Shinichi Aotsuka

A simple and rapid microenzyme-linked immunosorbent assay for antibodies to poly(ADP-ribose) in systemic lupus erythematosus

A simple and rapid microenzyme-linked immunosorbent assay has been developed for determination of anti-poly(ADP-ribose) antibodies in humans using a combination of protein A-alkaline phosphatase conjugates and poly(ADP-ribose)-coated polyvinyl microplates. After a 1-h treatment of the plates with 100 microliters of poly L-lysine (PLL) solution (50 micrograms/ml), an aliquot of the solution containing 100 ng poly(ADP-ribose) (50 microliters) was added to the PLL-treated plates and evaporated at 37 degrees C overnight to facilitate the adherence of poly(ADP-ribose) to the plates. Nonspecific binding of diluted test sera from patients with systemic lupus erythematosus (SLE) or from normal individuals to the PLL-coated plates was minimized by exposure of the plates for 1 h to Tris-buffered saline (pH 7.4) containing 0.01% bovine serum albumin (BSA). This method was also applicable to the determination of anti-double-stranded DNA antibodies in humans. The present assay is advantageous over those reported so far as it saves time and antigen.

C1q solid-phase radioimmunoassay: Binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.

Anti-double strand (ds) DNA antibody formation by NZB/W (F1) spleen cells in a microculture system detected by solid-phase radioimmunoassay

A solid-phase radioimmunoassay method was devised to detect mouse anti-double strand (ds) DNA antibody. This method could easily detect the anti-dsDNA antibody in 1 : 10,000 dilutions (1 unit) of pooled 9-10-month-old female NZB/W F1 sera. The sensitivity was about 10(3)- and 10(2)-fold higher than that of the modified Farr method and of the double antibody technique respectively. NZB/W mice developed high titer anti-dsDNA antibody as they grew older. Spleen cells brought to a microculture system using flat-bottomed polystyrene plates produced anti-dsDNA antibody clearly detectable by solid-phase radioimmunoassay. Anti-dsDNA antibody produced in vitro (y units) was in close correlation with the anti-dsDNA antibody titer of the spleen donor (x units) (y = 4.8 X 10(-2) x -65, gamma = 0.94, P less than 0.001). A combination of the microculture system and solid-phase radioimmunoassay was recommended for the characterization of anti-dsDNA antibody-forming cells.

Suppressive effect of anti-rheumatic drugs on interleukin-1β release from human peripheral blood monocytes

We developed an ELISA system for human IL-1 alpha and -beta release from silica-stimulated monocytes from healthy volunteers and tested the effect of several anti-rheumatic drugs including nonsteroidal anti-inflammatory drug (Ibuprofen). Anti-rheumatic drugs including Auranofin and Sulphasalazine suppressed IL-1 beta release significantly at therapeutic concentrations, whereas Bucillamine, Lobenzarit, D-Penicillamine and Ibuprofen did not. These results suggest a possible immunotherapeutic effectiveness of some anti-rheumatic drugs on rheumatoid arthritis through their inhibition of IL-1 beta release.

Monocyte-mediated suppression of rheumatoid factor production in normal subjects

The regulatory role of normal monocytes in the production of rheumatoid factor (RF) was investigated. Monocyte depletion from normal mononuclear cells (MNC) resulted in an elaboration of IgM RF in 18 of 20 subjects. The increased RF production was inhibited by the addition to the cultures of adherent cells or their culture supernatants. These observations demonstrate the suppressive role of normal monocytes in the production of RF. Supernatants obtained from normal monocytes cultured with indomethacin could suppress the RF production, suggesting that prostaglandin E2 may not be involved in the regulation of IgM RF production.

Classification tree criteria of pulmonary hypertension in mixed connective tissue disease

Tree criteria for the classification of pulmonary hypertension (PH) in mixed connective tissue disease (MCTD) were developed by stratifying patients into groups according to the physician’s diagnosis, mean pulmonary artery pressure (mPA) and prognosis, respectively. A classification tree for PH diagnosed by the physician was constructed with two criteria: dilatation of the pulmonary artery segment evident on chest roentgenography (or an accentuated pulmonic sound as a surrogate) and shortness of breath on exertion, which demonstrated a sensitivity of 96% and a specificity of 99%. A classification tree for PH diagnosed by mPA was also constructed with almost similar criteria: an accentuated pulmonic sound (or dilatation of the pulmonary artery segment evident on chest roentgenography as a surrogate) and shortness of breath on exertion, which demonstrated a sensitivity of 100% and a specificity of 100%. The prognostic classification tree was constructed with four criteria: an accentuated pulmonic sound, systolic pulsation at the left sternal border, shortness of breath on exertion and retro-sternal pain on exertion, which demonstrated a sensitivity of 62% and a specificity of 98%. The classification tree criteria for the diagnosis and prognosis of PH in MCTD were found to be accurate and useful for the screening of PH.

Interleukin-1β release from human peripheral blood monocytes associated with phagocytosis of carbonyl-iron or erythrocytes

Interleukin-1 beta (IL-1 beta) release from human peripheral blood monocytes during the incubation with carbonyl-iron or sheep red blood cells was investigated. The incubation of purified monocytes with carbonyl-iron or sheep red blood cells enhanced IL-1 beta release, while their compounds, hemoglobin, globin and ferric citrate did not. The mechanisms of IL-1 beta release by carbonyl-iron or sheep red blood cells may be related to their phagocytosis, as non-phagocytic monocytes did not release IL-1 beta.

Occurrence of Antibody against Calmodulin in Chronic Liver Diseases

SummarySera from patients with liver diseases, collagen diseases, and malignant neoplasms were examined for autoantibody to calmodulin (anti-CaM). Calmodulin (CaM) was fixed to a polystyrene plate, incubated with appropriately diluted sera, and then the presence of IgG, IgM, and IgA bound to fixed CaM was determined with horseradish peroxidase-conjugated rabbit anti-human IgG, IgM, and IgA serum. IgG class anti-CaM was detected in none of the normal healthy subjects, whereas IgM and IgA class antibodies were detected in 5.0% and 4.3% of them, respectively, though their titres were low. IgG class antibody showed no disease specificity, although it was more frequently found in patients with liver diseases (51.2%), collagen diseases (25.2%), and malignant neoplasms (41.2%) than in healthy subjects. The frequencies of IgM class antibody were 36.6%, 3.9%, and 0% in the patients with liver diseases, collagen diseases, and malignant neoplasms, respectively. Thus, IgM class antibody was shown to be specific for liver diseases. Among the patients with liver diseases, the frequency was the highest in those with liver cirrhosis. The frequency of IgA class antibody was significantly higher in patients with liver diseases (28.9%) and collagen diseases (15.5%) than in healthy subjects (4.3%), whereas that in the patients with malignant neoplasms was not. Pretreatment of sera with CaM decreased the titer of anti Ca-M, whereas pretreatment with actin or calf thymus DNA did not.

The significance of autoantibody against calmodulin in chronic liver diseases, collagen diseases and malignant neoplasmas.

慢性肝疾患,膠原病,悪性腫瘍における仔牛より精製したカルモデュリン(CaM)に対する自己抗体の出現を報告した.抗CaM抗体の測定は酵素抗体法によった.抗体陽性血清と,CaM結合アガロースとを反応させ,酸性グリシンによる溶出分画中に,抗CaM抗体活性を認めた.抗体活性は,アクチン,仔牛胸腺DNAによる吸収では,低下しなかったが,CaM,家兎肝細胞膜分画および肝ホモジュネート77,000g上清分画による吸収で,低下した.IgGクラスの抗体は,肝疾患51.2%,膠原病25.2%,悪性腫瘍41.2%に認め,正常人(0%)より高頻度であった.IgMクラスの抗体は,それぞれ36.9%, 3.9%, 0%, 5%で,肝疾患,特に肝硬変は高頻度であった(p<0.02). IgAクラスの抗体は,肝疾患(28.9%),膠原病(15.5%)で,正常者(4.3%)より高頻度であった.抗平滑筋抗体の出現とは相関しなかった.