Synovial nerve fiber density decreases with naturally-occurring osteoarthritis in horses.

OBJECTIVE: To measure the nerve fiber density in synovial membranes from healthy and OA equine joints and to investigate the relationship between synovial innervation and OA severity, synovial vascularity and synovitis. DESIGN: Twenty-five equine metacarpophalangeal joints were collected post-mortem. The joints were dissected and the macroscopic lesions of the articular cartilage were scored. Synovial membrane specimens (n = 50) were harvested, fixed, sectioned and scored histologically. Immunohistochemical staining and immunofluorescence with S-100 protein, that identifies nerve fibers, and ⍺-actin, that stains vascular smooth muscle, were also performed on site-matched specimens and the relationships between these tissues was interrogated. RESULTS: The nerve fiber density was higher in the superficial layer (≤200 µm) of the synovium when compared to the deeper layer in control equine joints (mean difference (95% C.I.): 0.054% (0.018%, 0.11%)). In osteoarthritic joints, synovial innervation decreased in the superficial layer with increasing macroscopic OA score (ß (SEM), 95% C.I.: -0.0061 (0.00021), -0.0011, -0.00017). The blood vessel density was also higher in the superficial layer of the synovium compared to the deep layer in the control (mean difference (95% C.I.): 1.1% (0.36%, 2.3%)) and OA (mean difference (95% C.I.): 0.60% (0.22%, 1.2%)) equine joints. Moreover, considering all synovial specimens, higher nerve fiber density in the deep layer positively correlated with blood vessel density (ß (SEM), 95% C.I.: 0.11 (0.036), 0.035, 0.18). CONCLUSION: The reduction in nerve fiber density with advanced cartilage degeneration suggests that peripheral neuropathy is associated with equine OA. Whether this link is associated with neuropathic pain, requires further investigation.

The proteins and the formation of gallstones.

Cholesterol supersaturation of bile requires assistance to form gallstones. Proteins have been proposed as candidates either to facilitate or hinder the formation of stones. It is assumed that the identity of these stone proteins should be revealed in order to assess their role in the process. We have used electrodialysis of crushed stones to extract the proteins followed by 2D electrophoresis and N-terminal amino acid sequencing to characterize them. The discovery of bacterial proteins in some stones adds evidence to the importance of an inflammatory process and the deconjugation of bilirubin in mixed gallstone formation.

Urinary stone proteins: an update.

The discovery of an organic component in kidney stones dates back to 1684. More than 150 years elapsed before the incrustation of this organic component, which is now called the matrix, was proposed as the mechanism of stone formation. The composition of the matrix remained largely unknown until the development of electron microscopy and the advances in biochemistry combined in the 1950's to usher in the modern era of renal stone matrix investigation. Composed mainly of selectively incorporated proteins generally characterized by high glutamic and aspartic acid content and the frequent occurrence of gamma-carboxyglutamic acid, the matrix displays a variable and complex composition and shares a few proteins in many stones. The embryonic stone may first appear in the renal tubules where it can acquire the blood and cell membrane proteins recently found by analysis of stone protein extracts. The combination of supersaturation, an appropriate environment, the availability of calcium binding proteins which may be abnormal, and the incorporation of proteins extracted from leukocytes and cell wall membranes may induce stone formation.

Sequencing of proteins extracted from stones.

Proteins from urinary tract and gallbladder stones were extracted and characterized to determine the composition of the matrix and possibly unravel the role of the organic phase in stone formation. Proteins from crushed stones were extracted by electrodialysis and concentrated in the Amicon centricon cartridge or by lyophilization after dialysis against distilled water. Aliquots were first analyzed by isoelectric focusing in gel and if suitable subjected to two-dimensional (2D) electrophoresis. The most promising spots were harvested and the N-terminal amino acids sequenced, thus providing maximum information with minimum expenditure of material. The 2D separations and amino acid sequences of several protein extracts demonstrated similarities and differences in composition and achieved the identification or demonstration of previously and recently detected polypeptides.

A cationic protein from a urate-calcium oxalate stone: isolation and purification of a shared protein.

A protein extracted from a urate-calcium oxalate stone by electrodialysis is also excreted in the urine which served as the source material for its purification by FPLC after separation on an ACA44 column. It has an amino acid composition appropriate for a cationic protein. One peptide obtained by cyanogen bromide cleavage has significant (approximately 60%) homology with CD59 protein (protectin). Both proteins have wide distribution, the unknown having been found in bile, cholesterol gallstones, and the wall of the aorta. However, the two proteins appear to be immunologically different.

The matrix of urinary tract stones: protein composition, antigenicity, and ultrastructure.

We have extracted proteins from urinary tract stones by electrodialysis and have developed antisera to the core and the shell of a renal stone. The protein composition varies between stones but is identical in the core and the shell of the same stone. One stone antigen is present in the urine of normal individuals and stone formers, as well as in cholesterol gallstone extracts. Electron microscopy of the core of a urate-calcium oxalate stone before and after demineralization reveals a fibrillar structure associated with mineral deposits, as well as aggregates of crystals.

Physical chemical studies of calcium oxalate crystallization.

The physical chemical approach to the investigation of the calcium oxalate (CaOx) crystallization and urolith formation is the systematic examination of the various aspects of mineral precipitation and growth in pure solution, in the presence of individual urinary components, and in whole urine media. Recent experimental studies have indicated that while small urinary ions such as citrate, magnesium, and phosphocitrate retard the mineralization rate of CaOx, urinary macromolecules may act either as inhibitors of growth or promoters of nucleation. Some CaOx mineralization inhibitors have also been found to influence the growth mechanism of the phase and its flocculation properties. Therefore, urinary macromolecules that are adsorbed on the mineralizing crystals and incorporated into the developing stone may play a significant role in urolithiasis.

The influence of urinary macromolecules on calcium oxalate monohydrate crystal growth.

The Constant Composition (CC) kinetics method has been used for studying the mineralization of calcium oxalate monohydrate (COM) at sustained supersaturations in the presence of pre-bladder urine and macromolecules isolated from normal urine and kidney and bladder stones. The method is especially sensitive for investigating the inhibitory activities of these urinary macromolecular components (UMMC) and matrix macromolecular components (MMMC) with a coefficient of variation in growth rate of approximately 2%. Significant COM mineral inhibition was observed in a wide molecular weight region of urine components. Urine removed directly from the kidney showed appreciable inhibitory activity towards COM crystallization. Normal urinary proteins and the dissolved precipitate resulting from urine centrifugation were fractionated by gel filtration. The resulting solutions were mostly COM mineralization inhibitors. Electrodialysis was utilized to isolate the MMMC (greater than 7000 d) of renal and bladder calculi. While these solutions inhibited COM crystallization, they were also found to be calcium binders as measured by the calcium electrode.

Pharmacokinetic interaction between amitriptyline and neuroleptics.

The influence of amitriptyline on the plasma level of various neuroleptics was studied in 25 chronic schizophrenic patients. The study lasted 20 weeks. Patients were kept first 4 weeks on their former neuroleptic medication, with amitriptyline added for 12 subsequent weeks, and withdrawn during the last 4 weeks when only the neuroleptic medication was continued unchanged. The plasma level of neuroleptics was assayed by gas-liquid chromatography, once weekly throughout the study. The amitriptyline plasma level was also evaluated once weekly during the 12 weeks of its administration. The mean neuroleptic plasma values for each 4-week period were pooled together in three groups: aliphatic, piperdine and piperazine phenothiazine derivatives. Amitriptyline provoked some increase of the plasma level of all phenothiazine derivatives. This augmentation was significant only transitorily, however. The putative mechanisms of this neuroleptic tricyclic antidepressant interaction are discussed.

The isolation and identification of the P-component of normal human plasma proteins.

A normal human plasma protein called the P-component, which has a reaction of identity with the pentagonal structure found in amyloid-laden organs, has been isolated and identified with a recently characterized protein, the 9.5S alpha(1)-glycoprotein.