Widespread dissemination of metal debris from implants.

In a post-mortem study, we compared subjects with metal implants with and without visible wear with an age-matched control group to determine the extent and effects of dissemination of wear debris. In subjects with stainless-steel and cobalt-chrome prostheses metal was found in local and distant lymph nodes, bone marrow, liver and spleen. The levels were highest in subjects with loose, worn joint prostheses and the main source of the debris was the matt coating. Metal levels were also raised in subjects with implants without visible wear and, to a less extent, in those with dynamic hip screws. Necrosis of lymph nodes was seen in those cases with the most wear, and potential damage to more distant organs such as the bone marrow, liver and spleen in the long term cannot be discounted. The consequences for the immune system and the role of metal dissemination in the possible induction of neoplasia are discussed.

Submicroscopic crystals in osteoarthritic synovial fluids.

OBJECTIVES: To investigate the hypothesis that synovial fluid (SF) from patients with osteoarthritis (OA) may contain calcium phosphate crystals that are either too small, or too few in number to be identified by conventional light microscopy techniques. METHODS: Twelve SF from 11 patients with established knee OA, five SF from patients with rheumatoid arthritis (RA), and two control samples of SF from patients with pseudogout were subjected to an enzyme/hypochlorite extraction procedure. The patients with OA and RA had no radiographic evidence of chondrocalcinosis, or SF crystals on polarised light microscopy. Extracted material was examined and analysed by analytical electron microscopy (AEM) and x ray powder diffraction (XRD). RESULTS: Mineral was found in 11 of 12 OA samples, ranging from 2-120 micrograms/ml SF. Analytical electron microscopy revealed calcium pyrophosphate dihydrate (CPPD) crystals in five (confirmed by XRD in three) and basic calcium phosphates (BCP) in eight (five on XRD). Two samples with confirmed CPPD contained some rods with a mean length below 100 nm. The majority of BCP clusters were also less than 100 nm in diameter. BCP was detected in 1/5 RA samples. Control samples contained CPPD crystals of the expected size range of 0.42-17.9 microns. CONCLUSIONS: The data indicate that many OA SF may contain CPPD or BCP crystals which are too small or too few in number to be identified by conventional techniques. Crystal deposition is not an 'on-off' phenomenon in OA.

A method for isolating rabbit atrioventricular node myocytes which retain normal morphology and function.

This report describes a method for isolating single rabbit atrioventricular (AV) node myocytes which retain their normal morphology when exposed to millimolar levels of calcium. Previous attempts to isolate cells from the AV node have produced myocytes that "round up" (i.e., go into contracture) when exposed to calcium. We show that the cells isolated with our technique possess properties similar to those described for intact AV nodal tissue. We find that single AV node myocytes are shorter and thinner (mean dimension = 103.5 +/- 2.3 by 7.8 +/- 0.2 microns; mean +/- SE, n = 90) than atrial or ventricular cells. Many of the cells produced by this isolation procedure generate spontaneous action potentials (188 +/- 9 beats/min; n = 6), which resemble action potentials recorded previously from the intact AV node. Voltage-clamp recordings from spontaneously active cells revealed similar membrane currents to those seen in intact tissue: fast sodium current and a L-type calcium current, followed by a delayed outward current. However, we found little evidence for the hyperpolarization-activated current (I(f)). Because the cells responded normally to concentrations of acetylcholine and isoproterenol within the physiological range, their cholinergic and adrenergic receptors appear to be well preserved by the isolation procedure. The ability to isolate morphologically and functionally normal AV myocytes may represent a significant advance for the investigation of nodal physiology at the cellular level.

Systemic distribution of wear debris after hip replacement. A cause for concern?

The production of particulate wear debris is a recognised complication of joint arthroplasty, but interest has concentrated on local tissue reactions and a possible association with implant loosening. The fate of wear products in the body remains unknown, although some of the metals used in the construction of orthopaedic implants are known to have toxic and oncogenic properties. We report histological and electron-microscopic evidence from two cases which shows that metallic debris can be identified in the lymphoreticular tissues of the body distant from the hip some years after joint replacement. The increase in the use of total arthroplasty in younger patients, the development of new alloys and the use of porous coatings must raise concern for the long-term effects of the accumulation of wear debris in the body.

Analysis of 'clean' air samples by scanning and transmission electron microscopy and X-ray probe micro-analysis.

Air samples taken from the clean air over Bristol were examined using scanning, transmission and X-ray probe micro-analysis. A diversity of material was identified demonstrating the importance of man-made pollutants which have the potential to produce lung damage in addition to pollen moulds.

Ultrastructural localization of the allergen Der p I in the gut of the house dust mite Dermatophagoides pteronyssinus.

The sites and concentrations of Der p I within the house dust mite were determined. Highest concentrations were found in the epithelium of the posterior end of the mite stomach, implying that this is the site of Der p I synthesis and secretion.

Effect of aerosol challenge with sensitising antigen on the permeability of the surface of the rat trachea in life.

A rat model of immediate pulmonary hypersensitivity was used to investigate the permeability changes in the tracheal epithelium produced by aerosol challenge with antigen. The rats were sensitised by the intraperitoneal injection of antigen (dinitrophenyl (DNP19) ovalbumin). Sensitised and control animals were then challenged for 60 minutes with an aerosol of the same antigen, which also contained the electron dense pore marker lanthanum. Histological examination and x ray probe microanalysis showed a greater intercellular concentration of lanthanum in the tracheal epithelium in sensitised than in control animals. The results show that in sensitised rats increased intercellular penetrance of antigen can occur after antigen challenge.

Changes in peripheral progestagen levels in early pregnant gilts following injection of human chorionic gonadotrophin.

The capacity of human chorionic gonadotrophin (hCG) to raise plasma progestagen levels during the first 8 days of gestation in gilts was examined. The effective half-times of hCG in gilts treated with 500 and 5000 i.u. hCG were 29.1 and 26.3 h respectively (P greater than 0.05). Neither 500 nor 5000 i.u. hCG caused rises in peripheral concentrations of progesterone or pregnenolone sulphate, and plasma pregnenolone concentrations declined (P less than 0.05) following hCG treatment. Apart from diminished total corpus luteal weights in gilts treated with 500 i.u. hCG (P less than 0.05) and lower peripheral progesterone levels in gilts treated with 5000 i.u. hCG (P less than 0.05), ovarian and plasma steroid characteristics of hCG-treated animals between 23 and 25 days of gestation were similar to control values. Furthermore, treatment with hCG did not affect embryo survival during the first 4 weeks of pregnancy, and plasma oestrone/oestrone sulphate levels provided no evidence for differences between control and treated animals in trophoblastic outgrowth. These results challenge the rationale for the treatment of early pregnant sows with hCG in order to reduce the levels of embryonic wastage in early pregnancy.

Antigenic challenge via the airway alters the permeability of the trachea in sensitized rats.

Inbred rats were sensitized by intraperitoneal injection of DNP19 ovalbumin (DNP-OA) or saline. The rats were subsequently challenged by exposure to an aerosol of DNP-OA or bovine gamma globulins (BGG) for 10 min. Tracheal tissue was processed immediately after challenge in fixative containing 1% lanthanum. Planimetry of transmission electron micrographs showed 59% of the intraepithelial membranes were stained with lanthanum in the DNP-OA-sensitized and DNP-OA-challenged group; 39% in the saline-sensitized, DNP-OA-challenged group, and 26% in the DNP-OA-sensitized, BGG-challenged group. X-ray static probe microanalysis confirmed that most lanthanum penetrated in the DNP-OA-sensitized and DNP-OA-challenged group. In this group, the magnitude of the mechanical response, measured by body plethysmography, correlated directly (r = 0.74) with the lanthanum penetration. Our results confirm that increased permeability of the intercellular spaces occurs in immediate pulmonary hypersensitivity. They suggest that this change in permeability may be involved in producing the mechanical response.

Lanthanum penetration of the trachea after the immediate response of sensitized rats to aerosol antigen.

Inbred rats were sensitized by the intraperitoneal injection of 10 micrograms DNP19-ovalbumin (DNP-OA). These rats were compared both in their responses to 10 min of aerosol challenge with DNP-OA or bovine gamma globulins (BGG) and with other rats similarly 'sensitized' with saline and subsequently challenged with DNP-OA. Lanthanum (1%) in the fixative permitted the identification by transmission electron microscopy of electron-dense lanthanum in the tracheal epithelium. Planimetry showed 59% of the intercellular membranes in the tracheal epithelium of the group sensitized and challenged with DNP-OA were stained with lanthanum versus 39% in the saline-sensitized and 26% in the BGG-challenged. X-ray energy probe microanalyses confirmed that significantly more lanthanum had penetrated in the group sensitized and challenged with DNP-OA than in the other two groups. In this group the magnitude of the change in respiratory pattern produced by challenge was directly related to the quantity of the lanthanum in the epithelium. Our findings show that increased permeability of the intercellular spaces occurs in immediate pulmonary hypersensitivity, probably as a result of the opening of the tight intercellular junctions and suggests that this change of permeability may be involved in producing the physiological response.

Enzootic pneumonia of pigs in South Australia--factors relating to incidence of disease.

Environmental and management conditions of 15 herds with a high-prevalence (greater than 70%) of enzootic pneumonia of pigs at slaughter were compared with 16 herds with a low-prevalence (less than 30%) to determine factors commonly predisposing pigs to enzootic pneumonia in South Australia. Comparisons were made of herds having greater than 100 sows (fully intensive units) and also small herds having 20 to 70 sows (sideline units). Half the herds were visited in summer and half in winter to detect seasonal factors. In small herds, factors commonly found associated with a high prevalence of enzootic pneumonia were larger numbers of pigs per shed section (p less than 0.001), larger group sizes (p less than 0.01) and draughty farrowing and weaner accommodation (p less than 0.01). In large herds, factors associated with a high prevalence were higher pen stocking rate (p less than 0.05) and airspace stocking rate (p greater than 0.05), and a trend toward higher atmospheric ammonia levels in summer (p less than 0.1).

Effect of enzootic pneumonia of pigs on growth performance.

Growing pigs were naturally infected with a field strain of Mycoplasma hyopneumoniae to assess the effect of enzootic pneumonia on production. Both the initial ("breakdown") and endemic stages of infection were evaluated. The pigs were reared under environmental and management conditions commonly found on commercial piggeries in South Australia. Growth rate of pigs held in-contact with inoculated pigs was reduced by 12.7% (p less than 0.01) between 50 to 85 kg bodyweight. In the second trial inoculated gilts were used to naturally infect piglets during suckling. Growth rate of infected pigs was reduced by 15.9% (p less than 0.001) between 8 to 85 kg bodyweight, while feed conversion was depressed by 13.8% (p less than 0.05) between 10 to 25 kg bodyweight. At current feed and production costs this reduced performance added approximately $2.80 to the cost of every pig produced. These losses were recorded in groups of pigs in which enzootic pneumonia was present. At slaughter, 40% of lungs contained gross lesions of enzootic pneumonia which were free of significant secondary bacteria. The nature of the infection was established by gross and microscopic pathology and confirmed by the detection of specific complement fixing antibody in infected pigs and the demonstration of M. hyopneumoniae by direct immunofluorescent staining of lung sections.

An analytical ultrastructural study of the iron-rich surface layer in rat-incisor enamel.

Electron-dense particles, approximately 4 nm (40 A) in diameter were present. Evidence is offered that these particles represent iron probably in the form of amorphous hydrous ferric hydroxide. From X-ray probe analytical data, it is suggested that this iron component is accommodated through the process of adsorption rather than by direct substitution of calcium and phosphorus. The iron-rich surface layer was more resistant to acid etching than subsurface enamel. Reported areas of reduced mineral density of enamel at the enamel-dentine junction appear to be preparation artifacts.

An ultrastructural study of glycosaminoglycans associated with collagen and other constituents of the cat anulus fibrosus.

It has been confirmed that glycosaminoglycan is associated with collagen and other constituents of the anulus fibrosus. In cells, it appears to be present on the rough endoplasmic reticulum and is removed by hyaluronidase digestion and thus may consist of chondroitin sulphate. In pericellular material it appears as fibrils and granules, is unaffected by hyaluronidase and may consist of keratan sulphate or dermatan sulphate. Collagen fibrils contain hyaluronidase-digestable glycosaminoglycan (such as chondroitin sulphate) in the form of peripheral rings that appear as bands in longitudinal sections of the fibrils.

Correlation between Alcian Blue stainig of glycosaminoglycans of cat nucleus pulposus and TEM x-ray probe microanalysis.

The nucleus pulposus of cat intervertebral disc was examined after staining glycosaminoglycans with Alcian Blue and the results correlated with TEM X-ray probe microanalysis. In unstained sections a difference in copper levels between tissues and resin was detected. In tissue stained with Alcian blue before embedding, the copper levels were slightly increased and the morphological appearance of the intercellular material was amorphous. In sections restained after cutting, the relative levels of copper in the resin were considerably increased and tissue levels were significantly higher than in the resin. Moreover, the morphology of the intercellular material was altered from a rather amorphous material to a network. Sulphur levels behaved in similar manner to copper levels but any correlation between the elements was due to factors unrelated to glycosaminoglycan staining and probably resulted from contaminating sulphur.

Stimulus-secretion coupling: role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland.

1. The role of adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3':5'-phosphate (cyclic GMP) as second messengers for the enzyme secretory response evoked by the autonomic neurotransmitters, noradrenaline and acetylcholine, is examined in this in vitro study on the guinea-pig submandibular gland. 2. Noradrenaline increased enzyme (kallikrein) secretion. The initial stimulation of enzyme release appeared to be dose-dependent. The time course of cumulative kallikrein secretion revealed a complex pattern. Isoprenaline and phenylephrine were almost as potent as noradrenaline in releasing kallikrein. Both propranolol and phentolamine were required to fully inhibit the noradrenaline-stimulated enzyme secretion. 3. The cumulative secretion of kallikrein evoked by acetylcholine was dose-dependent. The onset of secretion showed a significantly greater time-lag than that observed with noradrenaline. Atropine effectively blocked the release of kallikrein by acetylcholine. 4. Dibutyryl cyclic AMP stimulated enzyme secretion. Dibutyryl cyclic GMP caused an initial increase which was not maintained. 5. The cyclic nucleotide phosphodiesterase inhibitors, theophylline and papaverine, increased basal kallikrein secretion. The action of the cyclic phosphodiesterase inhibitors on the secretory response to noradrenaline, acetylcholine, dibutyryl cyclic AMP and dibutyryl cyclic GMP was complex. In general, the increase in enzyme release produced by the secretagogues was additively enhanced by both inhibitors. 6. Omission of calcium inhibited both acetylcholine and dibutyryl cyclic GMP stimulated kallikrein release, but to a lesser degree than that of noradrenaline and dibutyryl cyclic AMP. High concentrations of extracellular calcium (10 mM) appeared to enhance the action of acetylcholine. 7. Noradrenaline produced a rise in the intracellular level of cyclic AMP. The increase preceded the stimulated secretion of kallikrein. Of the various adrenergic agonists, noradrenaline and isoprenaline were the most potent, whereas phenylephrine was significantly less effective in raising basal cyclic AMP values. Acetylcholine was without effect, even in the presence of a cyclic phosphodiesterase inhibitor. 8. Acetylcholine and noradrenaline raised intracellular levels of cyclic GMP only when the tissue incubations were performed in the presence of a cyclic phosphodiesterase inhibitor. The increase in cyclic GMP produced by acetylcholine preceded enzyme secretion. 9. Morphological data substantiated the finding that the in vitro release of kallikrein evoked by the secretagogues was associated with the depletion of secretory granules and vacuolations in acinar cells of the gland slices. 10. The molecular mechanisms which control enzyme secretion in the exocrine submandibular gland are discussed. Models are presented for the role of transmitter-specific cyclic nucleotides and calcium in stimulus-secretion coupling.