Tissue levels of histamine, PAF-acether and lysopaf-acether in carrageenan-induced granuloma in rats.

The tissue concentrations of several inflammatory mediators were determined from day 0 to day 60 in granuloma induced in rats (n = 105) by injection of carrageenan in the fascia of the latissimus dorsi muscle. Noncollagen proteins (NCP) and the number of polymorphonuclear leukocytes (PMN) and mast cells were also assessed. In comparison with the tissue at time 0, we noted in the inflamed tissue (at 4 h) an increase in total proteins (4.0 +/- 3.0 vs. 84 +/- 12.0%, mean +/- SEM) and PMN (0.0 +/- 0.0 vs. 43.3 +/- 13.4%), and a fall in histamine concentration (from 30.0 +/- 9.0 to 9.0 +/- 4.0 ng/ml). A partial disappearance of mast cells and an increase of PAF-acether (PAF) levels (1.0 +/- 1.0 vs. 30.0 +/- 22.0 ng/ml) was noted at 16 h, whereas lysopaf remained unchanged (3.7 +/- 4.0 vs. 3.5 +/- 1.0 ng/ml). During evolution towards chronic inflammation (day 10-60), NCP decreased, PMN disappeared and mast cells reappeared; the histamine level rose to 11.0 +/- 2.0 mg/ml, thus not reaching back baseline values. Lysopaf rose to 7.1 +/- 12.2 ng/ml and PAF levels increased further to reach 240.0 +/- 153.0 ng/ml at day 10. This study suggests that PAF may contribute to the acute phase of an inflammatory state such as the carrageenan-induced granuloma and that it is also present during the chronic process.

Effects of long-term treatment with D-penicillamine on antigen-induced arthritis in rabbits: studies on in vivo articular chondrocyte damage and in vitro collagen biosynthesis by cultured chondrocytes.

The effects of a long-term (120 days) treatment with D-penicillamine (DP) (50 mg/kg/day; i.v.) on antigen-induced arthritis were studied in rabbit. They were investigated by the terminal histological examination of the joints of different groups of rabbits (unimmunized treated or untreated, immunized treated or untreated) and the study of collagen and non-collagen protein biosynthesis by cultured chondrocytes obtained from articular cartilage of the same groups of animals. Treatment with D-penicillamine diminished the intensity of the erosions of cartilage and subchondral bone, the severity of the inflammatory synovitis, and the loss of chondrocyte clusters found in cartilage sections. In cultures of chondrocytes obtained from immunized treated rabbits, a partial or complete inhibition of the decreased biosynthesis of collagen and non-collagen proteins seen in culture of chondrocytes obtained from immunized untreated animals was observed. These results show that DP could be effective in preventing damage of chondrocytes and inhibition of collagen biosynthesis in them, phenomena important in cartilage destruction induced by a chronic immunological inflammation.

The induction of human antinuclear antibodies by D-penicillamine: activation of inducer helper T cells in the absence of irradiation sensitive suppressor T cells.

The capacity of D-Penicillamine (DP) to induce or to potentiate the production of antinuclear antibodies (ANA), detected by immunofluorescence (IF), was investigated in vitro, using peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE) and normal individuals. Except in one patient with SLE, DP did not enhance ANA synthesis when using unseparated PBMC. In contrast, when B cells were cocultured with irradiated T cells or irradiated enriched T4+ subset, DP induced or potentiated the production of ANA. These results indicate that DP acts by stimulating T4+ helper cells to promote ANA synthesis in the absence of radio-sensitive suppressor T cell function contained within the T4+ population.

D-penicillamine: a modulator of anti-DNA antibodies production.

The effect of D-Penicillamine (DP) on the in vitro production of anti-DNA antibodies by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE) and from healthy individuals was studied. Anti-DNA antibodies were measured in culture supernatants using a sensitive microenzyme-linked immunoassay technique. The results of this investigation suggest that DP can act as an immunomodulator capable of potentiating or initiating anti-DNA antibodies synthesis as well as suppressing it. Although PBMC from both SLE patients and controls were responsive to this thiol compound, our results indicate that PBMC from patients with SLE were more susceptible to the enhancing effect of DP than did PBMC from controls. The cellular mechanism by which this drug can modulate anti-DNA antibodies production is discussed.

A study of microvasculature in normal and inflammatory synovial membranes in the rabbit using light and electron microscopy and freeze fracture.

The Authors made a study of microvasculature in normal and inflammatory synovial membranes in the rabbit, by light and electron microscopy and with the use of freeze fracture. The synovial membrane of the knee was studied in 15 normal rabbits and in 22 rabbits in whom immunization with mycobacterium tuberculosis crude cytoplasmic water soluble extract had provoked inflammatory synovitis. A comparison between normal and inflammatory synovia by light microscopy showed, in inflammatory synovitis, a significant increase in the number of congested and obliterated microvessels (p less than 0.001) and in the number of endothelial nuclei per microvessel (p less than 0.0001). By electron microscopy no specific alteration of synovial microvessels was observed. A significant increase was found in the number of venules in inflammatory synovitis (p less than 0.01). The number of simple interendothelial tight junctions, defined by 1 or 2 interendothelial contacts, and of complex interendothelial tight junctions, defined by 3 or more interendothelial contacts, was similar in venules and in capillaries of inflammatory synovitis and of normal controls. The mean number and mean spacing of junctional strands in interendothelial tight junctions was similar in the microvessels of inflammatory synovitis and of normal controls. The Authors conclude that inflammation does not provoke specific alterations in synovial microvasculature but could induce an adaptative state resulting in increased synovial permeability.

[Xanthine oxidase activity: NAD+-dependent and O2-dependent forms in carrageenan granuloma in the rat]. / Activité de la xanthine oxydase: formes nad+-dépendante et o2-dépendante dans le granulome à la carragénine chez le rat.

Xanthine oxidase activity: NAD+-dependent form (D) and O2-dependent form (O) were carried out in cytosol supernatants of connective tissue growth (T.C.N.F.), skin tail, liver and plasma of carrageenan induced granuloma in the Rat. The specific activities of skin, liver and plasma were normal in animals with a granuloma. The total specific activity (D + O): 7.53 +/- 0.98 mU/mg protein, and the percentage of form O: 51.6 +/- 5.1 of the granulomatous tissue as compared to the tail are significantly increased. These results suggest the likely function of xanthine oxidase during the inflammatory response.

[Xanthine oxidase activity: O2-dependent and NAD+-dependent forms in the liver, in rats with adjuvant arthritis and hepatitis]. / Activité de la xanthine oxydase: formes O2-dépendante et NAD+-dépendante du foie, chez le rat atteint d'arthrite et d'hépatite à adjuvant.

Xanthine oxidase activity: O2-dependent and NAD+-dependent forms, were carried out in cytosol supernatant of Rat liver homogenat with adjuvant and hepatocytes induced arthritis and hepatitis. Both forms were increased without modification of their ratio. These results suggest that xanthine oxidase was implicated in the inflammatory reaction.

Comparison of effects of D. Penicillamine and dexamethasone on the articular and non articular lesions in immune connective tissue disease induced in rabbits.

The effect of D. Penicillamine (DP) at the dose of 50 mg/kg/day, on an immune induced connective tissue disease in rabbit, is compared to that of dexamethasone (Dexa) at the doses of 0.15 and 0.075 mg/kg/day. This model includes polyarthritis and lesions of connective tissue of liver, kidneys and lungs. The result of immunization is initially a non-specific macrophage infiltration and secondarily a specific lymphocyte and plasma-cell infiltration. In short treatment, high dose of Dexa inhibits the non-specific and specific responses while DP modifies only non specific response. In long treatment, Dexa at low dose and DP inhibit the two responses. Data suggest that, in vivo, macrophages is the target cell of DP.

[N1-substituted 1H-indazole-3-ethyl carboxylates and 1H-indazole-3-hydroxamic acids]. / 1H-Indazole carboxylates d'éthyle-3 N1-substitués et acides 1H-indazole hydroxamiques-3.

Sixty five new derivatives of ethyl-1H-indazole-3-carboxylate are described; they contain in N1 various aliphatic or aromatic acyl radicals. Moreover halogens or methyl groups are present as substituents at the 5 position or methyl groups at 5,6. The synthesis of seven 1H-indazole-3-hydroxamic acids, substituted at 6 and/or 5 as above, is also described. Some of the synthesized derivatives have preliminarily been tested on rats to investigate acute toxicity, possible antiarthritic effects on primary or secondary arthritis, and their action on weight gain. Some of these indazole derivatives had an antiarthritic effect at doses much lower than the toxic ones; among the compounds tested up to now, the ethyl-5-methyl-N1-p-chlorobenzoyl-1H-indazole-3-carboxylate gave the best results. Weight gain as not affected by any of the examined compounds.

[Chemical reactivity and biological properties of a series of aminochlorambucil derivatives]. / Réactivité chimique et propriétés biologiques dans la série de l'aminochlorambucil.

The substitution of aminochlorambucil by a methyl group increased the chemical reactivity in IV b (n = 1) and IV b (n = 2) but their cytotoxicity remained low. The immunosuppressive effect (adjuvant arthritis in Rats and tuberculin related skin reaction) was observed with IV b (n = 2). Aminochlorambucil was effective on adjuvant arthritis only and IV b (n = 1) had no activity. Aminochlorambucil, IV b (n = 1) and IV b (n = 2) were devoid of any non-specific anti-inflammatory activity.

A new experimental model of systemic connective tissue disease in immunized rabbit.

In rabbit, the immumizations with whole Mycobacterium Tuberculosis injected intradermally and its crude cytoplasmic W.S.E. injected intraarticulary induce not only a self-perpetuating synovitis in the stimulated knee but also chronic systemic lesions of connective tissue (non-stimulated knee, both shoulders, aortic artery adventitial valvular endocardium, liver, kidneys and lungs). W.S.E. stimulates precipitating antibody synthesis, skin reaction and lymphoblastic transformation of lymphocytes in vitro. Furthermore, rabbits develop tuberculin skin test and lymphocytic response to P.P.D. The intensity of inflammation in stimulated knee is in direct ratio to intensities of humoral and cellular immunological responses. On the contrary, the incidence of systemic lesions is in inverse ratio to immune responses and intensity of inflammation in stimulated knee.

The failure of long term effect of cyclophosphamide on systemic immunologic connective tissue disease in rabbit.

Cyclophosphamide is given intravenously in the high dose of 12 mg/kg/day during 2 months from the beginning of immunological systemic connective tissue disease in rabbits, according to the modified Glynn's model. The effects of this alkylating drug are studied after the termination of treatment. At short term (15 days), cyclophosphamide depresses the inflammation of synovia in stimulated and non-stimulated joints, the humoral and cellular immunities, but the drug does not modify the other systemic lesions. At long term (between 1 and 6 months), these effects of cyclophosphamide disappear completely.

Immunofluorescence of synovial membrane multifactorial analysis of the results.

Synovial membrane taken by needle biopsy from the knee joint of 61 patients with various rheumatic diseases were studied using immunofluorescence methods. Staining techniques and their controls were detailed. Classical statistical tests and principal components multifactorial analysis of the data emphasized some differences between the pathological groups. Connective tissue diseases seemed to be characterized by plasma cells fluorescence and mixed immunoglobulins and complement deposits. These were mostly localized to extracellular spaces in sero-positive rheumatoid arthritis and to blood vessels in sero-negative rheumatoid arthritis and systemic lupus erythematosus. On the contrary, isolated immunoglobulins without complement were mostly found in the other inflammatory arthritis, while negative results were obtained in non inflammatory arthropathy. Immunoglobulin classes did not seem to have any diagnostic value. On the contrary, rheumatoid factor was specific for rheumatoid arthritis, whatever the serological pattern was, and it was particularly frequent in patients suffering from rheumatoid arthritis associated with a Sj5AOGREN SYNDROME. A strict relationship between classical histological findings and immunofluorescence results was not always found; so, immunological methods can be aquivocal.

Study of the mode of cyclophosphamide action on the rabbit arthritis according to Glynn's model.

A knee joint arthritis is induced in rabbits previously immunized by an intradermal injection of emulsified egg albumin (E.A.) a complete adjuvant, then by an intra-articular injection of E.A. alone. Histologically, the synovium shows at first an Arthus phenomenon and later an immune cellular reaction. Several immunological parameters are studied in these rabbits. Cyclophosphamide (CY), injected intravenously, in a high dose, during a short period from the day of the first immunization, inhibits in the same time the arthritis index, E.A. skin test, humoral and synovial anti-E.A. antibodies and lymphocyte response to P.H.A. Cy, injected I.V., in a low dose, for a long period from the day of the intra-articular immunization, decreases only the arthritis index, but does not modify the various immune reactions to E.A. These results suggest that, according to the timing and the dose, CY inhibits synovial inflammation either by a strict anti-inflammatory action, or by an immunosuppressive effect on the initial phase of the immune reaction inducing subsequently a synovial inflammation.

Physiopathological aspect of Glynn's type of experimental arthritis.

Knee joint arthritis is induced among rabbits previously immunized by intradermal injection with egg albumin (EA) emulsified in adjuvant containing either M. tuberculosis or M. butyricum, then by intra-articular injection with EA. Arthritis evolution involves two phases, an early one during the first 2 months and a late one from 3 months to 1 year. During the early phase, arthritis intensities are similar no matter which Mycobacterium is used. However, during the late phase, only rabbits immunized with M. tuberculosis develop self-perpetuating arthritis. Among more than 50% of arthritic rabbits, immunological lesions of aortic artery and cardiac valvules are found. Among the rabbits immunized with M. tuberculosis, the humoral anti-EA antibody level remains constant during the whole arthritis evolution; but, among the rabbits immunized with M. butyricum, the arthritis intensity decreases from 3 months of evolution. The correlation between arthritis index (AI) and humoral antibody level is only significant among the rabbits with early arthritis. The intradermally immunized rabbits show a positive skin test with EA and tuberculin. The more intense the cutaneous reactions, the greater the chances of developing self-perpetuating arthritis after the EA intra-articular injection. The fluorescent anit-EA antibodies in the synovia and spleen are found only among the early arthritis. After 2 months of evolution, fluorescent antibodies disappear whatever the immunization may be. Among the immunized rabbits, it is probable that antigenic EA does not persist in the synovia. Indeed, the autologous inflamed synovia transplantation, from the donor-challenged knee joint, does not develop an inflammatory reaction in the non-challenged knee joint. The fluorescent immunoglobulins IgG and IgM in the synovium of arthritic rabbits are found with the same percentages as fluorescent anti-EA antibodies. The lymphocyte response to EA, PHA and PWM are positive whatever the immunization and arthritis evolution may be. There is no correlation between AIs and lymphocyte responses to specific and nonspecific mitogens. It is probable that self-perpetuation depends closely on M. tuberculosis whose adjuvant power is much superior than M. butyricum and not on antigenic EA whose essential role would be to trigger the inflammatory process.