Biomedical subjects
Publications and source records attributed to O Kohashi.
Explore the source record for details and available documents.
Our previous finding, that germ-free F344 rats develop severe adjuvant-induced arthritis, whereas specific pathogen-free rats develop mild disease, prompted us to investigate the role of bacterial flora in promoting the development of this disease. Germ-free rats given Escherichia coli experienced disease suppression. Germ-free rats treated with 3 strains of Lactobacillus experienced an enhancement of the disease, although it was not significant. Germ-free rats treated with a combination of E coli and lactobacilli had disease suppression similar to that of E coli monoassociated rats. Thus, these findings suggest that E coli may play a dominant role in modulating the development of the disease in this particular strain of F344 rats, possibly through its lipopolysaccharide (as evidenced by positive results on limulus tests). These findings also suggest that microflora play an important role in modifying the development of joint disease.
The analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), 6-O-stearoyl-MDP (L18-MDP), and N-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N-stearoyl-L-lysine (MDP-Lys-L18) and N-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) induced acute polyarthritis in both euthymic rats and in athymic nude rats after a single or multiple systemic administrations of their aqueous forms; water-in-oil emulsions did not induce acute polyarthritis. MDP and the adjuvant-active analogs MDP-O-nBu (MDP butyl ester), N-(3-hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) and the adjuvant-inactive analogs [L18-MDP (L)] or MDP-Lys-L18 (L), which is 6-O-stearoyl-N-acetylmuramyl-L-alanyl-L-isoglutamine, or N-(N-acetylmuramyl-L-alanyl-L-isoglutaminyl)-N-stearoyl-L-lysine, did not induce disease, at the dosages used. Histological examination of the acute exudative inflammatory reactions around joints revealed hypertrophy of both synovial villi and tendon sheath, infiltration of polymorphonuclear leukocytes (PMN) and fibrin deposition of the affected joint spaces. Most of these lesions rapidly subsided after cessation of the adjuvant injections. During multiple systemic injections of acyl-MDPs, this acute arthritis became chronic in euthymic rats but not in athymic nude rats; the chronic polyarthritis consisted of mononuclear cell infiltration and proliferation of the connective tissue growth and severe new bone formation of the shaft of the bone. It remains uncertain whether chronic polyarthritis results from the repeated flare of acute inflammatory reactions to the continuing stimulation of the adjuvants or whether it may require some immunologic process to such exogenous substances as bacterial fragments or autoantigens modified by exogenous substances such as type II collagen.
Germfree (GF) F344 rats developed severe adjuvant-induced arthritis with a 100% incidence after a single intradermal injection of heat-killed Mycobacterium bovis (BCG). Specific pathogene-free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that gram-negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; gram-positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and E. coli may play a dominant role in modulating the development of adjuvant-induced arthritis.
Explore the source record for details and available documents.
Muramyl dipeptide (MDP), a minimal structure in bacterial cell walls essential for their adjuvant activity, was incorporated in a water-in-oil emulsion and injected into the footpads of nude rats devoid of functional T cells. MDP thus injected evoked massive epithelioid granulomas in the draining lymph nodes, indicating that MDP induced epithelioid granuloma formation requires no T cells. This finding with other data available strongly suggest that epithelioid granulomas can be induced without immunologic reactions.
A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), induced severe polyarthritis in euthymic rnu/+ rats. The rnu/+ rats were the most susceptible to MDP-induced arthritis among various rat strains tested, whereas congenitally athymic nude rats (rnu/rnu), males or females, did not develop the disease. This disease was clinically and histologically indistinguishable from classic adjuvant-induced arthritis in terms of clinical course, clinical signs, and histologic features such as (1) an initial acute exudative reaction observed primarily in stroma of the synovial membrane, periarticular tissue, about the tendons, tendon sheath, along the periosteum, between muscle bundles and in the subcutaneous tissue; (2) hypertrophy of the synovial villi, hyperplasia of the synovial lining cells; (3) the very active periosteal new bone formation; and (4) granulation tissue growth either in the articular tissues or in liver, lymph nodes, and capsule of the spleen. Thus, it is postulated that MDP-induced arthritis is basically the same disease as classic adjuvant-induced arthritis. The thymus may play an important role in promoting the development of the disease, possibly through some immune mechanisms to undetermined antigen(s). We believe that nonimmune mechanisms may also be involved in some part of acute and chronic inflammatory reactions to MDP molecules. This new model of MDP-induced arthritis will be a very useful tool to elucidate the underlying mechanisms of adjuvant-induced arthritis.
A synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), produced extremely severe polyarthritis with almost 100% incidence in Rowett euthymic rnu/+ rats, but the same dose of MDP (100 microgram) did not produce the disease in athymic rnu/rnu rats. Five hundred micrograms of MDP or 0.2 mg of heat-killed Mycobacterium bovis BCG, however, produced mild and transient polyarthritis in nude rats with very low incidence. We have not yet succeeded in reconstituting the disease susceptibility of nude rats by using thymus cells from normal rnu/+ rats. After intradermal inoculation of 100 microgram of MDP, nude rats developed small granulomas with a little necrosis and very few multinucleated giant cells only in the regional lymph nodes, whereas, in addition to the development of polyarthritis, euthymic rnu/+ rats developed typical granuloma with massive necrosis accompanied by numerous polymorphonuclear leukocytes and sparse multinucleated giant cells in the regional lymph nodes. Thymus cell-reconstituted rnu/rnu rats developed granuloma with sparse giant cells, relatively large areas of necrosis, and many polymorphonuclear leukocytes. Neonatal thymectomy may depress adjuvant-induced arthritis in the high-responder Lewis rats and enhance the disease development in the low-responder F344 rats. These findings suggested that (i) thymus plays an important role in promoting the development of MDP-induced arthritis; (ii) MDP-induced granuloma formation does not require thymus functions; (iii) the thymus functions may however be involved in the development of massive necrosis surrounded by considerable polymorphonuclear leukocyte infiltration, the mechanisms of which remain to be determined; and (iv) there is no direct correlation between granuloma formation and development of adjuvant arthritis.
Explore the source record for details and available documents.
A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), failed to produce polyarthritis with a wide dose range in a water-in-oil emulsion of mineral oil such as liquid paraffin, Drakeol, or heavy mineral oil. MDP, however, produced moderate to severe arthritis with almost 100% incidence in a water-in-oil emulsion made up of Difco incomplete adjuvant, which consists of Bavol F as an oil vehicle and Arlacel A as an emulsifier. N-acetylmuramyl-L-alanyl-L-isoglutamine did not produce arthritis, whereas 4,6-diacetyl-MDP produced the disease. Bacterial peptidoglycans, such as disaccharide peptides which were N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine and N-acetylglucosaminyl-6,o-acetyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine, also produced polyarthritis with low incidence in Difco oil but not in the other mineral oils described above. MDP and bacterial disaccharide peptides were able to produce the disease even in the latter mineral oil only when the concentration of Arlacel A was increased from 15% to 20 to 30% in the oil. We concluded that one of the minimal essential structures responsible for development of this disease is MDP, although the role of the oil vehicle remained uncertain, and there is no direct correlation between granulona formation and arthritogenicity of MDP.
Explore the source record for details and available documents.
Lewis rats developed polyarthritis after a single injection of a water-in-oil emulsion containing various peptidoglycans (PGs) derived from Lactobacillus plantarum. A copolymer of polyriboinosinic acid and polyribocytidylic acid markedly potentiated the arthritogenicity of these PGs. The synthetic adjuvants N-acetylmuramyl-L-alanyl-D-isoglutamine (MurNAc-L-Ala-D-isoGln) and MurNAc-L-Ala-D-Gln were non-arthritogenic, but they did produce severe arthritis when mixed in a water-in-oil emulsion with a copolymer of polyriboinosinic acid and polyribocytidylic acid. Substitution of either L-isoGln or D-isoAsn for the D-isoGln in the MurNAc-L-Ala-D-isoGln markedly reduced its capacity to induce the disease. Taken together with the results of skin testing against various PGs and MurNAc-L-Ala-D-isoGln in the diseased rats, the present results suggest that (i) a minimal essential structure required for development of polyarthritis is related to a larger molecule than either MurNAc-L-Ala-D-isoGln or a monomer of PG, probably to a dimer of PG, and (ii) an antigenic determinant(s) for the delayed-type skin hypersensitivity to PGs exists on a common structure shared among these PGs, possibly somewhere on a monomer of PG not on N-acetylmuramyl peptides.
Germfree F344 rats developed severe arthritis with 100% incidence after a single intradermal inection of either squalane containing 0.5 mg of heat-killed Mycobacterium bovis BCG or a water-in-oil emulsion containing 0.2 mg of peptidoglycan derived from Staphylococcus epidermidis. Conventional F344 rats developed less-severe arthritis with 20% incidence for heat-killed BCG and 0% incidence for peptidoglycan. Specific-pathogen-free rats showed an intermediate susceptibility between germfree and conventional rats. Interestingly, both unimmunized specific-pathogen-free and conventional rats. but not unimmunized germfree rats, showed weak delayed-type hypersensitivity reactions to peptidoglycans derived from either S. epidermidis or Lactobacillus plantarum, suggesting that a bacterial flora may furnish a stimulus for induction of cell-mediated immunity to ubiquitous bacterial peptidoglycans. It is thus possible that although a bacterial flora is not necessary for development of adjuvant arthritis, it may have some suppressive effect on the development of the disease in specific-pathogen-free and conventional F344 rats, possibly through modulation of the immune response.
Cell wall lytic enzyme (Kyowa lytic no. 2 enzyme) liberated arthritogenic hydrosoluble peptidoglycans from both arthritogenic and non-arthritogenic bacterial cell walls. From these cell walls, mutanolysin (peptidoglycan-degrading enzyme) also liberated hydrosoluble peptidoglycans which, however, lacked arthritogenicity. Based on the chemical composition of these peptidoglycans, it was suggested that their arthritis-inducing ability depends on a relatively long chain of glycan units that consists of repeated units of N-acetylglucosaminyl-N-acetylmuramic acid. However, the glycan chain lengths on these peptidoglycans appeared to be related to their adjuvancy rather than to an antigen(s) responsible for development of arthritis in rats.
We confirmed that, when immunized with a conventional complete Freund adjuvant (water in oil), Lewis rats were highly susceptible to adjuvant arthritis, Fisher rats were less susceptible, and Buffalo rats were much less susceptible. However, mycobacterial delipidated cells in squalane (squalane-type adjuvant) produced severe arthritis with almost 100% incidence even in the less susceptible rat strains except for Buffalo rats. With regard to an immune response, Freund complete adjuvant induced strong delayed hypersensitivity to purified protein derivative (PPD) and peptidoglycan (PG) in all rat strains used, Whereas the squalane-type adjuvant induced these hypersensitivities only in Lewis and Buffalo rats, but not in Fisher and Brown Norway rats. No correlation was found between development of arthritis and delayed hypersensitivity to either PPD or PG, or both. It seems that PPD hypersensitivity may be inherited differently from PG hypersensitivity.
Cell walls of Mycobacterium smegmatis were able to produce much more severe arthritis in rats than the delipidated cells, whereas cell envelope and cell membrane fractions were unable to produce the disease. The lysozyme-solubilized product was able to produce mild disease with only 30% of incidence with an optimum dose, whereas the higher and the lower doses did not produce the disease. The rats immunized with cell envelope, cell membrane fraction and nonarthritogenic doses of lysozyme-solubilized product were protected against the subsequent homologous and heterologous challenge of delipidated cells. It was discussed that this preventative effect can be the result of antigenic competition between the arthritogenic and nonarthritogenic components of M. smegmatis. On the other hand, all the fractions separated here were able to serve as an immunoadjuvant in terms of inducing delayed hypersensitivity to ovalbumin in guinea pigs.
Arthrigenicity of Mycobacterium smegmatis subfractions appeared to be remarkably potentiated in oil vehicles such as squalane or mineral oil, while water-in-oil emulsions containing Arlacel A appeared to decrease or suppress their arthritogenicity. It seems that Arlacel A can exert a suppressive effect on the arthritogenicity of the subfractions. Poly I:C and acetylated wax D potentiated the arthritogenicity of lysozyme-solubilized product, while cord factor was unable to do so. When given together with either cell membrane fraction or cell envelope, the lysozyme-solubilized product produced much more severe disease than that of lysozyme-solubilized product alone. Cell walls lost much of their arthritogenicity when mixed with lysozyme-solubilized product.
Bacterial cell walls from Str. bovis, Str. lactis, Str. mutans, Str. thermophilus, Str. salivarius, and Str. pyogenes were able to produce polyarthritis in rats but Str. faecalis cell walls were nonarthritogenic. S. aureus cell walls produced extremely severe disease. It was also shown that cell walls from S. epidermidis, B. megaterium, and M. lysodeikticus were nonarthritogenic. A close correlation was observed between development of arthritis and the delayed hypersensitivity to bacterial peptidoglycans but not with the PPD hypersensitivity. It was suggested that the adjuvanticity of bacterial cell walls is needed to induce the disease and that arthritogenicity requires a specific antigen in addition to the presence of an adjuvant-inducing agent.