Hideo Igarashi

Study of the Biological Activities of Toxic Shock Syndrome Toxin-1

The mitogenic and interleukin 2 (IL 2) production‐inducing effects of toxic shock syndrome toxin‐1 (TSST‐1) on murine lymphocytes were investigated. TSST‐1, an exotoxin produced by Staphylococcus aureus recovered from patients with toxic shock syndrome (TSS), is thought to be a causative agent of the syndrome.

Sensitive enzyme-linked immunosorbent assays for the detection of bacterial superantigens and antibodies against them in human plasma.

Enzyme‐linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic‐shock syndrome toxin‐1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3 pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150 min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme‐linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.

Preparation of a superantigen-adsorbing device and its superantigen removal efficacies in vitro and in vivo

OBJECTIVE A new superantigen-adsorbing device (SAAD) was developed, and its characteristics and efficacy in septic animals were evaluated. METHODS The SAAD was prepared by stepwise chemical modification of a polystyrene-based composite fiber reinforced with polypropylene. Adsorption affinities for several factors and the biological effect of superantigen (SAg) removal were measured in vitro. Also, superantigen-infused rabbits were treated with SAAD, and the efficacy was evaluated in vivo. RESULTS When the SAAD was evaluated for its ability to adsorb SAg in human plasma (1 ng/mL each), the adsorption rates were 74%, 76% and 85% for staphylococcal enterotoxins A, B and C, respectively, and 80% and 72% for toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxin A, respectively. In addition, the SAAD showed some affinity towards other molecules, such as streptococcal pyrogenic exotoxin B, beta2-microglobulin, and vancomycin. Residual activities in whole blood samples containing TSST-1 (1 ng/mL) after incubation with the SAAD were 125 pg/mL for tumor necrosis factor alpha (TNF-alpha) production, and 359 pg/mL for interleukin-8 (IL-8) production (the initial activities: 194 pg/mL for TNF-alpha production, and 1029 pg/mL for IL-8 production). When TSST-1/lipopolysaccharide (LPS)-infused rabbits were subjected to extracorporeal blood purification with a SAAD column, 50% of the animals survived for a 14-day period after the infusion. In contrast, all control animals died within 3 days after the infusion. CONCLUSION These results indicate that the SAg-adsorbing device may be useful in treating SAg-related diseases.

Involvement of HLA class II molecules in acquisition of staphylococcal enterotoxin A‐binding activity and accessory cell activity in activation of human T cells by related toxins in vascular endothelial cells

Human umbilical vascular endothelial cells (HUVEC) express HLA class II molecules upon stimulation with recombinant human interferon‐gamma (IFN‐γ). Staphylococcal enterotoxin (SE) A (SEA)‐binding assay using [125I]‐SEA showed the presence of specific SEA binding in HUVEC stimulated with IFN‐γ but not in unstimulated HUVEC. Levels of HLA class II expression and SEA‐binding increased as the IFN‐γ concentration and the period of stimulation were increased. Binding of [1254I]‐SEA to the IFN‐γ‐stimulated HUVEC was reduced markedly by an anti‐DR/DP MoAb. T cells produced IL‐2 upon stimulation with a group of SEs (SEA, SEB, SEC, SED and SEE) in the presence HUVEC stimulated with IFN‐γ but not in the presence of control HUVEC. The level of accessory cell activity in the IFN‐γ‐stimulated HUVEC was related to the level of HLA class II expression and SEA‐binding activity. Antibodies to HLA class II molecules almost completely inhibited the response. These results indicate that HLA class II molecules are directly involved in the acquisition of these activities in HUVEC.

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A‐bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml−1 and 103 cfu ml−1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase‐negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at ≈ 50 cfu ml−1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.

Tumor necrosis factor production by human T-cells stimulated with bacterial superantigens

Tumor necrosis factor (TNF) production from T-cells stimulated with superantigenic exotoxins, staphylococcal enterotoxin B and streptococcal pyrogenic exotoxin A was investigated in the presence of cells bearing distinct isotypes of HLA class II molecules. The main T-cell subset for TNF production was investigated in parallel. Similarly high levels of TNF production were induced upon stimulation with the toxins in the presence of DR+ or DQ+ cells, but only marginal levels of TNF production were induced in the presence of DP+ cells. Although both CD4+ T-cells and CD8+ T-cells produced TNF-alpha and TNF-beta in response to toxin stimulation in the presence of HLA class II+ cells, the former T-cell subset was the major source of producers of TNF-alpha and TNF-beta.

Improved Reversed Passive Hemagglutination for Simple and Rapid Detection of Staphylococcal Enterotoxins A~E in Food

Detection and identification of staphylococcal enterotoxins in food or culture filtrates were performed using the reversed passive hemagglutination (RPHA) technique, with formalized sheep red blood cells (FSRBC) sensitized with immunoglobulins of anti‐A, B, C, D, and E rabbit hyperimmune sera fractionated by affinity chromatography. The FSRBC sensitized with anti‐A~E immunoglobulins showed a high level of reactivity and specificity in RPHA, against homologous types of purified enterotoxins and culture filtrates of toxin‐producing strains. No non‐specific reactions with various ingredients in foods nor cross‐reactions among enterotoxin types were observed. The minimum amount of enterotoxins in foods detected by RPHA was calculated to be 0.01 μg/g without concentration, and the recovery rate of experimentally added toxins was calculated to be about 80%.

Bindings of Toxic Shock Syndrome Toxin-1 and Staphylococcal Enterotoxins A, B, and C to Rabbit Spleen Cells

Toxic shock syndrome toxin‐1 (TSST‐1) and staphylococcal enterotoxins (SE) A, B, and C were studied on binding to rabbit spleen cells. The toxins showed remarkable mitogenic effects on the cells. Among them, SEA and TSST‐1 had much stronger mitogenic activities than SEB and SEC. Binding study showed that labeled TSST‐1 and SEA bound considerably to cells, but that labeled SEB or SEC was not observed to bind at a detectable level under the same conditions as TSST‐1 and SEA. Competitive binding analysis between toxins to cells proved that TSST‐1 and SEA clearly competed with each other in binding. Scatchard plots for TSST‐1 and SEA in binding were linear at the doses used. The Scatchard analysis for TSST‐1 and SEA gave a dissociation constant of 2.5 × 10−9 m and 7.6 × 10−8 m and the number of binding sites per cell of 5.3 × 103 and 1.0 × 105, respectively.

Heterogeneity of Staphylococcal Enterotoxin A on Isoelectric Focusing and Disc Electrophoresis

Heterogeneity of purified staphylococcal enterotoxin A, obtained from a culture supernatant of Staphylococcus aureus, strain 13N‐2909, was demonstrated by isoelectric focusing. The toxin was composed of three immunologically identical fractions with isoelectric points of 6.5, 7.0 and approximately 8.0. Heterogeneity of the toxin was also shown by disc electrophoresis. At pH 8.0 and 9.4 two major bands and a faint minor band were observed, while at pH 4.3 only one band was observed. The faster‐moving band for the anode in disc electrophoresis at pH 9.4 was found to correspond with the pI 6.5 component from isoelectric focusing, while the slower‐moving band corresponded with the pI 7.0 component. From the results of the electrophoretic migration tests of the toxin, the components corresponding to the two major bands found in disc electrophoresis at pH 9.4 were considered to be charge isomers.

Shigella dysenteriae strains possessing a new serovar isolated from imported diarrheal cases in Japan

Two Shigella strains (93-119 and 95-619) isolated from stool cultures of imported diarrheal cases in Japan, did not react to any antisera of the established SHigella serovars. These strains had the typical biochemical characteristics of Shigella dysenteriae, and were biochemically identical to each other. Both strains were positive in the Serény test and other tests for invasiveness; these indicate that they can cause shigellosis in humans. The results of antigenic analysis showed that they did not belong to any of the recognized or provisional serovars, and were serologically indistinguishable. Strain 93-119 is designated as the test strain for this new serovar.