generally have a uniform effect on the activity of the different proteinases in vitro. While aminopeptidases do not show any great changes in activity, the peptidases hydrolysing N(2,4)-dinitrophenyl-peptide exhibit a distinct increase in activity in the late phase in culture medium without fetal calf serum. In the presence of fetal calf serum this effect is not observed and enzyme activity is diminished. Our experiments do not show whether such a phase-bound increase in the activity of proteinases in vitro is only one finding in a much broader range of effects of electromagnetic fields, or whether it is a specific effect of weak pulsed magnetic fields of 285 +/- 33 nT on enzyme activity after single irradiation. This question requires further elucidation.
Amino Acid Sequence↗
Pre- and postnatal development of the primary visual cortex of the common marmoset. I. A changing space for synaptogenesis.
The primary visual cortex of Callithrix jacchus occupies a large portion of the occipital neocortex and can be safely delineated from fetal stages onwards. In 20 animals ranging in age from fetal to adult age the morphological development of area 17 was evaluated and compared with the growth of whole brain, skull, and head size. Cortical thickness, surface area, and volume of the area were determined in addition to predominant growth directions. The volume of area 17 approximately doubles between birth (241 mm3) and three months of age (506 mm3). This maximum value marks an overshoot in growth (volume: 180%, surface area: 150%, thickness: 122%), which is followed by a considerable reduction before adult values (100%) are reached. Although these values seem to indicate that the overall reduction in size is fairly isometric, growth and regression are locally anisometric. For example, layers II-IVc contribute disproportionately to the overshoot; thickening is less pronounced than tangential growth and follows a slightly different time course. These data suggest that the developing visual cortex represents a highly dynamic distribution space for the developing synaptic junctions which should be taken into account in studies on synaptogenesis. By comparison it is suggested that this growth dynamic is not restricted to area 17 but also occurs in some other parts of the cerebral cortex. In contrast, most subcortical brain regions apparently do not undergo overshoot growth. Structural changes of the skull compensate the overshoot in cortex growth, so that head size increases steadily.
Animals↗
Pre- and postnatal development of the primary visual cortex of the common marmoset. II. Formation, remodelling, and elimination of synapses as overlapping processes.
During ontogenesis changes in the numerical density of synapses are usually assumed to depend essentially on variations in the formation of synapses. Only the final adjustment to adult synapse densities is thought to include the elimination of synapses in some brain regions of certain species. Here, we focus attention on quantitative aspects of synapse elimination throughout development of area 17 of marmoset monkeys (Callithrix jacchus). Mature synapses, various precursor forms, and indicators of lysosomal degradation of synapses were quantitatively analysed by electron microscopy and morphometric methods. A total number of about 135 x 10(9) synapses was calculated for area 17 in each adult hemisphere corresponding to a volume density of 600 x 10(6) synapses/mm3. At 3 months of age, the respective values were 508 x 10(9)/area and 1,159 x 10(6)/mm3, while at birth these values were 69 x 10(9)/area and 328 x 10(6)/mm3. Consequently, at least three out of four synapses are eliminated between 3 months and adulthood. However, the real number of synapses being eliminated during development is probably much larger if the time course of lysosomal degradation is additionally taken into account. The frequency of lysosomes in presynaptic endings is highest before net-elimination of synapses occurs, i.e., between 1 and 3 months. This suggests that lysosomal degradation is not directly responsible for the majority of synapses removed during ontogenesis but apparently represents a second mechanism for synapse remodelling and elimination. Thus, it appears from this study that remodelling and elimination of synapses are quantitatively as important as their formation, and accompany synaptogenesis from its very onset onwards.
Animals↗
Electron and fluorescence microscopic investigations on composition and structure of the epithelial basement membrane of the human inferior nasal concha.
Light and electron microscopic as well as immunohistochemical investigations were performed on the basement membrane (BM) of the nasal mucosa from the inferior nasal conchae of 20 patients, aged 15-50 years. Two of the patients served as controls. Clinical disorders requiring the turbinectomies were hyperplastic nasal inferior conchae (17 patients) and immobile cilia syndrome (1 patient). In all cases light microscopy demonstrated a 10-15 microns thick homogeneous BM underneath an intact epithelium. Electron microscopic findings revealed a typical subepithelial basal lamina (BL). The remaining parts of the BM consisted of single, isolated 25-mm-thick collagenous fibrils. Only a few cells and small unmyelinated nerves occurred in this layer. Immunohistochemical investigations showed BL components (collagen type IV, laminin, nidogen and heparan sulfate proteoglycan) directly underneath the epithelium. Collagen types I, III, V, and VI could also be demonstrated immunohistochemically in the remaining parts of the BM. Collagen type VII was allocated to the anchor filaments beneath the BL. This special BM is presumed to contribute to mechanical stabilization of the epithelium. The significance of the BL and the subepithelial connective tissue for the behavior of the epithelium--e.g. direction of differentiation--are discussed.
Adolescent↗
Localization of collagen type VI in articular cartilage of young and adult mice.
Collagen type VI was demonstrated immunomorphologically in articular cartilage (distal femur) of young (2-8 weeks) and adult mice by fluorescence and electron microscopy (gold-labelled second antibody--sandwich method) using pre- and post-embedding techniques. This collagen type was mainly seen in the vicinity of chondrocytes, and in larger amounts in adult cartilage. Electron-microscopic inspection (pre-embedding technique) revealed labelling above plaques that were 40-160 nm in size, and from which up to 7 fine filaments (< or = 10 nm) per unit sectional plane radiated. Using the post-embedding technique, only labelled plaques could be demonstrated; fine filaments were not perceptible. This was partly a result of the low contrast. It is assumed that the globular ends of up to 20 of the fine type VI filaments are anchored in one plaque and that the antibodies bind to the non-collagenous globular domains. Filaments radiated from the plaques and formed a three-dimensional network that stabilized the structures of the cartilaginous matrix. Antibodies against fibronectin also labelled similar plaques. The ends of the type VI filaments are possibly linked into the plaques by fibronectin.
Aging↗
Cell shape and arrangement of cultured aortic smooth muscle cells grown on collagen gels.
Aortic smooth muscle cells (SMC) grown on conventional plastic culture dishes have morphological and functional properties of dedifferentiated cells in subculture. We examined the influence of collagen gels on the cell shape and arrangement. The cells grown on collagen gels showed a multilayered growth with formation of nodules. When the edge of the collagen gels was detached from the culture dish, the shape and arrangement of cells on the edge differed from that of the central, still attached region. The cells grown on floating collagen gels exhibited a spindle-like shape and were arranged in concentric circles. These findings suggest that the physical property of the substrate influences the cell shape and arrangement.
Animals↗
Inhibition of the effects of rheumatoid synovial fluid cells on chondrogenesis and cartilage breakdown in vitro: possible therapeutical conclusions. A morphological--biochemical study.
Short-term co-cultivation of blastemal cells from 12-day-old mouse limb buds and human rheumatoid synovial fluid cells in high density cultures (Trowell culture system) resulted, depending on when co-cultivation started, either in (1) an inhibition of chondrogenesis (co-cultivation right from the start) or in (2) an extensive breakdown of cartilaginous matrix (co-cultivation after formation of embryonic cartilage). These synovial effects were markedly impeded if Avarol (a dioxygenase inhibitor) was applied singly or in combination with PAI-2 (a u-PA-inhibitor). PAI-2 alone, however, had no effect on the synovial-induced inhibition of chondrogenesis, but produced a pronounced inhibitory effect on matrix breakdown. The effects of both inhibitors were studied electron microscopically and biochemically (determination of sulfated-glycosaminoglycans in the high density cultures by Alcian Blue binding assay). The results of this study are consistent with the presumption that rheumatoid synovial cells are capable of inhibiting chondrogenesis and enhancing the breakdown of the cartilaginous matrix. Amongst others, the possible mediators involved are prostaglandins and plasminogen activators. The response to the inhibitors Avarol and PAI-2 is compatible with their mode of action. The chondroprotective action of these substances may be useful in developing potential antirheumatic drugs.
Animals↗
[Intraepithelial mast cells in the vocal cords and nasal mucosa].
Leukocytes and mast cells have been described in the human nasal mucosa and epithelium of the lower airways (Bonneville 1988, Goodman and Lefrancois 1989, Guy-Grand 1991, Denburg 1992). In mast cells, heterogeneity of mucosal and connective tissue mast cells has been observed. In the present electron microscopic study, the epithelium of the upper airways (larynx and nose) of patients, suffering from chronic laryngitis, was examined with special regard to intraepithelial mast cells. Numerous mast cells were found in the stratum spinosum of the true vocal cords showing extensive elongated surface folds that had little contact to epithelial cells. Desmosomes were absent. Mucosal mast cells are characterised by four types of membrane-bounded granules, whereas serosal mast cells only showed two different types of granules and resembled blood basophiles. Migration of connective tissue mast cells into the epithelium could not be observed. According to our findings, mucosal and serosal mast cells belong to different cell lines.
Adult↗
Type I collagen synthesis in cultured aortic smooth muscle cells from spontaneously hypertensive rats and normotensive control, Wistar-Kyoto rats.
An inhibition ELISA was used to quantify the amount of type I collagen synthesized in culture media and cell layers from aortic smooth muscle cells (SMCs) of spontaneously hypertensive rats (SHR) and normotensive control, Wistar-Kyoto rats (WKY). Cultured cells were also observed by electron microscopy. Collagen content in the culture media was strongly increased after 6 days in both cultures. Collagen and protein contents in the medium and cell layer from SHR were significantly higher than those in WKY at day 14. However, cell density in SHR-derived cells was also higher than that of WKY. No significant differences were detected in the rates of collagen content between SHR and WKY on a per cell basis. The main differences between SHR and WKY in collagen and protein levels may be due to the greater number of SHR cells and increased amounts of extracellular matrix components. The assay system outlined here should be useful for studying the control of extracellular-matrix synthesis.
Animals↗
Integrins in ageing cartilage tissue in vitro.
Matrix-cell interactions are of great importance for numerous cell functions whereby integrins play an essential role as transmitters of extracellular signals. In cultures of ageing cartilage tissue (organoid or high density cultures) cartilage cells occur on the surface of which thick fibrils of collagen type I are deposited. Since integrins, in their role as receptors, cause an interaction between matrix components and cell membrane, we tried to demonstrate immunomorphologically (light and electron microscopically) the corresponding integrin receptors for collagen type I (beta 1 alpha 1 and beta 1 alpha 2) on the surface of these ageing cartilage cells. Cultures of normal, i.e. young cartilage tissue exhibit only beta 1 alpha 3- and beta 1 alpha 5-receptors; labelling against the integrins beta 1 alpha 1 and beta 1 alpha 2 is not possible in this case. Our results show that after the occurrence of thick fibrils cartilage cells express new receptors (beta 1 alpha 1 and beta 1 alpha 2) on the cell membrane. Thus, in ageing or dedifferentiating cartilage tissue it is not only the synthesis programme of matrix components (e.g. instead of collagen type II >> collagen type I) which changes but also the integrins (instead of alpha 3/beta 1, alpha 5/beta 1 >> alpha 1/beta 1, alpha 2/beta 1) so that new collagen types can be bound. These findings may also serve for a better understanding and interpretation of cartilage changes in vivo during ageing and under pathological conditions.
Animals↗