John McClure

The normal human chondro-osseous junctional region: evidence for contact of uncalcified cartilage with subchondral bone and marrow spaces.

BackgroundThe chondro-osseous junctional region of diarthrodial joints is peculiarly complex and may be considered to consist of the deepest layer of non-calcified cartilage, the tidemark, the layer of calcified cartilage, a thin cement line (between the calcified cartilage and the subchondral bone) and the subchondral bone. A detailed knowledge of the structure, function and pathophysiology of the normal chondro-osseous junction is essential for an understanding of the pathogenesis of osteoarthrosis.MethodsFull thickness samples from human knee joints were processed and embedded in paraffin wax. One hundred serial sections (10 μm thick) were taken from the chondro-osseous junctional region of a block from the medial tibial plateau of a normal joint. They were stained with haematoxylin and eosin and photographed. For a simple physical reconstruction images of each 10th sequential tissue section were printed and the areas of the photomicrographs containing the chondro-osseous junctional region were cut out and then overlaid so as to create a three-dimensional (3D) model of this region. A 3D reconstruction was also made using computer modelling.ResultsHistochemical staining revealed some instances where prolongations of uncalcified cartilage, delineated by the tidemark, dipped into the calcified cartilage and, in places, abutted onto subchondral bone and marrow spaces. Small areas of uncalcified cartilage containing chondrocytes (virtual islands) were seen, in two-dimensional (2D) sections, to be apparently entombed in calcified matrix. The simple physical 3D reconstruction confirmed that these prolongations of uncalcified cartilage were continuous with the cartilage of zone IV and demonstrated that the virtual islands of uncalcified cartilage were cross-sections of these prolongations. The computer-generated 3D reconstructions clearly demonstrated that the uncalcified prolongations ran through the calcified cartilage to touch bone and marrow spaces and confirmed that the apparent entombment of chondrocytes was a 2D artefact.ConclusionThis study demonstrates that the chondro-osseous junctional region is more complex than previously described. The tidemark is a clearly defined boundary delineating uncalcified from calcified cartilage. It is not a straight line across a joint, but a complex three-dimensional structure that follows uncalcified cartilage prolongations dipping down through the calcified cartilage to abut onto subjacent bone or marrow spaces.

The tidemark of the chondro-osseous junction of the normal human knee joint

SummaryThe chondro-osseous junction includes the junction between calcified and non-calcified cartilage matrices often referred to as the tidemark. A detailed knowledge of the structure, function and pathophysiology of the chondro-osseous junction is essential for an understanding both of the normal elongation of bones and of the pathogenesis of osteoarthrosis. In this study the molecular anatomy of the tidemark was studied using histochemical techniques, including lectin histochemistry, on blocks of normal cartilage from human knee joints. The tidemark stained with H&E, picro-sirius red, toluidine blue, safranin O and methyl green, but not with alcian blue in the presence of magnesium chloride at 0.05 M or above. It stained with only four lectins, those from Datura stramonium, Maclura pomifera, Erythrina crystagalli and Helix pomatia, out of the 19 used. Therefore, it is rich in collagen and contains hyaluronan, but appears to lack the glycosaminoglycans of ‘conventional’ proteoglycans and it expresses a very limited and distinctive lectin staining glycoprofile, which is probably attributable to specific glycoproteins. In addition, the tidemark had a distinct microanatomical trilaminate appearance. From all of these results it is clear that this part of the chondro-osseous junctional region is chemically more complex and distinctive than has previously been described.

Lectin Histochemistry of Normal Human Lung

This study aimed to identify and specify the glycotypes of cell populations in normal human lung including types I and II pneumocytes, alveolar macrophages and mast cells, and also in the larger tissue structures of lung, including blood vessels and bronchi/bronchioles, using lectin- and immuno-histochemistry on paraffin-embedded tissue from 11 normal cases. The alveolar macrophages were anti-CD68 positive whereas the cells lining the alveolar walls were positive for cytokeratins. The alveolar macrophages in normal lung tissues showed a broad spectrum of staining for different subsets of N-linked saccharides, N-acetylgalactosamine, N-acetylglucosamine, terminal β-D-galactose and sialyl groups. This study showed that some lectins could be used as specific markers for some cell types i.e. Galanthus nivalis and Narcissus pseudonarcissus lectins for macrophages, Psophocarpus tetragonolobus lectin-II for capillary endothelium, Dolichos biflorus agglutinin for bronchial epithelial cells, Lycopersicon esculentum, Phytolacca americana or Triticum vulgaris (succinylated) for type I pneumocytes and Hippeastrum hybrid or Maclura pomifera lectins for type II pneumocytes. Patchy staining of type I pneumocytes by peanut agglutinin indicated the possibility of two distinct populations of these cells or a pattern of differentiation that is unapparent morphologically.

Application of solid-phase micro-extraction technology to drug screening and identification

Benzodiazepines, tricyclic antidepressants and local anaesthetics are frequently involved in poisoning episodes and fatalities. A specific, sensitive and rapid procedure for identifying and quantifying such drugs in postmortem matrices has been developed using solid-phase micro-extraction (SPME) and gas chromatography-mass spectrometry. Very clean extracts were obtained in one step using SPME. The most commonly used fibre coatings were tested to select the best coating for SPME of the drugs. The appropriate fibre coating for most drugs was polyacrylate, followed by Carbowax-divinylbenzene. A Hewlett-Packard 5890 gas chromatograph in combination with a Trio 2000 mass spectrometer was used to analyse the samples. Temperature, time, pH and addition of sodium chloride were optimized to obtain consistent extraction. The between-day and within-day coefficients of variation were less than 16% and less than 6%, respectively.

Lectin histochemistry of normal human gastric mucosa

Information about the saccharides expressed in gastric mucosa is mostly limited to the glycan content of gastric mucins and there are only a few studies of the glycoprofiling of the constituent cells and their components. Knowledge of the glycan expression of normal gastric mucosa is necessary for the interpretation of the significance of changes of expression in disease.A lectin histochemical study of normal human gastric (body) mucosa was performed using 27 lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans.There were marked differences in staining reactions in the various microanatomical structures of the mucosa, particularly between pits and glands with the former more closely resembling the surface epithelium. A notable feature was the degree of difference in the staining between a substantial sub-population of cells within the neck region and the epithelium of both the pits and glands. These neck cells resembled the pit cells with some lectins, glandular cells with some others and neither with some other lectins. Overall, the differences between the pit, gland and neck epithelia were diverse and numerous, and could not be explained by altered activity of a small set of glycosyltransferases. Widespread alterations of glycans must have occurred (affecting terminal and internal parts of their structures) and the very different glycotypes of the pit, neck and gland epithelia are, therefore, suggestive of the existence of three cell lineages within normal gastric epithelium. Published in 2004.

The effect on chick osteoclasts of infection with paramyxoviruses.

The detection of virus in osteoclasts from Pagetic patients is now well known, but it has yet to be shown convincingly that the presence of virus in Pagetic osteoclasts influences their behaviour. In this study, osteoclasts from embryonic chick tibiae were infected with canine distemper virus or measles virus and compared with mock‐infected controls. Infection was confirmed using virus‐specific fluorescent antibodies. It was found that virus infection did not alter osteoclast morphology or tartrate‐resistant acid phosphatase (TRAP) activity. Both infected and mock‐infected osteoclasts produced resorption pits on bovine bone slices; these could be divided into two distinct size classes with a computer‐based measuring system. Virus infection significantly increased the proportion of the larger size class of resorption pit. These results suggest that virus infection can increase bone resorption by osteoclasts, lending further support to the hypothesis that viruses play a role in Pagets disease of bone.

Antibody to human lymphocyte actin regulates immunoglobulin secretion by an EBV-transformed human B-cell line

Antibody against actin isolated from a human EBV-transformed lymphoblastoid B-cell line exerted an inhibitory effect on in vitro IgM secretion by a different lymphoblastoid B-cell line, LA350. This effect was dose dependent showing from 24-40% inhibition at a dilution of 1:100 and 68-80% inhibition at a dilution of 1:50. This effect was noted in the absence of changes in either total cell count or [3H]-thymidine incorporation and was reversed by co-incubation with purified rabbit thymus actin (100 micrograms/ml) but not bovine serum albumin at the same concentration. These data demonstrate regulation of immunoglobulin synthesis by antibodies against human lymphocyte derived actin in a lymphoblastoid B-cell line.

Anti-rabbit thymus actin antibody inhibits proliferation of Epstein-Barr virus-transformed human B cell line LA350: Augmentation by cyclic AMP

Antibody to actin isolated from the rabbit thymus gland exerted a dose-dependent inhibitory effect upon the proliferation of an Epstein-Barr virus-transformed human B cell line, LA350. Purified rabbit thymus actin specifically reversed the inhibitory effect of antibody by competing with surface actin on LA350. For example, LA350 proliferation at 48 hr was inhibited 90% at a 1:10 antibody dilution (p less than 0.001) but only 48% and 20% in the presence of 190 and 380 micrograms/ml of rabbit thymus actin, respectively (p less than 0.001 for reversal). Cyclic AMP augmented in a dose-related fashion the inhibitory effects of antibody, e.g., a 1:20 dilution of antibody inhibited 10, 19, 41, and 67% at 0.0, 0.5, 1.0, and 2.0 mM cAMP, respectively (p less than 0.001). We conclude that anti-actin antibody recognizes surface actin on a human B cell line and produces a functional inhibition of proliferation by a process that is augmented by cAMP.

Lectin and other histochemical studies of the articular cartilage and the chondro-osseous junction of the normal human knee joint

There are few studies on normal, adult diarthrodial joints which look in detail at the histochemical properties of the chondro-osseous junctional region. This study of the normal human knee joint was performed using lectin and other histochemical techniques. There were differences in the reactions of mineralised cartilage compared to those of hyaline cartilage with the former demonstrating more collagen and less glycosaminoglycans. Lectin histochemistry revealed more accessible terminal 2-deoxy,2-acetamido-α-d-galactose and more N-acetyllactosamine but less fucosyl and α-2,6-linked-sialyl termini in the mineralised cartilage. The hyaline cartilage chondrocytes stained for N-glycans but those of mineralised cartilage did not. The staining patterns of prolongations and islands of uncalcified cartilage running through the calcified layer to abut bone and marrow spaces were distinct, resembling the patterns of the hyaline cartilage but with some unique features. A possible relationship was revealed between the presence of the Maclura pomifera ligand (Galβ1,3GalNAcα1-) and mineralisation. Subchondral bone had a markedly restricted glycoprofile.

A comparative study of lectin binding to cultured chick sternal chondrocytes and intact chick sternum

Cultured chondrocytes derived from the caudal and cephalic ends of embryonic chick sterna have been compared with each other and with whole sternum, by using a panel of 21 lectins to probe the distribution of oligosaccharides in glycoconjugates of cells and matrix at various times of culture or development. On culture in collagen gels, the cells changed their morphology with time, degrading glycan in the surrounding culture medium and depositing new matrix, the glycan content of which reflected the site of origin of the cells, indicating that the glycan phenotype of both cells and matrix (‘glycotype’) was predetermined and persistent. Sterna of embryonic chicks showed unexpected complexity in their distribution pattern of glycan, containing at least six distinct regions. Major regional temporal differences were evident among saccharides terminating in α-N-acetyl galactosamine and β-galactose, while changes in glycans terminating in fucose, sialic acid and α-mannose were somewhat less marked. Subsets of complex N-glycans changed little.