Noboru Tamura

Platelet adhesion and complement activation studies on poly(N-alkyl mono and disubstituted) acrylamide derivatives

Poly(N-alkyl mono and disubstituted) acrylamide derivatives were synthesized from poly(acryloyl chloride) by monomer analogous reaction. The polymers were characterized by FTIR-ATR and GPC. The contact angle measurements were performed to evaluate hydrophobic/hydrophilic characters of these polymers. The N-alkyl substituents changed contact angle between 55 and 75 degrees. In vitro platelet adhesion studies showed that surfaces of poly(N-alkyl substituted) acrylamides are prone to adhere platelets. Platelet spreading was more on poly(N-benzyl-N-ethyl acrylamide) surfaces in comparison to that on poly(N-benzyl-N-propionic acid ethyl ester acrylamide) and poly(N-benzyl acrylamide) surfaces. As a result of modification of amino group with N-alkyl substituents, the activations of C3a and C5a complements were suppressed 9-20% and 5-6% of native poly(acrylamides), respectively.

Determination of C4b•C4-bp complex formed by the activation of classical complement pathway using an enzyme-linked immunosorbent assay

We developed a quantitative enzyme-linked immunosorbent assay (ELISA) for the detection of C4b.C4-bp complex by incubating the sample on anti-C4-bp-coated plate and then developing with HRP-labeled anti-C4. The amount of C4b.C4-bp complex, generated in vivo by the interaction of purified C4b with C4-bp or normal human serum with aggregated human IgG, was measured by the ELISA. The complex, however, rapidly decreased in serum by the action of factor I. Six out of the 100 plasma samples from patients with various diseases were found positive in the ELISA. One plasma sample from a patient with SLE showed high level of C4b.C4-bp complex with decreased levels of factor I, C4, C4-bp and CH50. These results suggest that the detection of C4b.C4-bp complex is useful for monitoring the diseases in which the classical pathway activation is expected.

An immunoglobulin g inhibiting lactate dehydrogenase activity

We found extremely low (6 U/l) serum lactate dehydrogenase (LDH, EC 1.1.1.27) activity in a patient with uterine myoma. The patients serum inhibited purified LDH isoenzymes. One milliliter serum neutralized 73 U purified LDH-1 isoenzyme, equivalent to 300-500-fold the amount of LDH in 1 ml of normal serum. The serum inhibitor was purified by ordinary procedures using chromatographies and identified as IgG which contains both kappa- and lambda-chains. The IgG is very unique in showing higher affinity to the H- than M-subunit of the LDH tetramer, in contrast with the IgGs already reported. After removal of the myoma, the anti-LDH activity gradually decreased with a half-life of 20 days corresponding to that of IgG and finally almost disappeared. This indicates a possibility that the myoma cells produce some factors such as B-cell growth and differentiation factors.

A solid-phase anti-C3 assay for detection of immune complexes in six distinguished forms

A solid-phase anti-C3 assay detecting immune complexes associated with C3 fragments is described. Two monoclonal antibodies against C3 fragments are used; one recognizes an epitope expressed on C3b, C3c and C3i, the other recognizes an epitope expressed on C3dg and iC3b. Combining these monoclonal anti-C3s with antibodies against class-specific immunoglobulins in enzyme-linked immunosorbent assay (ELISA), immune complexes (ICs) are detected in six distinguished forms; C3b-IgG-IC, iC3b/C3dg-IgG-IC, C3b-IgA-IC, iC3b/C3dg-IgA-IC, C3b-IgM-IC and iC3b/C3dg-IgM-IC. About three-fourths of the plasma samples from patients with active SLE or IgA-nephritis were found positive in one or more types of ICs. IgA-type ICs were found very frequently in patients with autoimmune or renal diseases.

Abnormal expression of complement receptor (CR1) in IgA nephritis: increase in erythrocytes and loss on glomeruli in patients with impaired renal function.

The number of complement receptor for C3b (CR1) molecules in erythrocytes from patients with renal diseases was measured by an immunoradiometric assay using monoclonal antibodies against CR1. IgA nephritis patients with high serum creatinine value (Scr) showed markedly elevated levels of CR1, whereas patients with normal Scr had normal CR1 levels. A similar increase in CR1 number was observed in membranoproliferative glomerular nephritis with high Scr. CR1 of these patients functioned normally as a cofactor of C3b inactivator in cleaving immune complex-bound C3b. In contrast, a high frequency (5/6) of negative staining of glomerular CR1 was observed in IgA nephritis patients with high Scr by immunofluorescence study. We postulate that the disease-associated, acquired factors at least in part contribute to the abnormal expression of CR1: elevated levels in erythrocytes and defective expression on glomeruli.

Experimental allergic encephalomyelitis induced by basic protein with synthetic adjuvant in comparison with Freund's complete adjuvant. Role of antibodies in correlation with the clinicopathological features.

Experimental allergic encephalomyelitis (EAE) was induced in guinea pigs with bovine myelin basic protein (BP) with adjuvant of either synthetic muramyl dipeptide (Mdp) or Mycobacterium tuberculosis (Tbc). The following results were obtained: (1) The body temperature of the animals was studied serially after sensitization and its elevation was shown to be an early sign of EAE. (2) Several animals developed the clinical and histological signs of hyperacute EAE. (3) An optimal combined dosage of BP and adjuvant was found for induction of clinical EAE and for the the production of complement fixing (CF) antibodies. (4) Little passive hemagglutinating (PH) antibody was produced by single immunization. These results displayed no essential difference in EAE induced by either adjuvant. (5) Detectable PH antibodies developed later in addition to CF antibodies in a few animals immunized with Tbc adjuvant. These animals were skin-tested to BP, and had recovered from body weight loss or limb weakness. The results suggest that humoral antibodies play a role in modifying the disease process, even if they are not essential in production of EAE.

Silkworm haemolymph factor(s) interacting with vertebrates complement system

Two complement-related activities were found in the haemolymph of the silkworm, Bombyx mori. One was detected as a ‘classical C3-like’ activity (CC3LA) to cooperate with mammalian or anuran ‘classical R3’ reagent to induce enhanced lysis of rabbit IgM-rich haemolysin-sensitized sheep erythrocytes. Another is detected as an alternative pathway inhibiting activity (APIA). Silkworm haemolymph (SWHL) was fractioned by DEAE cellulose, CM cellulose and Sephadex G-200 chromatographies. In every step of the fractionation procedure, these two activities were eluted in the same regions, suggesting that these two activities are carried on a single factor.