A simple method for quantitation of cell-bound protein A (SpA) on organisms of Staphylococcus aureus was successfully devised by using hemagglutination between staphylococcal organisms and a series of sheep erythrocyte suspensions sensitized with different amounts of anti sheep erythrocyte rabbit antiserum. The validity of the principle and the reproducibility of the method presented here were precisely analyzed and the details of the method are presented. The hemagglutination was quantitatively inhibited both by normal rabbit serum and by soluble SpA. Using the method presented here, 376 strains of S. aureus freshly isolated from clinical materials were subjected to SpA quantitation in cell-bound form. According to the results, there was an unexpected distribution profile in the amounts of cell-bound SpA among the clinical isolates, which showed a two-peak pattern. A possible usefulness of the method presented here in clinical investigations is briefly discussed also.

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Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. The surface of a M. luteus cell consisted of two or three areas with borders-the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area. Gold particles were observed uniformly and densely on the whole cell surface. However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A-gold particles. Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M. luteus IFO 3333.

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Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti-coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6-amino caproic acid, polyethylene glycol 6, 000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type-specifically inhibited in the presence of type-specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.

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Streptococcal erythrogenic toxin type A (ET-A) was purified from culture filtrate of Streptococcus pyogenes strain NY-5 grown in a chemically defined synthetic medium NCTC-135. We succeeded in simplifying the purification procedure, and obtained a highly purified preparation of ET-A. The purification procedure was the combination of ultrafiltration with Amicon PM-10 and YM-10 membranes, chromatofocusing with PBE-94 exchanger (pH 4.0-6.0), and gel filtration through Sephacryl S-200. The purified toxin protein showed a single band with M_r 28, 000 on SDS-PAGE and had pI 5.2 on agarose IEF. HPLC chromatography pattern of the toxin revealed one symmetric peak. The result of amino acid analysis of the toxin was in accordance with that of Gerlach et al and with Weeks and Ferretti who reported the nucleotide sequence of the spe A gene. Biological activities of the purified toxin were remarkably potent. The mitogenic activity for rabbit lymphocytes and one skin test dose in rabbit were found at the lower dose of 10pg and 1ng of the toxin, respectively.

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Two strains of hemorrhagic fever with renal syndrome (HFRS) virus from Rattus, SR-11 and KI-262, showed virtually identical antigenicity but differed from prototype strain Hantaan 76-118 (Apodemus origin) in a neutralization test. Wistar newborn rats inoculated intraperitoneally (i.p.) with SR-11, which was isolated from a laboratory rat associated with an outbreak of HFRS, developed clinical signs such as ataxia and limb paralysis and died at about 18 days after inoculation. The LD₅₀ of SR-11 in 1-day-old rats was 10^1.2 focus-forming units (FFU). In contrast, the animals inoculated i.p. or intracerebrally with 10⁴ FFU of KI-262, which was from a wild rat in a dumping-ground area-an enzootic focus where no human cases have been recorded-did not show any significant clinical signs. The susceptibility of rats to SR-11 fatal infection was age-dependent. Virus titers in brains, lungs, kidneys, and livers of the rats inoculated with SR-11 were significantly higher than those in the same organs of the animals infected with KI-262. Necrosis of neurons in the brain tissue occurred in the rats infected with SR-11, while it was mild in the animals infected with KI-262.

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During the three-year period from 1984 to 1987, 506 acute gastroenteritis outbreaks involving 14, 383 patients were reported to the Bureau of Public Health, Tokyo Metropolitan Government. Eighty (4, 324 patients) of 150 outbreaks (4, 860 patients) from which etiologic agents were not identified were subjected to virological investigation. Spherical particles of 28-32nm in diameter with capsomere-like structures on the surface were detected in patients' stool specimens. Buoyant density of the particles appeared to be 1.36 to 1.40g/ml in CsCl. Seroconversion to the particles was observed in patients by immune electron microscopy. From these observations, we concluded that the detected particles were members of small round structured virus (SRSV), and that they were implicated in the etiologically ill-defined outbreaks encountered. Prevalence of SRSV infections in these outbreaks was examined by electron microscopy. SRSV was positive in 83.8% of the outbreaks, and 96.4% of the cases. SRSV-positive outbreaks usually occurred during winter in contrast to bacterial outbreaks which often occurred in the summer season. Of 80 outbreaks examined, 53 were associated with the ingestion of oysters, and the remaining 27 mostly with food other than oysters. Oyster-associated outbreaks usually occurred on a small scale, while unassociated ones on diverse scales ranged from family clusters to large outbreaks.

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Water extraction method was applied to isolate the cell membrane from line 10 hepatoma cells and normal liver cells in strain 2 guinea pig. The materials isolated by this method were further analyzed by different immunochemical techiques including SDS-PAGE, crossed immunoelectrophoresis and crossed affino immunoelectrophoresis to demonstrate the major components and their antigenicities. Five major glycoproteins of apparent molecular weights of 44, 46, 62, 64, and 68 kDa were prominent in line 10 tumor cell materials, whereas one band of molecular weight of 82kDa was prominent in the materials from normal liver cells. Also four minor components from line 10 tumor cells were found to be glycoprotein in nature.

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The role of interleukin 1 (IL1) in causing IL2 and interferon-γ (IFN-γ) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-γ when stimulated with phytohemagglutinin (PHA) or with a combination of phorbolmyristate-acetate (PMA) and Ca²⁺ ionophore, ionomycin, showed marked IL2 and IFN-γ production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-γ production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-γ mRNA than IL2 mRNA. In contrast to the IL2 and IFN-γ gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.

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A B-cell subpopulation (B_M-A cell) responding to an antigen with the production of IgM and IgA plaque-forming cells but not of IgG plaque-forming cells was isolated from neonatally bursectomized chickens and was examined for the mode of activation by B-cell mitogens. The B_M-A cells did not elevate both glucose consumption and protein synthesis with the B-cell mitogens, in striking contrast to normal B cells. The B_M-A cells were also not activated by an activator of protein kinase C, phorbol myristate acetate. Both anti-Ig and a calcium ionophore, A23187, however, primed the B_M-A cells to increase intracellular free calcium ion as well as normal B cells. From these results it is conceived that the lack of protein kinase C activation may be responsible for the failure of activation of B_M-A cells.

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