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Biomedical subjects

V Everts

Publications and source records attributed to V Everts.

At least 73 records · Page 4Linked to original sources
PubMed1993

Failure of calcitriol treatment in a patient with malignant osteopetrosis.

In an attempt to stimulate bone resorption, a 10-week-old infant with malignant infantile osteopetrosis was treated with high doses of calcitriol, a potent bone resorption stimulatory agent, combined with a low calcium diet to prevent hypercalcaemia. Although calcitriol administration was initiated at this very young age, our patient did not show any clinical, radiological, or histological improvement. Despite reports of positive results of this treatment in the literature, our patient did not reveal any signs of bone resorption. She eventually died from the complications of osteopetrosis at the age of 6 months after 88 days of therapy.

Bone and Bones↗
PubMed1993

The release of tissue inhibitor of metalloproteinases by calvarial bone explants and its immunolocalization.

Tissue inhibitors of metalloproteinases (TIMPs) play an important role in the regulation of the activity of matrix metalloproteinases (MMPs) such as collagenase, stromelysin and gelatinase. Although it has been shown that upon culturing bone tissue releases relatively large amounts of TIMP, little is known as to the source of the inhibitor. In an attempt to investigate this in more detail calvarial bone explants from young rabbits were cultured in serum-free medium. The explants were cultured with or without adhering periosteum. In some experiments solitary periosteal fragments were maintained in the absence of bone. Media were analyzed for the presence of TIMP by immunoblotting and ELISA as well as for their capacity to inhibit the activity of collagenase. In addition, TIMP was immunolocalized in cryosections of the explants. The data demonstrated that bone-conditioned medium contained significantly more (2-10 times) collagenase inhibitor than periosteum-conditioned medium. Removal of the (convex and/or concave) periosteum from the calvariae did not significantly affect the amount of inhibitor released. Immunoblots and ELISA showed the presence of TIMP in the media, being more in bone- than in periosteum-conditioned medium. In immunolabeled cryosections TIMP appeared to be present in osteoblast-like cells lining both the outer bone surface as well as the endosteal spaces. Label was also found in a number of osteocyte lacunae. The periosteum was almost negative. It is suggested that TIMP contributes to the regulation of MMP-activity involved in the remodeling and turnover of bone.

Animals↗
PubMed1993

Interleukin-1 alpha and epidermal growth factor synergistically enhance the release of collagenase by periosteal connective tissue in vitro.

The effects of recombinant human interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on the release of collagenase were studied in an in vitro model system using periosteal explants from rabbit calvariae. Following an incubation period of 72 h it was shown that IL-1 alpha in combination with EGF (IL-1 alpha + EGF) induced a synergistic increase in the amount of collagenase released by periosteal explants. This increase appeared to be at least 10-fold. Most of the enzyme was present in a latent form since the increase in enzyme activity was only detectable after activation by APMA and the molecular weight as determined in immunoblots corresponded to the latent form of this enzyme. Incubations carried out with IL-1 alpha alone resulted in a 2- to 4-fold increase of total enzyme activity, whereas the amount of collagenase in media of EGF-treated periosteal did not surpass control values. A neutralizing anti-IL-1 alpha antibody completely blocked the enhanced release of collagenase as induced both by IL-1 alpha and by IL-1 alpha + EGF. Indomethacin partially prevented the IL-1 alpha + EGF-induced increase in enzyme release, suggesting the involvement of prostaglandins. The amount of tissue inhibitor of metalloproteinases (TIMP) as determined by ELISA was slightly elevated in culture media obtained from all cytokine-treated explants. Comparable results were obtained by Western blot analysis as well as by a functional bioassay. It is suggested that the concomitant presence of the cytokines IL-1 alpha and EGF may play an important role in collagenase-mediated degradation of collagen.

Animals↗
PubMed1993

A quantitative enzyme histochemical analysis of the distribution of alkaline phosphatase activity in the periodontal ligament of the rat incisor.

The spatial distribution of alkaline phosphatase (ALP) activity was examined in the periodontal ligament of the continuously growing rat incisor. With the indoxyl-tetrazolium salt method, enzyme activity was demonstrated in undecalcified cryosections, and the amount of reaction product was quantified. ALP activity appeared to be distributed heterogeneously. Its highest activity was found in the bone-related compartment of the ligament. In the tooth-related compartment and the supracrestal extension of the ligament, enzyme activity was significantly lower, but still higher than in the lamina propria of the gingiva. In the part of the ligament bordering the cementum, highest activity was found in the apical region just occlusal to Hertwig's epithelial root sheath, where formation of acellular cementum begins. From there toward the incisal edge, the activity of the enzyme gradually decreased. It is suggested that differences among the various parts of the periodontal ligament are related to local variations in phosphate metabolism and cementum deposition.

Alkaline Phosphatase↗
PubMed1992

Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases.

The site of action of cysteine-proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP-inhibitor trans-epoxy-succinyl-L-leucylamido (4-guanidino)-butane (E-64) and the MMP-inhibitor N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide (Cl-1) in an in vitro model system of PTH-stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor-treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor-treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E-64 and Cl-1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs participate in the degradation of the collagenous bone matrix.

Animals↗
PubMed1992

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A.

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.

Amino Acid Sequence↗
PubMed1991

Selective inhibition of cysteine proteinases by Z-Phe-AlaCH2F suppresses digestion of collagen by fibroblasts and osteoclasts.

Effects of the selective inhibitor of cathepsins B and L, Z-Phe-AlaCH2F were studied on the degradation of fibrillar collagen by fibroblasts and osteoclasts in cultured rabbit calvariae at the electron microscopic level. Periosteal fibroblasts from inhibitor-treated explants showed a dose-dependent increase of the volume fraction of vacuoles containing cross-banded collagen fibrils. This was a 7-fold increase over control fibroblasts and the ratio of intracellular and extracellular collagen increased from 2 to 43. The presence of collagen-containing vacuoles was also found in some osteoclasts from inhibitor-treated explants (1 microM or more). The inhibitor appeared to have cytotoxic effects at a concentration of 100 microM. It was concluded that this selective inhibitor exerts its effects intralysosomally in living cells, indicating possibilities for in vivo inhibition of protein degradation.

Animals↗
PubMed1991

Morphologic features of bone in human osteopetrosis.

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells. In all patients there were large numbers of osteoclasts. Numbers of osteoblasts, bone lining cells, and bone marrow stromal cells were extremely low in all patients with malignant osteopetrosis. In six of the patients a mineralized layer of amorphous organic material lacking collagen fibrils was seen covering large areas of the bone or cartilage matrix. We suggest that this layer represents a pathological calcification on which subsequently organic material has accumulated. The abnormalities in osteopetrotic bone are discussed in the light of the pathogenesis of this disease.

Bone and Bones↗
PubMed1990

Elastic and collagenous fibers in the temporomandibular joint capsule of the rabbit and their functional relevance.

This study deals with the form and function of the temporomandibular joint capsule and compares the histological structure of the capsule and kinetics of condylar and disc movement in rabbit and man. The morphology of the capsule of the jaw joint of the rabbit was studied by means of histological sections of isolated capsules, celloidin sections of decalcified heads, and cryostat sections containing the joint, fixed in jaw open and closed positions. General staining methods and selective methods for elastin and collagen were used. The volume density of the elastic fibers was determined morphometrically. The capsule consists of a portion connecting disc and skull and a portion connecting disc and mandible. The former portion has a fibrous outer layer of dense connective tissue. It contains numerous bundles of specifically oriented collagen but hardly any elastic fibers. Few irregularly arranged elastic fibers are present in the lateral and medial areas of the capsule. In contrast, the portion connecting disc and condylar process contains not only oriented collagen but also large amounts of elastic fibers. Anteriorly, there is a thin elastic band that is stretched greatly at jaw opening. Posteriorly, the capsule takes the appearance of a large fibro-elastic pad that covers the posterior extension of the condyle. This part is somewhat stretched when the jaw is closed. The findings correlate well with the results of jaw movement studies, showing a remarkable capability for condylar protrusion and retrusion. In contrast to the situation in humans, this movement occurs mainly between condyle and disc. It is made possible by the rather tight connections between skull and disc and flexible ones between disc and condyle.

Animals↗
PubMed1990

Interleukin 1 increases the production of collagenase but does not influence the phagocytosis of collagen fibrils.

The aim of the present study was to determine whether interleukin 1 alpha (Il-1), a cytokine known to have a stimulatory effect on collagenase production, also influences the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. Mouse long bones and calvariae both with surrounding periosteum were cultured for 24 or 48 hours in media containing varying concentrations of the cytokine. The periostea were subjected to morphometric analysis in order to assess the volume density of phagocytosed collagen fibrils in fibroblasts. The results indicated that neither in calvarial nor in long bone periosteum the uptake and intracellular degradation of collagen by fibroblasts was influenced by Il-1. However, between both tissues the amount of collagen phagocytosed differed considerably. It appeared that within 48 hours periosteal fibroblasts of calvariae ingested at least three times more fibrillar collagen than those of long bone periosteum. This finding suggests intrinsic differences between these connective tissues as to the phagocytic behaviour of the fibroblasts. Analysis of collagenase activity in the media demonstrated that under the influence of Il-1 collagenase release increased about 1.5- to 2-fold, most of the enzyme being in a latent form. The media also proved to contain an inhibitor of collagenase, its production not being affected by Il-1. It is concluded that under the conditions tested Il-1 does not seem to play a role in the regulation of the intracellular pathway of collagen digestion.

Animals↗
PubMed1990

Formation of acellular root cementum in relation to dental and non-dental hard tissues in the rat.

After the periodontium of the rat was wounded, the formation of acellular extrinsic fiber cementum (AEFC) did not appear to be restricted to the hard dental tissues (pre-existing cementum, dentin, and enamel). Layers resembling AEFC were also deposited along the inner wall of the alveolar bone. At the time of observation (six weeks after being wounded), cells other than fibroblast-like cells could not be distinguished close to the newly formed AEFC-like layers. We suggest that the deposition of this material was related to the activity of periodontal ligament fibroblasts.

Alveolar Process↗
PubMed1989

Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts.

The effect of various metalloproteinase-inhibiting compounds on collagen phagocytosis by fibroblasts was studied in cultured periosteal tissue. Evidence is presented indicating that neither anti-collagenase nor anti-stromelysin interfere with the uptake of collagen fibrils from the extracellular space and their intracellular digestion. Similar results were obtained with tissue inhibitor of metalloproteinases (TIMP). In the presence of the proteinase inhibitor leupeptin, a compound which strongly inhibits the intracellular degradation of phagocytosed collagen, a time-dependent increase in the amount of internalized collagen was found. This increase proved to be similar in explants treated as well as in those not treated with the metalloproteinase-inhibiting compounds. It is concluded that enzymes, such as collagenase and stromelysin, do not play a crucial role in the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. If these enzymes are involved it must be prior to these events. Based on the morphometric data the intralysosomal degradation time of collagen was calculated to be about 30 minutes. A comparison with findings in the literature on collagen metabolism in the periodontal ligament of the rat molar suggests that all collagen degraded may pass through the phagolysome pathway during physiological turnover and remodelling.

Animals↗
PubMed1988

Effects of the proteinase inhibitors leupeptin and E-64 on osteoclastic bone resorption.

To determine the possible involvement of cysteine-proteinases in bone matrix degradation by osteoclasts, the effects of the proteinase inhibitors leupeptin and E-64 were studied in an in vitro system using mouse bone explants. It was observed that in explants treated with the drugs, the amount of demineralized matrix opposing the ruffled border of the osteoclasts increased about 20-fold within 6 hours. This suggests that demineralization had proceeded whereas matrix degradation had been retarded. It was further noticed that in 12 of 287 osteoclasts, cytoplasmic vacuoles were present containing collagen fibrils that could not be distinguished from those in cartilage or bone. Their intracellular localization was proved by the study of serial sections. Finally, a significant reduction was shown as to the relative surface density of electron-translucent vacuoles; this would seem to suggest reduced endocytic activity of the cells. Our observations support the view that cysteine-proteinases play an important role in osteoclastic bone resorption. It was further noticed that the in vitro effects of leupeptin and E-64 in certain respects resemble ultrastructural features of pycnodysostosis, an osteopetrosislike bone disorder. The data are in line with the hypothesis that this disease is caused by insufficient activity of osteoclastic cysteine-proteinases.

Animals↗
PubMed1987

Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5-nitrosalicylaldehyde.

The histochemical fluorescence method using 5-nitrosalicylaldehyde for the demonstration of cathepsin B activity has been used. Precipitation of the fluorescent final reaction product was analysed continuously during incubation for cathepsin B activity. Unfixed cultured human fibroblasts as well as cryostat sections of mouse metacarpal bone explants were used. Continuous monitoring of the formation of the fluorescent reaction product showed that after a certain lag phase, depending on the enzyme activity in the tissue, discrete granules appeared which became increasingly fluorescent with incubation time. Subsequently, recrystallization and redistribution of the final reaction product started to occur. It is concluded that the coupling reaction with 5-nitrosalicylaldehyde is sufficiently fast for a proper localization of proteinase activity and can be used for 'kinetic' analysis of enzyme activity. The method provides indications of relative amounts of cathepsin B activity in different cell types within a tissue section. It appeared from the study on metacarpal bone explants that fibroblasts in perichondrium and periosteum contained a relatively high cathepsin B activity whereas chondrocytes showed a low but distinct activity. This observation suggests that cysteine proteinases are not only involved in collagen degradation by fibroblasts but that they also play a role in the intracellular digestion of collagen by chondrocytes.

Aldehydes↗