Pioglitazone,a peroxisome proliferator–activated receptor γ agonist,reduces the progression of experimental osteoarthritis in guinea pigs

Objective

To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator–activated receptor γ (PPARγ) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP‐13) and interleukin‐1β (IL‐1β) in articular cartilage.

Methods

The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP‐13 and IL‐1β in OA cartilage chondrocytes were evaluated by immunohistochemistry.

Results

OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP‐13 and IL‐1β in cartilage from placebo‐treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP‐13 and IL‐1β in OA cartilage.

Conclusion

This is the first in vivo study demonstrating that a PPARγ agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP‐13 and IL‐1β, which are known to play an important role in the pathophysiology of OA lesions.

     

Correlation of morphologic and biochemical changes in the natural history of spontaneous osteoarthrosis in guinea pigs

Objective. To study how the concentrations of proteoglycans (PGs) and collagen change in various parts of tibial articular cartilage during aging, and to evaluate the development of spontaneous osteoarthrosis (OA) in guinea pigs. Methods. PGs were extracted from guinea pig cartilage samples using 4M guanidine hydrochloride, and the amount of hydroxyproline was determined in the extraction remainder. The molecular size and aggregation of PGs were analyzed by electrophoresis, and the glycosaminoglycan composition was assessed by highperformance liquid chromatography. Results. The PG concentration was proportional to the load distribution. However, when OA became histologically manifest, the PG concentration decreased by 50% (from a mean of 44 μg to 22 μg per mg fresh tissue) and the collagen level decreased by 40% (from a mean of 17 μg to 10 μg per mg fresh tissue), while the proportion of water increased by 13% (from a mean of 710 mg to 800 mg per mg fresh tissue). Conclusion. Unmineralized cartilage can, within physiologic load limits, respond to increased mechanical demands by increasing the PG and collagen concentrations. Beyond a certain limit, however, the cartilage can no longer compensate for further increases in stress, which results in cartilage degeneration and losses of matrix constituents. These losses seemed to appear earlier in the disease process than has been described in previous animal models of secondary OA.     

A calcitonin radioimmunoassay: in vivo evaluation of calcitonin secretion in the pig

A radioimmunoassay capable of measuring porcine calcitonin (CT) in unextracted porcine plasma has been developed. A guinea pig antiserum to partially purified porcine CT was highly specific for CT and was capable of detecting 0.025 ng/ml of porcine CT. The antiserum cross-reacted significantly with sheep CT and beef CT but not with monkey CT or rabbit CT. The mean fasting CT concentration in 25 samples from the internal jugular vein of four pigs was 0.93 ng/ml (range, 0.46–1.61 ng/ml). An infusion of calcium ($\text{10–15}\text{mg}\text{/}\text{kg}\text{30}\text{min}$) was followed by an increase in plasma CT 3–10 ng.ml above base line in “responsive” animals and a rise of 0.5–1 ng/ml in less sensitive pigs.     

Ouabain binding and inotropy in acute potassium depletion in guinea pigs

In hypokalaemia the incidence of cardiac toxicity with digitalis is increased, possibly through changes in the affinity or capacity of the digitalis receptor in the heart. Previous studies have reported an increased ouabain binding capacity or Na+-K+ ATPase activity after hypokalaemia in human erythrocytes and rabbit and guinea pig hearts and no change or decreases in rabbit or rat skeletal muscles with no changes in ouabain affinity. The present study determined (a) the effect of potassium on 3H-ouabain binding to normokalaemic guinea pig cardiac cell membranes, (b) the effect of acute hypokalaemia induced by a potassium deficient diet for 14-18 days in guinea pigs on 3H-ouabain binding to erythrocytes and cardiac and skeletal muscle homogenates, and (c) ouabain induced inotropy in isolated contracting guinea pig left atria, right ventricular papillary muscles, and soleus muscle strips from normokalaemic and hypokalaemic guinea pigs. 3H-ouabain binds to cardiac cell membranes in the presence of magnesium and inorganic phosphate with an affinity (KD value) of 1.13 X 10(-7) mol X litre-1. Potassium decreased this affinity without changing the binding capacity. Erythrocytes and heart muscle homogenates showed the same affinity as cardiac cell membranes, whereas soleus muscle homogenates had a higher affinity for ouabain (KD 5.1 X 10(-8) mol X litre-1). After hypokalaemia, the ouabain affinity did not change in any tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin-like immunoreactivity in mammalian thyroid glands: contents and partial characterization

Somatostatin (SRIF)-like immunoreactivity (SLI) in the thyroid glands of human and several animal species were compared, and the SLI peptides were characterized chromatographically and immunologically. All specimens were extracted with 2 M acetic acid, and the SLI content determined by RIA. The SLI concentrations in guinea pigs [34.3 +/- (SE) 4.8 ng/mg protein] and rabbits (9.4 +/- 0.8 ng/mg protein) were much greater than those in other mammals: dogs, rats, mice, and humans. On gel filtration of extracts of the guinea pig, rabbit and dog thyroids, the major peak of SLI (1.6 K SLI) coeluted with synthetic SRIF-14 (S-14). Two other forms of SLI ("big" SLI and 3 K SLI) were also detected, although their relative proportions to total SLI were small (2.3 to 8.2%). The 3 K SLI and 1.6 K SLI from guinea pig and rabbit thyroids contained peptides coeluting with synthetic SRIF-28 (S-28) and S-14, respectively, on reverse-phase high performance liquid chromatography. The dilution curves of the two molecular forms of SLI, i.e. 3 K SLI and 1.6 K SLI, were parallel to the displacement curves of S-28 and S-14 in the SRIF RIA. It is concluded 1) that the thyroid contents of SLI varied greatly from species to species, with the highest content being found in guinea pig thyroids; 2) that in guinea pigs, rabbits, and dogs, the predominant form of thyroid SLI is 1.6 K SLI; and 3) that the 3 K SLI and 1.6 K SLI peptides from guinea pig and rabbit thyroids are immunologically and chromatographically indistinguishable from S-28 and S-14, respectively.     

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

OBJECTIVE: A dramatic loss of aggrecan proteoglycan from cartilage is associated with osteoarthritis. The fate of residual G1 domains of aggrecan is unknown, but inefficient turnover of these domains may impede subsequent repair and retention of newly synthesized aggrecan. Thus, the objective of this study was to determine whether ITEGE- and DIPEN-containing G1 domains, generated in situ, are internalized by articular chondrocytes, and whether these events are dependent on hyaluronan (HA) and its receptor, CD44. METHODS: ITEGE and DIPEN neoepitopes were detected by immunofluorescence staining of bovine articular cartilage chondrocytes treated with or without interleukin-1alpha (IL-1alpha). Additionally, purified ITEGE- or DIPEN-containing G1 domains were aggregated with HA and then added to articular chondrocytes, articular chondrocytes transfected with CD44delta67, or COS-7 cells transfected with or without full-length CD44. Internalized epitopes were distinguished by their resistance to extensive trypsinization of the cell surface. RESULTS: Both ITEGE and DIPEN were visualized within the extracellular cell-associated matrix of chondrocytes as well as within intracellular vesicles. Following trypsinization, the intracellular accumulation of both epitopes was clearly visible. IL-1 treatment increased extracellular as well as intracellular ITEGE epitope accumulation. Once internalized, the ITEGE neoepitope became localized within the nucleus and displayed little colocalization with HA, DIPEN, or other G1 domain epitopes. The internalization of both ITEGE and DIPEN G1 domains was dependent on the presence of HA and CD44. CONCLUSION: One important mechanism for the elimination of residual G1 domains following extracellular degradation of aggrecan is CD44-mediated co-internalization with HA.     

Phosphocitrate blocks calcification‐induced articular joint degeneration in a guinea pig model

Objective

Calcium deposition occurs frequently in osteoarthritic (OA) joints. However, evidence for a causal role of calcification in cartilage degeneration is inferential. The present study was undertaken to examine the role of calcification in OA disease progression and to evaluate a formulation of phosphocitrate (PC) as a potential therapeutic agent.

Methods

We have identified a guinea pig OA model in which meniscal calcification appears to correlate with aging and disease progression. We synthesized a new formulation of PC, [CaNa(PC)₂(H₂O)]_n (CaNaPC), which is a potent antimineralization agent and a specific inhibitor of crystal‐induced biologic effects. After weekly treatment of guinea pigs with experimental OA with CaNaPC for 3 months, we examined calcification in menisci and cartilage degeneration. As a control, we examined whether similar CaNaPC treatment had any therapeutic effect in a hemi‐meniscectomy model in which there is no known crystal involvement.

Results

Meniscal calcification correlated with cartilage degeneration in this animal model. PC treatment led to significant reduction of calcium deposits and arrested OA disease progression. Similar treatment had no effect in the hemi‐meniscectomy model.

Conclusion

CaNaPC diminishes mineralization in a cutaneous calcergy model and a model of OA in which intraarticular mineralization is a prominent feature. In the OA guinea pig model, inhibition of calcification is accompanied by diminished cartilage degeneration. CaNaPC has no therapeutic effect in the hemi‐meniscectomy model. We conclude that pathologic calcification may initiate or amplify processes leading to cartilage degeneration and that CaNaPC may interrupt such a pathway.

     

Evidence for the Presence of M Cells in the Guinea Pig Ventricle

M Cells in the Guinea Pig. Introduction: Recent studies have described the presence of M cells in the deep layers of the canine and human ventricle displaying electrophysiologic and pharmacologic features different from those of epicardial (EPI) and endocardial (ENDO) cells. The M cell is distinguished electrophysiologically by the ability of its action potential to prolong disproportionately to that of other myocardial cells with slowing of the stimulation rate and pharmacologically by its unique sensitivity to Class III antiarrhythmic agents. The present study was designed to test the hypothesis that similar cells are present in the guinea pig ventricle. Methods and Results: We used a dermatome to obtain thin strips of left ventricular free wall from the hearts of guinea pigs (8 to 14 weeks old) and standard microelectrode techniques to record transmembrane activity. Action potential duration measured at 90% repolarization (APD₉₀) was significantly longer in mid-myocardial (MID) cells than in surface EPI or ENDO cells at all basic cycle lengths (BCLs) tested. At a BCL of 300 msec, APD₉₀ was 102 ± 21, 136 ± 9, and 95 ± 15 msec in EPI, MID, and ENDO cells (mean ± SD; n = 12). At a BCL of 5000 msec, APD₉₀ was 133 ± 14, 185 ± 24, and 135 ± 13 msec in EPI, MID, and ENDO cells ([K⁺]₀= 4 mM). Thus, APD-rate relations were more pronounced in the MID cells. MID cells were also more sensitive to agents with Class III actions (e.g., d,I-sotalol: 10 to 100 μM), exhibiting a greater APD prolongation than EPI or ENDO. d,I-Sotalol also induced early afterdepolarizations in MID cells hut not in EPI or ENDO cells. The rate of rise of the action potential upstroke (V_max) was significantly greater in MID cells: 129 ± 13, 240 ± 42, and 192 ± 28 V/sec in EPI, MID, and ENDO cells (n = 10 to 18). Conclusion: Our results demonstrate the existence of important transmural electrical heterogeneity in guinea pig ventricular myocardium. The study provides data in support of the existence of M cells in the mid-myocardial layers of the guinea pig ventricle exhibiting longer APDs and a greater sensitivity to agents with Class III antiarrhythmic action.     

Diverse inotropic effects of 5-hydroxytryptamine in heart muscles of various mammalian species

Summary The inotropic effects of 5-hydroxytryptamine (5-HT) on mammalian heart muscles were investigated. 5-HT (10^–8–10^–3M) produced increases in the contractile tension of atrial and ventricular muscles isolated from guinea pigs, Japanese monkeys, and humans, but not in rat heart preparations. The maximum percent increase of contraction was largest in guinea pig ventricular muscles (142.0%), followed by monkey atrium (86.3%), human atrium (71.7%), guinea pig atrium (48.7%), and monkey ventricle (30.1%). The sensitivity to 5-HT, measured as the negative logarithm of the half-maximal inotropic molar contractions of 5-HT, i.e., –logEC₅₀, was highest in the human atrium (6.65 ± 0.20), followed by guinea pig atrium (5.53 ± 0.36), monkey ventricle (4.83 ± 0.28), guinea pig ventricle (4.56 ± 0.11), and monkey atrium (4.46 ± 0.16). The inotropic effects of 5-HT seen in the atrial and ventricular muscles of guinea pigs were abolished in the presence of the -receptor blocker, pindolol (8µM), while these effects in human atrial muscles and monkey atrial and ventricular muscles were abolished only in the presence of both pindolol (8µM) and of prazosin (1µM), an ₁-receptor blocker. 5-HT increased the V_max of the slow response recorded from guinea pig ventricular muscles exposed to high K⁺ (27mM) media, whereas this agent did not alter the calcium current of isolated guinea pig ventricular myocytes devoid of sympathetic nerve terminals. In reserpinized guinea pig hearts, 5-HT exerted no inotropic effect on ventricular muscle, yet it had an inotropic effect in the atrial muscle, although the latter effect was considerably depressed, compared to that seen in non-reserpinized atrial muscles. We conclude that the positive inotropic effects of 5-HT observed in the ventricular muscle of the guinea pig and in the atrial and ventricular muscles of the Japanese monkey can be attributed to the release of noradrenaline from sympathetic nerve terminals (indirect effect). In contrast, in human atrial muscles, the positive inotropic effect of 5-HT was apparently the result of stimulation of a specific membrane receptor for 5-HT (direct effect). In guinea pig atrial muscles, both direct and indirect effects of 5-HT were involved in the positive inotropism. An explanation for the lack of sensitivity of rat atrial and ventricular muscles to 5-HT awaits further studies.     

Cellular and humoral immune responses to mycobacterial stress proteins in experimental pulmonary tuberculosis

Objective: Immunity to mycobacterial stress protein antigens was studied in response to vaccination and/or virulent infection.Design: Guinea pigs, either vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), infected by the pulmonary route with virulent M. tuberculosis, or vaccinated then infected, were studied for the development of cellular and humoral immunity to two recombinant mycobacterial stress proteins, hsp 65 and hsp 70.Results: Recombinant hsp 70 stimulated good proliferation in blood lymphocytes and, to a lesser extent, spleen and bronchotracheal lymph node lymphocytes from BCG-vaccinated guinea pigs. The proliferative responses to hsp 70 were diminished in both the spleen and lymph node cells following subsequent pulmonary challenge alone, but were boosted significantly by prior vaccination. Recombinant hsp 65 was much less active at inducing the proliferation of spleen and lymph node cells, with lowest responses observed in blood lymphocytes occurring in the cells from BCG-vaccinated, aerosol-challenged guinea pigs. Using a semi-quantitative dot blot procedure, serum antibodies to both hsp 65 and hsp 70 developed gradually following BCG vaccination, with all guinea pigs studied exhibiting significant seroreactivity after 15 weeks post-vaccination. In guinea pigs exposed to virulent M. tuberculosis by aerosol, serologic reactivity to hsp 70 was consistently stronger 6 weeks post-challenge in both vaccinated and non-vaccinated guinea pigs. In fact, 6 weeks following pulmonary exposure to M. tuberculosis in previously naive guinea pigs, 3 out of 6 animals had no detectable serum antibodies to hsp 65. Somewhat surprisingly, antibody levels to both hsp 65 and hsp 70 were only slightly increased by prior BCG vaccination in guinea pigs exposed to virulent M. tuberculosis by the respiratory route.Conclusion: These results demonstrate that both hsp 65 and hsp 70 stimulate detectable humoral and cell-mediated immunity in guinea pigs vaccinated and/or infected under highly relevant conditions. There is little evidence that vaccination with BCG primes the guinea pig to make an anamnestic response to hsp 65 following virulent pulmonary challenge. The precise contribution of immunity to mycobacterial stress proteins to the pathogenesis of tuberculosis in this model remains to be elucidated.     

Aerosolized Polymerized Type I Collagen Reduces Airway Inflammation and Remodelling in a Guinea Pig Model of Allergic Asthma

Collagen-polyvinylpyrrolidone (Collagen-PVP) has been demonstrated to elicit immunomodulatory properties in different chronic inflammatory diseases. Nevertheless, its effects on asthma are still unknown. We have evaluated whether collagen-PVP could modulate airway inflammation and remodelling in a guinea pig model of allergic asthma. Sensitized guinea pigs were challenged with the allergen (ovalbumin) six times (at 10-day intervals). From the third challenge on, animals were treated every 5 days with saline aerosols containing 0.16, 0.33, or 0.66 mg/ml of collagen-PVP (n = 5, respectively). Some guinea pigs, sensitized and challenged with saline as well as treated with 0 or 0.66 mg/ml collagen-PVP, were included in the study as control (n = 7) and sham groups (n = 5), respectively. From the first challenge on, ovalbumin induced a transient airway obstruction, measured by barometric plethysmography, which was not modified by collagen-PVP treatments. After the last allergen challenge, guinea pigs were anesthetized to obtain bronchoalveolar lavage (BAL) and the left lung caudal lobe. As expected, BAL cell count from allergen-challenged guinea pigs showed abundant neutrophils and eosinophils, as well as numerous tumor necrosis factor (TNF)-α-expressing granulocytes and macrophages in airway wall (determined by immunohistochemical assay). Neutrophilia and TNF-α-expressing leukocytes, from collagen-PVP treated animals, diminished from 0.16 mg/ml, and eosinophilia from 0.66 mg/ml of collagen-PVP doses. Histological changes induced by allergen challenges include thickening of connective tissue below airway epithelium and vascular wall widening of airway adjacent vessels; these changes were reduced by collagen-PVP treatment. Collagen-PVP seems to have anti-inflammatory and antifibrotic properties in this guinea pig asthma model.     

Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, reduces the progression of experimental osteoarthritis in guinea pigs

OBJECTIVE: To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP-13) and interleukin-1beta (IL-1beta) in articular cartilage. METHODS: The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP-13 and IL-1beta in OA cartilage chondrocytes were evaluated by immunohistochemistry. RESULTS: OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP-13 and IL-1beta in cartilage from placebo-treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP-13 and IL-1beta in OA cartilage. CONCLUSION: This is the first in vivo study demonstrating that a PPARgamma agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP-13 and IL-1beta, which are known to play an important role in the pathophysiology of OA lesions.     

Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles

Objective. Transglutaminases (TGases) (E.G. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor β (TGFß), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined. Methods. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3–5 years old) and young (2–6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds. Results. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean ± SD activity level in the membrane fraction of lysed chondrocytes was 6.0 ± 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean ± SD TGase activity level was 1.23 ± 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFß to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFß effect. Conclusion. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage.     

Effects of growth factors and cytokines on proteoglycan and collagen synthesis by chondrocytes in guinea pigs with spontaneous osteoarthritis

We examined the effects of various growth factors and cytokines on proteoglycan (PG) and collagen synthesis by chondrocytes isolated from osteoarthritic and normal articular cartilage of Hartley strain guinea pigs. The guinea pig represents a useful animal model of spontaneous osteoarthritis (OA). Cartilage tissue samples were obtained from the knee joints of under-3-month-old guinea pigs (control group) as well as 5- to 8-month-old guinea pigs with OA changes (OA group). Chondrocytes were isolated enzymatically and maintained in suspension culture. Growth factor addition groups were then prepared from both the OA group and the control group, using the factors TGF-β, bFGF, and IGF-1 (1.25 ng/ml each). Cytokine addition groups were also prepared using IL-1α and IL-1β (10 ng/ml each). An addition group was also prepared for sodium hyaluronate (HA) (500 μg/ml). In each group, ³⁵S was added as a PG metabolic marker, ³H-proline was added as a collagen metabolic marker, and the groups were cultured. Next, ³⁵S and ³H-proline uptake was measured by a liquid scintillation counter. The results revealed that (1) both PG synthesis and collagen synthesis were promoted significantly more in OA chondrocytes than in normal chondrocytes; (2) with the addition of growth factors, PG and collagen synthesis was enhanced in OA chondrocytes; and (3) PG synthesis and collagen synthesis were inhibited in both normal and OA chondrocytes with the addition of IL-1α and -β. This result suggests that the repair function is activated more in OA chondrocytes than in normal chondrocytes, thereby promoting the synthesis of the cartilage matrix by chondrocytes. This synthesizing capability is enhanced and acts to effectively repair degenerative articular cartilage further through the addition of growth factors. Received September 20, 1999 / Accepted December 6, 1999     

Characteristics of anti-type ii collagen antibody binding to articular cartilage

Objective. Previous studies suggested that it was possible to characterize the intact surface of articular cartilage by probing it with antibodies against matrix macromolecules. The present studies were undertaken to investigate type II collagen (CII) epitope availability on the intact surface of articular cartilage. Methods. Normal bovine, rabbit, and human cartilage specimens were used to measure binding of anti-CII antibodies to the articular and cut surfaces of cartilage. Antisera were raised against the material obtained after brief extraction of the cartilage surface with 4M guanidine solution. Results. Anti-CII did not bind to the intact surface of rabbit articular cartilage when compared with control rat sera, but did bind significantly to the cut surface. With normal human articular cartilage, the cut surfaces bound approximately 4 times as much anti-CII antibody as the intact articular surfaces. These findings suggested that the CII epitopes are normally protected by the superficial cartilage layer. In experiments carried out to characterize this layer, binding of anti-CII antibodies was unchanged after exhaustive washing of bovine or rabbit cartilage with phosphate buffered saline or 1M NaCl solution, whereas it was significantly increased after brief exposure to 4M guanidine solution or after incubation with neutrophil elastase. No restoration of the protection of CII epitopes in guanidine-treated cartilage could be achieved by incubation with undiluted normal bovine synovial fluid; however, 8-day culture of elastase-treated cartilage explants resulted in partial restoration of protection of the CII epitopes. Rat and rabbit antisera raised against the cartilage surface material extracted with 4M guanidine contained antibodies that bound to the cartilage surface. By Western blotting, rat antibodies identified 50–65-kd protein bands present in the guanidine extract, but not present either in the material obtained from brief digestion of cartilage with neutrophil elastase or in synovial fluid. The rabbit antisera identified a broad 70–95-kd band. Exposure of elastase-treated cartilage to the guanidine-extracted material resulted in a partial decrease of anti-CII antibody binding. Conclusion. These results suggest that CII on intact cartilage is protected from antibody binding, and that the protective material is at least partly proteinaceous in nature, is unlikely to be derived from synovial fluid, is noncovalently bound to the underlying intercellular matrix, and is synthesized by resident chondrocytes. Further characterization of the protective material may provide important information on the mechanisms of early cartilage damage in inflammatory arthritis.     

Dark neurons in the ageing cerebellum: their mode of formation and effect of Maharishi Amrit Kalash

Dark neurons are considered a manifestation of neuronal injury and although they cover various grades of damage their mode of formation is not yet clear. Age-dependent alterations in a dark purkinje neuronal population of guinea pigs (10 months and 32 months old) and rats (3 months, 6 months, 12 months, 15 months and 28 months) were studied. Light microscopical and electron microscopical observations revealed a significant increase (P < 0.05) in the number of dark purkinje neurons with age in both the guinea pigs and rats. Extraction of lipids from the cerebellum sections before processing for his to chemical reaction resulted in a reduction of the dark neuronal population. In another set of experiments, significant age-dependent increase in the cathepsin-D activity and lipid peroxidation was documented in the guinea pig cerebellum. Treatment of guinea pigs with Maharishi Amrit Kalash (MAK) (500 mg/kg body wt/day, for two months) significantly inhibited (P < 0.05) the activity of cathepsin-D and lipid peroxidation, and decreased the number of dark neurons. These findings suggest that the number of dark neurons increases with age and MAK prevents the conversion of light to dark purkinje neurons due to its inhibitory effects on cathepsin-D activity and antioxidant properties. We suggest that the conformational changes in the normal protein structure due to higher proteolytic activity and peroxidation of lipid in the aging cerebellum endangers are dundant capability for various staining agents and the Osimic acid molecules to react with proteins, lipids and other molecules, leading to an intensified cyto- and karyoplasms electron density. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of antiinflammatory drugs on arthritic cartilage: A high‐frequency quantitative ultrasound study in rats

Objective

To evaluate the ability of 55‐MHz quantitative ultrasound (US) to detect the in vivo effects of experimental arthritis, as well as those of two antiinflammatory drugs, naproxen (NPX) and dexamethasone (DEX), on cartilage and subchondral bone.

Methods

Arthritis was induced in both knees of 108 rats by intraarticular injection of zymosan (ZYM). Two groups of arthritic rats (n = 36 per group) were treated daily with either NPX (10 mg/kg/day) or DEX (0.1 mg/kg/day). Using a 3‐dimensional US microscope, patellae were explored in vitro on days 5, 14, and 21 after injections. US assessment included the analysis of quantitative indices of local modifications involving cartilage and bone: integrated reflection coefficient (IRC) from the cartilage surface and apparent integrated backscatter from the cartilage internal structure (cartilage matrix) (AIB_cartilage) and the cartilage–bone interface (AIB_bone).

Results

ZYM induced articular surface fibrillation that resulted in a decrease in IRC at all times (P < 0.02) and in an increase in AIB_bone on days 5 and 14 (P < 0.005). Fibrillation was not changed by NPX administration, while it disappeared following DEX treatment. Cartilage–bone interface alterations were prevented by DEX and partially compensated for by NPX. Cartilage matrix echogenicity decreased with time in all groups due to maturation (P < 0.05), except in DEX‐treated rats.

Conclusion

Quantitative 55 MHz US allowed detection of early cartilage and bone lesions due to experimental arthritis, and also allowed detection of the effects of antiinflammatory drugs. NPX seemed to have an effect on subchondral bone lesions, but not on cartilage. DEX appeared to repair articular surface and bone, but prevented animal growth and cartilage maturation.

     

Pharmacological Study of Oyster Mushroom (<Emphasis Type="Italic">Pleurotus Ostreatus</Emphasis>) Extract on Isolated Guinea Pig Trachea Smooth Muscle

Mushroom farm workers suffer from respiratory symptoms during the farming of mushrooms. The objective of this study was to analyze the effects of oyster mushroom (Pleurotus ostreatus) extract (OME) on isolated guinea pig tracheal smooth muscle in vitro. Isolated guinea pig tracheal tissue from 27 nonsensitized guinea pigs were studied. The OME was obtained from indoor mushroom growing fields and prepared as a 1:10 w/v aqueous solution. Dose-related contractions of nonsensitized guinea pig trachea were demonstrated using these extracts. The OME contained significant quantities of bacterial components (eg., endotoxin: 43,072.92 EU/mg). Parallel, pharmacological studies were performed by pre-treating the tissues with mediator-modifying agents including atropine, indomethacin, pyrilamine, BPB, acivicin, NDGA, captopril, TMB8 and capsaicin. Atropine consistently and strikingly reduced the contractile effects of this extract. These observations suggest an interaction of the OME with parasympathetic nerves or more directly with muscarinic receptors. Pretreatment with TMB8 (inhibitor of intracellular calcium mobilization) also significantly blocked the constrictor effect of OME, indicating a role of calcium mobilization in the constricting effect of OME. Inhibition of contraction by blocking of other mediators was less effective and varied depending on the drug. We conclude that OME causes a dose-related airway smooth muscle constriction by nonimmunological mechanisms involving a variety of airway mediators and possibly cholinergic receptors. This effect is not dependent on pre-sensitization of the guinea pigs.     

Early stages of gallstone formation in guinea pig are associated with decreased biliary sensitivity to cholecystokinin

The purpose of this study was to measure differences in gallbladder sensitivity to cholecystokinin (CCK)in vivo during the early stages of gallstone formation and to correlate these findings to gallbladder CCK receptors. Guinea pigs were placed on either a normal diet or a two-week cholelithogenic diet, after which gallbladder emptying pressure to exogenously administered CCK was measuredin vivo, according to the presence or absence of gallstones. At all doses of CCK tested (except 10^–10 mol/kg), the gallbladder response to CCK of guinea pigs that did not develop gallstones (on the cholelithogenic diet) was more sensitive than that of guinea pigs that did develop gallstones. Neither group was different from guinea pigs on a normal diet. In a second experiment, CCK receptors were measured on gallbladder muscularis from guinea pigs after two weeks on the same diet as in the first experiment. Those guinea pigs that did not develop gallstones had greater concentrations of CCK receptors (149±9 fmol/mg protein) than those that did develop gallstones (70±23 fmol/mg protein). Neither group was different from normal diet guinea pigs (119±57 fmol/mg protein). At the time point measured, there were no differences in the lipid chemistry, or protein concentrations of gallbladder bile between the guinea pigs on the cholelithogenic diet that did or did not develop gallstones, or those on normal guinea pig chow. We conclude that the early stages of gallstone formation in guinea pigs are associated with decreased gallbladder sensitivity to CCK and that this change may be due to a lower concentration of CCK receptors on the gallbladder smooth muscle.Supported by the Wellcome Foundation, Ethicon Foundation, and British Digestive FoundationSupported by grants from the National Institutes of Health (5R37 DK 15241-19, P01 DK 35608, and CA 38657)