The antigenicity of lateral (L-) flagella of two marine vibrios, Vibrio alginolvticus and V. harveyi, was studied, and the two species were found to have common antigenicity of their flagella. Antisera against L-flagella were prepared by immunizing rabbits with highly purified L-flagellar filaments. H-Agglutination tests with the anti-L-flagella antisera showed that four H-serovars existed in these species and that two of them were shared by the two species. Cross reactivity between H-serovars of these two species and other vibrios having lateral flagella, such as V. parahaemolyticus, V. campbellii, V. proteus, or V. fluvialis, was not observed in the H-agglutination test, although partial common antigenicity was observed in the gel diffusion test with flagellin monomers. These observations suggest that surface antigenic determinants of the lateral flagella of V. alginolyticus and V. harveyi are specific to these two species but internal antigenic determinants buried in the flagellar filaments are partially shared with other vibrio species.

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The immunomodulating effects of staphylococcal enterotoxins on in vivo immune responses in C57BL/6 mice were examined. Of the five serological types A (SEA), B, C, D, and E (SEE), only SEA and SEE markedly suppressed the antibody response to sheep red blood cells (SRBC) when injected 1 day before or on the day of immunization with SRBC.
Further study of SEA revealed that it did not affect the antibody response to a thymus-independent antigen, salmonella flagella, but did affect the T-cell-mediated immune response. Contact sensitivity to dinitrofluorobenzene (DNFB) was suppressed when SEA was injected before sensitization or before challenge with DNFB, indicating that SEA affected both the afferent and efferent phases of DNFB contact sensitivity. As the suppression of DNFB contact sensitivity could be transferred by anti-Thy-1.2 antibody-sensitive spleen cells of SEA injected donors into normal or DNFB-sensitized recipients, the suppression was thought to be an active one. However, SEA could augment the DNFB contact sensitivity when injected on the third day after sensitization with DNFB.
These results indicate that the immunomodulating effects of SEA can be mediated by the T-cell function.

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Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T-types, we found that the patterns were quite characteristic for their T-types, especially in the prevalent T-types 12 and 1, and that the isolates of T-types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti-DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T-types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T-types, suggesting the physicochemical taxonomic value of these properties.

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Klebsiella O3 lipopolysaccharide (KO3 LPS) was found to exhibit extraordinarily strong adjuvant activity in augmenting antibody responses and delayed-type hypersensitivity (DTH) to protein antigens in mice. The O-specific polysaccharide chain of KO3 LPS consists of α-mannoside. We investigated the effect of concanavalin A (Con A) or succinyl Con A, which is known to bind to α-mannoside, on the adjuvant activity of KO3 LPS in augmenting DTH to ovalbumin. When KO3 LPS was mixed with Con A prior to injection, the strong adjuvant activity of KO3 LPS in augmenting DTH was inhibited and the degree of inhibition depended upon the dose of Con A. An equal amount of Con A elicited nearly complete inhibition of the adjuvant activity of KO3 LPS, Con A at 1/10 the amount of LPS elicited partial inhibition, and Con A at 1/100 the amount of LPS showed no inhibition. An equal amount of succinyl Con A, which induced less marked aggregation of KO3 LPS than Con A, elicited inhibition of the adjuvant activity of KO3 LPS to an extent similar to that by Con A. On the other hand, Con A or succinyl Con A bound to KO3 LPS did not impair in any way the lethal toxicity of KO3 LPS for mice which is known to be due to the lipid A moiety. From these findings it is concluded that the strong adjuvant activity of KO3 LPS does not solely depend upon the lipid A moiety but the O-specific polysaccharide moiety plays an important ole in expression of the adjuvant activity.

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Both a saltd-ependent form and an active form of glucose dehydrogenase [EC 1.1.1.47] were isolated from germinated spores of Bacillus subtilis disrupted in deionized water and 100mM phosphate buffer (pH 6.6), and most of the enzyme isolated from young vegetative cells was the active form regardless of the conditions for breakage by sonication. The molecular weight of the salt-dependent form of the enzyme was about 55, 000 and that of the active form was about 120, 000. From the above results and the results on the glucose dehydrogenase extracted from resting spores disrupted in deionized water and 100mM phosphate buffer (pH 6.6) reported in a previous paper, we propose that glucose dehydrogenase is present in resting spores as a monomeric form and is activated with association in vivo during germination and outgrowth.

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The ability of 33 compounds of L-alanine analogues over a wide range of concentrations to initiate germination of Bacillus subtilis spores was determined, and the inhibitory activity against L-alanine-initiated germination was determined for the above compounds and 22 of their D- and DL-isomers. Nineteen L-isomers were able to initiate the germination. The maximum germination rate and the apparent binding affinity of the germinant were obtained from concentrationgermination response curves. Not only D-isomers but also L-isomers of many alanine analogues showed inhibitory action on L-alanine-initiated germination. The apparent binding affinity of an inhibitor was calculated by Schild's method. D-Alanine, D-serine, glycine, D-2-amino-n-butyric acid, D-cysteine, D-norvaline, and D-threonine were competitive inhibitors for the L-alanine action. Analysis of the relation between the structure of the side chain of L- and D-alanine analogues and their apparent affinity suggested that there are separate binding portions, which differ in size and electrostatic nature, for germination and for inhibition on the receptor. Certain L-alanine analogues had a dualistic property of initiating germination at low concentrations and inhibitory activity at higher concentrations, i.e., autoinhibition. The autoinhibitory phenomenon might be explained by the above postulation of the presence of separate binding portions for germination and for inhibition.

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The fecal flora of 10 infants with vitamin K deficiency (VKD) and 10 healthy infants was examined. All the infants were breast-fed. In the infants with VKD, the total counts (P<0.01) and the numbers of bifidobacteria (P<0.001) were lower than in the healthy infants, whereas bacteroides, veillonella and enterococci were present in greater numbers in the feces of infants with VKD. The incidence of the Bacteroides fragilis group was higher (P<0.05) in the infants with VKD than in the healthy infants. A significant reduction (P<0.05) in Biftdobacterium breve was shown in the infants with VKD. The data emphasize the abnormal flora in infants with VKD.

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Splenic lymphocytes from BALB/c mice immunized with “cores” of influenza virus, obtained after bromelain cleavage of the surface glyeoprotein, were fused with the P3-NS1/1-Ag-1 mouse cell line to yield hybridoma cultures. Among 20 stable cloned hybrid cells secreting monoclonal antibodies, one was specific for the nucleoprotein (NP), 11 were specific for the membrane (M) protein and eight were specific for the hemagglutinin (HA). These “cores” used as immunogen contained only the internal proteins of the influenza virus, namely the three polymerases, the NP and the M protein and no HA when examined by standard procedures of SDS-PAGE, electron microscopy and hemagglutination activity. It thus appeared that a small amount of contaminating antigens can sensitize a sufficient number of mouse B cells to be selected as hybrid partners. These antibodies were provisionally assigned as anti-carbohydrate attached to the HA.

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In leprosy, the common etiologic agent is the same Mycobacterium leprae, but the clinical manifestations are various, including the tuberculoid and lepromatous types. In tuberculoid type leprosy, macrophages in the granuloma differentiate into epithelioid cells; in the lepromatous type, in contrast, they differentiate into lepra cells containing multiple M. leprae. Thus host factors, which regulate macrophage activities, determine the type of leprosy.
To understand such regulation of macrophage activities, we assayed superoxide production, hydrogen peroxide production and glucose consumption in monocytes in vitro.
Glucose consumption spontaneously increased, with lymphokine enhancing the consumption rate. Superoxide production increased spontaneously and decreased from the 4th day; lymphokine added on the 4th day supressed the decrease of superoxide production. Hydrogen peroxide production increased until the 3rd day of culture. Twenty-four hour incubation with lymphokine, from day 0 to the 1st day, had no effect on hydrogen peroxide production, while from the 2nd to 3rd day it enhanced such production. Supernatants of lymphocytes incubated with M. leprae were prepared from tuberculoid and lepromatous patients. Tuberculoid supernatant enhanced reactive oxygen production and glucose consumption, while that from lepromatous patients had no remarkable effect on glucose consumption or reactive oxygen production. The range of spontaneous increase and decrease of reactive oxygen production was greater than the regulatory effect of lymphokine on these activities.
These data show that rapid provision of new monocytes to the granuloma is one of the important factors in the defense mechanism, that lymphocytes separated from lepromatous patients are not activated in response to M. leprae antigen, and that they do not secrete corresponding lymphokines.

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Four monoclonal antibodies to Legionella pneumophila Philadelphia 1 were produced by the fusion of immunized BALB/c lymphocytes to a murine myeloma cell line. Two (Lp1-1 and Lp1-3) of the four monoclonal antibodies reacted with 14 L. pneumophila serogroup 1 strains, and the other (Lp1-2 and Lp1-4) reacted with only three out of 20 strains tested. These four monoclonal antibodies did not bind to any strains of L. pneumophila serogroup 2-7 and other Legionella species. In addition, it has been shown that these monoclonal antibodies may be useful not only for subserotyping of L. pneumophila but also for retrospective diagnosis using immunopathological methods.

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