Seiichi Okumura

Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C. difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty‐four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 104.1/g in young adults and 104.6/g in elderly adults, with a range of 102.0 to 106.9/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.

Myasthenogenic significance of synthetic α-subunit peptide 183–200 of Torpedo californica and human acetylcholine receptor

Synthetic peptides of the Torpedo californica and human acetylcholine receptor alpha-subunit, Gly183-Asp200, were studied. Both peptides bound alpha-bungarotoxin to a significant extent, and inhibited toxin-binding with Torpedo or human acetylcholine receptor. The human peptide was, however, less potent than the Torpedo peptide. One can assume, therefore, that this sequence participates in cholinergic binding. The Torpedo alpha 183-200 segment was immunogenic in the induction of experimental autoimmune myasthenia gravis in rats, accompanied by elevation of anti-peptide and anti-rat acetylcholine receptor blocking antibody and reduced amplitudes of miniature end-plate potentials. Sera did not accelerate the degradation of rat acetylcholine receptor. Thus, one may interpret this to be the animal model induced by an immunopharmacological blockade of the acetylcholine-binding site. Furthermore, this Torpedo peptide was antigenic in the detection of antibody in human myasthenic sera. The human alpha 183-200 segment had neither of these 2 properties, although the corresponding anti-peptide antibody was elicited in immunized rats. It is feasible that the synthetic segment of this human sequence may not be maintained in such a conformation to be immunogenic.

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic‐associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline‐resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.

Experimental autoimmune myositis in SJL/J mice produced by immunization with syngeneic myosin B fraction. Transfer by both immunoglobulin G and T cells.

An experimental autoimmune myositis (EAM) was produced by immunizing SJL/J mice with the myosin B (MB) fraction of the syngeneic muscle. Immunoblot analysis of the IgG of the EAM mice demonstrated antibodies against a variety of muscle proteins. The condition was then transferred to normal mice by both injecting immunoglobulin G or T cells from the EAM mice. This is the first model of transferrable EAM caused by syngeneic muscle fraction, and it has an unique resemblance to human inflammatory myopathies involving both humoral and cellular immune mechanisms.

Myasthenogenicity in the main immunogenic region of acetylcholine receptor as modified by conformational design: an approach to antigenic synthetic peptides

Myasthenogenic regions in the acetylcholine receptor (AChR) alpha-subunit were studied in view of the conformation-dependent B-cell epitope expected at beta-turn and the MHC class II-restricted T-cell epitope expected at alpha-helix. Torpedo AChR alpha 67-76 and alpha 107-116 were synthesized as the main immunogenic region and the site specific for T-cell epitope in Lewis rat, respectively. Model peptides, synthesized by combining these natural sequence segments or by intervening the segment aligned as Asn-Pro-Gly-Gly (NPGG) in natural sequence segments, were tested in terms of antigenic conformation. The model peptide, alpha 107-116.alpha 67-76.alpha 107-116, was immunogenic in the induction of the animal model of myasthenia, accompanied by the anti-peptide antibody cross-reactive with the native AChR. High antigenicity in antibody assays for various peptide- and native AChR-immunized rats was found when the model peptides, alpha 107-116.alpha 67-76 and/or alpha 107-116.NPGG.alpha 67-76 were used for measurement as antigens. Antigenic conformation for the induction of the disease may thus be different from that for the reactivity to antibody.

Conformational modification enhances myasthenogenicity in synthetic peptide of acetylcholine receptor α-subunit

The induction of myasthenia gravis depends on linked recognition of antigenic sites of acetylcholine receptor (AChR) by B-cells and T-cells. The former is conformationally restrained, and the latter is under the MHC class II restriction. We synthesized an artificially formed peptide (model peptide) by coupling the alpha 190-195 selected as B-cell site and cholinergic binding site and the alpha-107-116 selected as T-cell site and agretope with the intervening chain segment aligned as Asn-Pro-Gly-Gly (NPGG) to adopt beta-turn conformation. This model peptide, alpha 107-116-NPGG-alpha 190-195, was potently immunogenic in Lewis rats to provoke anti-peptide antibody reactive with native AChR and to induce the animal model of immunopharmacologic blockade of acetylcholine (ACh)-binding site. Low immunogenicity compared with this was found when using natural peptides predicted as sequences of B-cell site or T-cell site and the peptide synthesized by linking both without intervention of NPGG. The alpha 190-195 had no function of cholinergic binding either as a single segment or as part of the conformation-modified peptides; results suggest that the conformation modified for high immunogenicity does not assume the bioactive conformation for ACh-binding.

Presynaptic modification of neuromuscular transmission by antiacetylcholine receptor antibody: myasthenic serum and monoclonal antibody produced by transformed lymphocytes.

When myasthenic serum or a monoclonal antiacetylcholine receptor (anti-AChR) antibody produced by human transformed lymphocytes and transferable to an animal was applied to rat diaphragms in vitro, presynaptic facilitation was demonstrated by changes in ACh quantal content of endplate potentials. The results correlated with ability of the antibody to block binding of α-bungarotoxin to AChR, but not with titers of anti-AChR antibody by immunoprecipitation assay and AChR degradation rate. Antibody to the receptor site near the ACh-binding site may act presynaptically to compensate for the postsynaptic failure in myasthenia gravis.

Presynaptic function modified by acetylcholine-receptor interaction in experimental autoimmune myasthenia gravis.

Rats with experimental autoimmune myasthenia gravis (EAMG) were studied to test if the postsynaptic receptor disease would be modified by a compensating presynaptic effect of acetylcholine (ACh) being mediated through the presynaptic receptor. In normal rat phrenic-diaphragms, the ACh quantum content was estimated using the cut-muscle technique by which the evoked release of transmitter can be measured in the absence of blocking agents; values were significantly increased when the ACh receptor was blocked by d-tubocurarine. In rats with EAMG, this presynaptic autoregulation acted to compensate the postsynaptic failure, but did not reach the range of d-tubocurarine-treated controls. Results may be explained by either the difference between the receptor sites for myasthenic antibodies and d-tubocurarine or the immunological property of the presynaptic receptor which may differ from the postsynaptic one.

Role of Interleukin 2 on Enhancement of Concanavalin A‐Induced Human Peripheral Blood Lymphocyte Proliferation by Murine B Cell Mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol‐extracted water soluble adjuvant (Bu‐WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP‐Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures.

In Vitro Proliferative Response and Polyclonal Antibody Production in Spleen Cells of Immunologically Defective CBA/N and C3H/HeJ Mice by Water-Soluble Adjuvant (Bu-WSA) Extracted from Bacterionema matruchotii

Mitogenicity and the polyclonal plaque forming cell(PFC)‐inducing property of a water soluble‐adjuvant extracted from Bacterionema matruchotii by butanol (Bu‐WSA) were examined in vitro in the spleen cells of hybrid (CBA/N female × BALB/c male) F1 mice and C3H strain of mice. The hybrid F1 male cells which expressed a CBA/N‐defect were unable to respond to Bu‐WSA, when assessed by the incorporation of [3H] thymidine into the cells and the generation of anti‐trinitrophenyl (TNP)‐PFC or autoantibody PFC defined by the anti‐bromel‐ain‐treated mouse erythrocyte PFC assay. However, hybrid F1 female cells with normal traits responded to Bu‐WSA. Cultured spleen cells of bacterial lipopolysaccharide (LPS)‐nonresponsive C3H/HeJ mice responded to Bu‐WSA as in the case of cells of LPS‐responsive C3H/He mice, and the [3H]thymidine‐uptakes and the numbers of PFC in these culture cells increased. Re‐extraction of Bu‐WSA by phenol did not affect its activities, while the activity of butanol‐extracted LPS on C3H/HeJ cells decreased after re‐extraction by the same procedure with phenol.