The addition of adjuvant (Bacillus Calmette-Guérin-cell wall skeleton; BCG-CWS) to the culture medium substantially increased the immune response of mouse spleen cells to immunization with heterologous erythrocytes and hapten-protein conjugate in vitro. Cell walls of mycobacteria, nocardia and corynebacteria and their cell wall constituents were used as adjuvants. In the present case, it was found that cell walls of mycobacteria, nocardia and corynebacteria markedly facilitated primary humoral response of mouse spleen cells to heterologous erythrocytes in vitro. The adjuvant effect of BCG-CWS was present only in the formation of 19 S antibody in both primary and secondary responses, but not in that of 7 S antibody in vitro. Primary antihapten response of mouse spleen cells against dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) also succeeded when BCG-CWS was added to the culture medium, and it was found that BCG-CWS increased the helper activity of carrier specific helper T cells in vitro where a double chamber system, separated by a cell-impermeable nucleopore membrane, was used. This result suggested that BCG-CWS acts on T cells, resulting in the release of soluble factor(s) from T cells capable of exerting an adjuvant effect. Furthermore, mucopeptide moiety of BCG-CWS retained some adjuvant activity, but other cell wall constituents, such as mycolic acid, arabinose mycolate, and arabinogalactan, did not show any adjuvant effect in vitro. These results strongly imply that mucopeptide moiety of BCG-CWS plays an important role in the development of adjuvanticity.

View full abstract

Two different growth media, one based on Eagle's minimum essential medium (MEM) and the other on Earle's balanced salt solution-lactalbumin hydrolysate-yeast extract (YLE), were used for growing primary chick embryo cells (CEC), and resistance to viral infection and interferon production induced by polyinosinic-polycytidylic acid (poly I·poly C) were compared. In CEC grown in Eagle's MEM, treatment with poly I·poly C at a concentration as low as 1.0 ng/ml was sufficient to induce a detectable resistance to infection with vesicular stomatitis virus (VSV), while more than 300-fold concentrated poly I·poly C was required to induce a similar resistance when the cells were grown in YLE. The cells grown in YLE did not produce an appreciable amount of interferon, whereas a significantly higher level of interferon was produced by the cells grown in Eagle's MEM. A similar phenomenon was observed in the interferon production of chick embryo cells treated with ultraviolet light (UV)-irradiated Newcastle disease virus (NDV) and in the induction of resistance to vaccinia virus in cells treated with poly I·poly C. It was found that the response of cells, bathing in one growth medium, to poly I·poly C was not affected by replacing it with the other at the same time with the addition of poly I·poly C, and that the response of CEC was strongly dependent upon the medium used for cultivation. These facts suggested that the observed difference in the response of cells to poly I·poly C was not due to a direct interaction between the inducer and medium components but to the physiological state of CEC established during their growth. Which component of YLE was responsible for such a lowered response of cells to poly I·poly C was also examined, and the marked reduction of PDD₅₀ by the replacement of lactalbumin hydrolysate of YLE with amino acids and the increase of PDD₅₀ by addition of lactalbumin hydrolysate to MEM suggested that lactalbumin hydrolysate might play an important role in this phenomenon.

View full abstract

Spores of Clostridium perfringens NCTC 8238 underwent so-called "ionic germination" under suitable conditions in aqueous solutions of either inorganic or organic salts. Chlorides of alkali metal ions except for Na⁺ were the most effective in inducing germination. Chlorides of alkali earth metal ions were effective to some extent, but those of heavy metal ions were rather inactive. Potassium salts of halogens, nitrogen, sulfur and phosphorus, as well as organic acids including fatty acids and polycarboxylic acids were also germinative. Preheating the spores at 80 C for 10 min was essential for rapid and complete germination by these salts. Maximum germination occurred at an incubation temperature of 40 C and at a fairly wide range of pH values (5.0-9.0). The germination was markedly inhibited by various uncoupling reagents such as carbonylcyanide m-chlorophenylhydrazone, pentachlorophenol, hexachlorophene (HCP), and dicumarol. This inhibition was reversible because removal of these reagents allowed germination to proceed. The inhibiting effect of HCP was highly dependent on pH, suggesting that a molecular dissociation of the reagent affects the inhibition. Furthermore, the inhibition of germination by HCP was competitively overcome at higher KCl concentrations. The possible mechanism of ionic germination and the inhibitory effect of uncouplers on the ionic germination are discussed.

View full abstract

Antiviral activity of scillarenin, a plant bufadienolide, and its mode of action were studied. Scillarenin selectively inhibited replication of picornaviruses, in particular, rhinovirus. At concentrations inhibiting rhinovirus replication, scillarenin had no effect on the synthesis of macromolecules in host cells. The action of scillarenin was not direct inactivation of virus. Scillarenin could inhibit virus replication when given at the latter half of latent period, although this treatment did not interfere with the normal level of viral RNA synthesis. The RNA synthesized in the presence of this compound showed the same sedimentation coefficient as normal viral RNA but possessed a reduced infectivity.

View full abstract

A new procedure of assaying interferon (IF) has been developed. Cell suspension was dispensed into liquid scintillation counting vials together with IF sample. During an overnight incubation, the cells adhered sufficiently to the bottom of the vials and all the subsequent procedures were carried out without transfer of the cells from the vials. Vesicular stomatitis virus was inoculated and virusspecific RNA was labeled by adding ³H-uridine and actinomycin D to the medium. Incubation was terminated prior to completion of a single-step growth of the virus and radioactivity of the labeled cells in each vial was determined. The reciprocal of the IF dilution which reduced the radioactivity in viral RNA by 50% was taken as the titer. The present procedure consists of simple manipulations and can be completed within 24 hr. Furthermore, it is quite reproducible and gives a titer almost identical to that obtained by the conventional plaque-reduction dose method. The procedure can be applied to mouse L cells, rabbit RK-13 cells and human FL cells, without modification.

View full abstract

Genetic analyses of the loci conferring resistance to antibiotics known to affect protein synthesis were made employing the conjugation system of Mycobacterium smegmatis. By the complementation tests, vic (viomycin-capreomycin-resistant) mutants were classified into two different groups; cistrons A and B. Neomycin-kanamyein-resistant (nek) mutants, on the contrary, fall into a single cistron. Erythromycin-resistance (ery) locus was linked to vic-nek region. The map order deduced was argB, argA, (vicA, vicB), nek, ery. Biochemical studies showed that strains resistant to streptomycin, neomycinkanamycin or viomycin-capreomycin had altered ribosomes.

View full abstract

Biological and serological comparisons of sequential isolates of Coxsackie A16 virus (Cox A16) in 1951 through 1970 were made to elucidate the mode of appearance of the mutant which caused a nationwide epidemic of hand-foot-and-mouth disease (HFMD) in 1970. Most strains isolated from feces of healthy children in 1964, 1966 and 1967 were serologically similar to the prototype strain isolated in 1951. However, some strains isolated from these healthy children in 1966 and 1967 were serologically and biologically related more closely to the epidemic strain isolated in 1970. Thus sequential isolates of Cox A16 could be roughly classified into two groups, i.e., the prototype strain-like and the epidemic strain-like groups.

View full abstract

Freeze-etching of Lactobacillus fermenti F-4 (NCTC 7230) revealed that the outer layer of the cell wall was composed of a regular array in which parallel lines ran obliquely to the longitudinal axis of the cell with an average distance between the centers of about 9.6 nm and were intersected by thinner lines with an average periodicity of approximately 6.2 nm at an angle of about 75°. Occasionally the direction of the striation was discontinuously shifted near one end of the cell, Beneath the regular array the middle cell wall layer packed with granules and the smooth inner cell wall layer were discernible and the mesosomes were also visible in the cytoplasm. When the ultrastructure of isolated outer cell wall fragments was examined by negative staining, the regular array appeared to be composed of subunits, about 3.6 nm in diameter, which were arranged in a tetragonal pattern. The tetragonal array consisted of the subunits in rows in two directions at an angle of about 75° to each other. The average spacing between the rows was about 9.3 nm in one direction and 5.5 nm in the other direction.

View full abstract