Methotrexate was developed in 1948 for neoplastic diseases such as leukemia. Recently low dose methotrexate has been widely used on nonneoplastic diseases such as rheumatoid arthritis. Clinical studies have shown that the administration of low dose methotrexate (5.0-7.5 mg/person, once a week) to patients with rheumatoid arthritis causes clinical improvement. Then low dose methotrexate (0.1-0.3 mg/head, 3 times a week) was given to animal experimental models for arthritis and evaluated its efficacy histopathologically and immunologically.
Spontaneous polyarthritis in MRL/Mp-lpr/lpr (MRL/l) mice and type II collagen induced arthritis (CIA) in DBA/1 J mice were studied. Two different studies were carried out in the CIA experiment. In one experiment, Freund's complete adjuvant (FCA) was given in both immunization and elicitation procedures. In the next experiment, FCA was used in immunization, and Freund's incomplete adjuvant (FIA) was applied in the elicitation period.
The experiments revealed evidences that methotrexate had appreciable effects on the spontaneous polyarthritis in MRL/l mice and FCA/FCA CIA in DBA/1 J mice. But it provided marked improvement on FCA/FIA CIA in DBA/1J mice in a dose dependent manner. Proliferative inflammatory articular lesion was suppressed and bone remodelling effect was observed under microscope. The bone remodelling effect indicates suppression of both bone absorpsion and new bone formation.
FACStar analysis provided data that methotrexate did not change number of immune responsive cells in the spleen of MRL/l mice.
The folic acid antagonist methotrexate blocks the activity of dihydrofolate reductase. The mouse enzyme has decreased rate of folate reduction. That is the reason why methotrexate had appreciable effect in mice. Scientists have to pay much attention to the metabolism of the animals, and to do effort to develop newer experimental animal models.

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Macrophages contain high amounts of lysosomal cysteine proteinases that are considered to be important in catabolic process of endogenous and exogenous proteins.
Addition of horseradish peroxidase to the cultured macrophages induced an increase in the density of lysosomes in parallel with the accumulation of peroxidase taken up the heavy lysosomes. Degradation of uptaken peroxidase was inhibited by addition of anticathepsin L antibody to the medium, but not by addition of anti-cathepsin B or H antibody. Treatment of cells with E-64 caused intracellular accumulation of endogenous proteins due to inhibition of lysosomal proteolysis, but pepstatin, a potent inhibitor of cathepsin D, showed much less effect.
Those results suggest a large contribution of cysteine proteinases, especially cathepsin L to the initiation of lysosomal degradation of both endogenous and exogenous proteins. The levels of mRNAs for cathepsin L and H and cystatin β in macrophages obtained from rats injected with sodium caseinate were several times as high as those in resident macrophages.

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Although peritoneal resident cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing serum amyloid A (SAA) to amyloid fibrils, the original residence peritoneum is a rare place of amyloid deposition. This is considered to occur due to deficiency of blood supply in the peritoneum. To increase blood supply in the peritoneum, ascitic fluid which contained about the same proteins as those in the serum was induced in mice.
Amyloidotic peritoneal cells were packed in a microchamber and cultured in ascites with additional inflammatory stimuli which increased the SAA concentration of ascites to about 1% of the highest serum level.
On the 7 th day, Congo red positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or-SAA immunostaining, amyloid deposits as well as cell surface of adjucent macrophages were positively stained. Immunologic depletion of T and B lymphocytes from amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers.
These results suggest that both ascitic fluid containing sufficient amount of SAA and peritoneal macrophages with high AEF activity are indispensable agents for AA amyloid fibrillogenesis in the peritoneum.

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Non-isotopic immunoassay for the human tumor necrosis factor α (TNFα) was established by employing the dissociated enhanced lanthanide fluoroimmunoassay (DELFIA) system, based on the timeresolved fluoroimmunoassay (TR-FIA) technique utilizing the europium-labeled antibody. In comparison with enzyme-linked immunosorbent assay and bioassay, the sensitivity and range of measurement could be significantly increased by applying the DELFIA system to TNF_α.
Biological samples, including serum, may affect some of the immunoassays. We overcome the serum influence to the measurement of TNF_α by DELFIA, Percent recovery of additional TNF_α in each diluted serum was determined, then the sliding standard curve of each serum sample was mathematically calculated from the recovery. Finally corrected TNF_α amount was detected by using the sliding standard curve. We suggest that more accurate, sensitive and wide ranged TNF_α immunoassay has been established by DELFIA with some revising calculation.

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The major basic protein (MBP) plays an important role in the antiparasitic and antibacterial activities of eosinophils. In this study, to elucidate the mechanism of the action of MBP, the antibacterial peptides of MBP were isolated and their structures were analyzed.
The antibacterial activity of MBP was not affected by the pyridylethylation of the thiol groups of MBP, suggesting that the disulfide bonds were not necessary for the antibacterial activity of MBP.
By the chymotryptic digestion of pyridylethylated-MBP (PE-MBP), 14 peptides (peptide I-XIV) were obtained, and 6 peptides (peptide V, IX, X, XII, XIII and XIV) showed antibacterial activities. Amino acid sequences of the 4 peptides (peptide V, IX, X and XIII) which had high antibacterial activities, were analyzed. Interestingly, all the 4 peptides proved to have both the hydrophilic regions which were rich in basic amino acids and the hydrophobic regions.
By the chymotryptic digestion of peptide V and X, their antibacterial activities were significantly reduced. Amino acid sequence analyses of the digested peptides (peptide V and X) showed that the hydrophilic regions were separated from the hydrophobic regions after the digestion.
These observations suggest that both the hydrophilic and the hydrophobic regions are important for the expression of the antibacterial activity of MBP.

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Platelet-activating factor (PAF) is a potent chemical mediator of diverse physiological and pathological processes. In the present study, we examined the role of endogenous PAF in development of gastric ulcers. Water-immersion stress of rats induced proo nged decrease in endogenous PAF levels and altered the molecular heterogeneities of PAF in their corpus and antrum in early and late stages of development of gastric lesions. These alterations were suppressed by the antiulcer drugs, atropine and dopamine, concomitantly with suppression of ulcer development Vagal stimulation of rats by carbamylcholine transiently decreased endogenous gastric PAF level, which was not associated with ulcer development. Surprisingly, intraluminal administration of 1-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine, a biologically potent and acetylhydrolase-resistant PAF analog, prevented the development of gastric lesions by water-immersion stress.
These observations indicate that endogeneous PAF is likely to function physiologically to prevent the initiation and development of gastric ulcers by water-immersion stress.

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The present study was undertaken to clarify the changes of bone metabolism in the femoral diaphysis of adjuvant arthritis (AA) rats. On 28 days after adjuvant inoculation to rats, calcium (Ca) and zinc (Zn) contents in the femoral diaphysis of injected hind leg were significantly decreased in comparison with those of control rats. Moreover, alkaline phosphatase activity (ALP) in the femoral diaphysis of AA rats was significantly increased. Deoxyribonucleic acid (DNA) and collagen contents in the femoral diaphysis were not significantly altered in comparison with those of control rats. Meanwhile, the serum A/G ratio, Ca and Zn concentration in AA rats were significantly decreased in comparison with those of control rats. Serum parathyroid hormon (PTH-C) was significantly increased in AA rats, although serum estradiol and calcitonin were not altered in comparison with those from normal rats. Serum adrenocorticotropic hormon (ACTH) level was increased but not significant in AA rats. Electron microscopic analysis showed a rough alteration of bone matrix and cartilage cell degeneration in the femoral epiphysis of AA rats.
These results suggest that AA rat has a disorder of bone metabolism in femoral diaphysis and these bone metabolism changes are different from that caused by immobilization or ovariectomized rats.

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Using a highly specific and sensitive radioimmunoassay for measurement of endothelin-1 (ET-1) and reverse-phase HPLC, the presence of ET-1-like immunoreactivity was demonstrated in human plasma, urine, cerebrospinal fluid and synovial fluid.
Determination of ET-1 levels and its molecular forms in biological fluid from several diseases should provide important clues to understanding its physiological and pathophysiological role (s) in man.

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The inhibitory effects of pemirolast potassium (TBX) and hydrocortisone on platelet activating factor (PAF) -induced human eosinophil chemotaxis were examined by a modified Boyden technique. The migration of eosinophils to lower wells was assessed by counting the number of cells and/or measuring total amounts of eosinophil cationic protein (ECP) . Human eosinophils were purified from nine atopic patients. TBX significantly inhibited PAF-induced eosinophil chemotaxis in a dose-dependent manner at concentrations from 1×10^-6 to 1×10^-4M. PAF-induced eosinophil chemotaxis was also inhibited by hydrocortisone in a dose-dependent fashion at concentrations from 1×10^-7 to 1×10^-5 M. The inhibition by these agents was not due to their cytotoxic effect as evaluated by trypan blue dye exclusion and by ECP release.
These results suggested that both TBX and hydrocortisone inhibited eosinophil chemotaxis at therapeutic concentrations. TBX and hydrocortisone could effect allergic diseases via their inhibition of eosinophil chemotaxis.

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Granulomas are focal, predominantly mononuclear tissue inflammations evoked by persistent irritants. Granulomatous inflammation can be classified as either a hypersensitive type (immunologic) or a foreign-body type (non-immunologic) response. The common histopathologic feature, predominant infiltra-tion of mononuclear cells, is observed in both types of lesions. Granulomatous inflammation has been difficult to study because existing assays were subjec-tive, time consuming, or only indirectly reflected changes in the lungs. Macrophages secrete a wide range of biologically active mediators such as cytokines and lysosomal enzymes in response to inflammatory stimuli. The purpose of this study was to determine whether lysosomal enzyme activities correlates with sizes of lung granulomas that are composed predo-minantly of macrophages. Foreign-body (dextran beads) and hypersensitivity (antigen-coupled agarose beads) lung granulomas were induced in BALB/c mice by the intratracheal injection of artificial micro-particles. Large granulomas developed, which reached peak intensity within 3 days and declined in size thereafter. Aqueous extracts of both granulomas contained a large amount of lysosomal enzymes N-acetyl-β-D-glucosaminidase and lysozyme. Lysosomal enzyme activities in the extracts correlated with granuloma sizes. Dispersed granuloma cells were able to produce these enzymes. These results suggest that lysosomal enzymes may reflect the activity/size of granulomatous inflammation.

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Treatment with a monoclonal antibody (R 73 mAb) against the T cell antigen receptor αβ (TCR αβ) suppressed markedly both induction and progression of adjuvant arthritis (AA) in rats. The mAb was also effective in preventing development of collagen-induced arthritis (CIA) . However, established CIA was not affected by R 73, although early CIA was partially suppressed by this antibody. The delayed type hypersensitivity response to arthritogenic antigens was suppressed in R 73-treated rats with AA and CIA and this was associated with marked depletion of αβ⁺ T cells in peripheral blood.
Thus, AA appears to be αβ⁺ T cell-dependent in its induction and progression. αβ⁺ T cells appear to have a critical role in induction of CIA, but do not in progression of established CIA. Treatment of patients with rheumatoid arthritis with anti-human TCR αβ mAbs may reveal subtypes of this disease with varying dependence on T cell action.

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Effect of cytokines on the production of the third component of complement (C 3) by human umbilical vein endothelial cells (HUVEC) was investigated. Interleukin-1 (IL-1), interferone-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and lipopolysaccharide (LPS) from E. coli stimulated the production of C 3 by HUVEC. The effect of TNF-β, one of the most effective substances to stimulate C 3 production by HUVEC, reached plateau at the concentration of 200 U/ml. The production of C 3 decreased significantly after 4 passages of cell strains established from the human umbilical cord vein.
The relationship between inflammation and complement production by HUVEC is now in investigation.

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