The dried cells of two strains of Vibrio parahaemolyticus were fractionated by extracting first with water and then with organic solvents. The hemolytic activity of the fractions was determined, and some of them were assayed for their effect in mice. The hemolytic agent present in the water-insoluble fraction was extractable in organic solvents such as 70% aqueous ethanol, chloroform-methanol-water (1:2:0.8) and acetone. The extracts showed no toxic effect in mice after intraperitoneal inoculation. No hemolytic activity was observed in the remaining cell residue, which bore the toxicity.

View full abstract

Clostridium perfringens type C strain CN 5384 produced a higher level of beta toxin in a controlled pH medium containing 1% glucose, starch, or sucrose than in media with dextrin, fructose, or raffinose. Toxin synthesis was not related to the growth yield.
The effect of glucose on beta toxin production by 11 strains was investigated with and without control of the culture pH at 7.5. Strain CN 5386 produced distinctly higher toxin when the pH of the culture was maintained at 7.5, compared with uncontrolled pH.

View full abstract

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/ or threonine as well as several other amino acids in OMZ 176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.

View full abstract

The process of cell separation and arrangement of Staphylococcus was investigated using a scanning electron microscope.
After two cycles of cell division, the Staphylococcal cells cultured on an agar medium were generally observed to be arranged in three morphological types : linear, square, and crooked arrangements. Results of the examination of cell surface structure revealed that separations had occurred in these clustered cells following two patterns. One type of second separation occurred parallel to the transversal axis of the preceding pair of the parental cells (X-type) and the other occurred tangential to it (Y-type). In the former type, the four daughter cells were usually arranged tetragonally after the separations, and in the latter type they were arranged either linearly or crookedly depending on the direction of the second separation. The final pattern of the cell arrangement was thus determined by the type of septal wall formation and the direction of cell separation. After several cycles of cell divisions, the cells were finally arranged in an irregular grape-like cluster, even though the cross walls were formed regularly at the rectangular face of the preceding cross walls.

View full abstract

Spores of Bacillus cereus T treated with trichloroacetic acid (6.1-61.2 mM) were compared with untreated spores, and as the concentration of the chemical increased, the following alterations in spore properties were found : (1) the extent of germination decreased irrespective of the germination medium used; (2) the spores became sensitive to sodium hydroxide (1 N) and hydrochloric acid (0.27 N), but not to lysozyme (200 μg/ml); (3) loss of dipicolinate increased on subsequent heating; and (4) the spores became more sensitive to heat. However, trichloroacetic acid-treated spores were still viable and there was no significant change in spore components. The mechanism of action of trichloroacetic acid is discussed.

View full abstract

Seroconversion to human cytomegalovirus (H-CMV) -specific antigens was observed in 8 out of 9 renal transplant recipients. In 7 of the 8 recipients antibody to “pre-early nuclear antigens” (PENA), which are detectable in human embryonic lung cells within 1 hr of H-CMV infection by anti-complement immunofluorescence staining, developed concomitantly with the increase in other antibodies including anti-early antigens (EA), anti-nuclear inclusions (NI), and complement-fixing (CF) antibody in 1-2 months after transplantation. About 1 year later, anti-PENA and anti-EA titers were concomitantly decreased in 2 recipients, whereas anti-NI and CF antibody titers were maintained at elevated levels in all the seroconverted recipients. These results support the idea that the development of antibody to PENA, like antibody to EA, may represent a current or recent infection with (or reactivation of) H-CMV. In one patient, antibody to PENA did not develop through the observation period despite increases in antibody to EA and other antibodies; this lends support to immunological distinctness of PENA from EA.

View full abstract

In vivo presensitization of donor mice of responding cells with third party cellular antigens augmented in vitro generation of cytotoxic T lymphocytes in allogeneic and xenogeneic combinations. In vitro induction of detectable cytotoxicity in presensitized responding cells required the incubation period needed for in vitro primary response. However, such cytotoxic T lymphocytes were generated after in vitro stimulation with monolayers of methylcholanthrene-induced tumor cells, UV-irradiated or heated spleen cells which had proved to be effective in secondary but not in primary response. Presensitized responding cells exposed to 600R-irradiation did not augment in vitro induction of cytotoxicity in normal responding cells. The augmenting effect of presensitized responding cells may be attributable to radiosensitive T cells which are in a transitional state in differentiation from typical unprimed cells to typical primed cells.

View full abstract

The mechanism for the infection-promoting effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) was investigated using the experimental system in which mice were infected intraperitoneally (i.p.) with a virulent strain of Salmonella enteritidis immediately after i.p. injection of CPS-K. In the peritoneal phagocytes of CPS-K-untreated control mice, approximately 70, 3, and 10% of phagocytized bacteria survived 6, 12, and 24 hr after challenge, respectively, when calculated from the ratio of the number of cell-associated viable bacteria, which was estimated by direct plate count, to the number of phagocytized bacteria, which was estimated by microscopic observation of stained smears. In contrast, almost all of the phagocytized bacteria were viable throughout the experimental period in mice treated with CPS-K. The electron microscopical findings of the phagocytes obtained 12 hr after challenge showed that in the cells of mice treated with CPS-K almost all of phagocytized bacteria were morphologically intact, with some of them in the stages of cell division, whereas in those of untreated control mice, almost all of the phagocytized bacteria underwent digestive changes. When the reaction product of acid phosphatase was examined by electron microscopy in the phagocytes obtained 12 hr after challenge, the enzyme activity in the phagosomes was very low in mice treated with CPS-K in comparison with that in untreated control mice. Enzyme assays of the lysosomal and extralysosomal fractions of peritoneal cells obtained at various times after challenge also showed that release of acid phosphatase from the lysosomal fraction to the extralysosomal fraction after bacterial challenge was inhibited in peritoneal cells of mice treated with CPS-K.

View full abstract

Interferon production stimulated by the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) in BCG-infected mice was compared with that by bacterial lipopolysaccharide (LPS). Prior infection with BCG increased the responsiveness of mice to the lethal effect of neutral CPS-K as well as to that of LPS. Associated with this, BCG-infected mice showed a markedly enhanced ability to produce interferon after stimulation not only by LPS but also by neutral CPS-K. In addition, a cytotoxic factor (cytotoxin) was found to be released in the serum of BCG-infected mice after injection of these inducers. The kinetics of production of interferon and cytotoxin stimulated by neutral CPS-K were very similar to those stimulated by LPS. The time pattern of cytotoxin production was not in parallel with that of interferon production. Interferon reached a peak 2 hr and cytotoxin 3 hr after injection with these inducers. Interferon and cytotoxin produced by neutral CPS-K showed essentially the same stabilities to heating at 56 C and to treatment at pH 2 respectively as those produced by LPS. Interferon was inactivated by heating at 56 C more rapidly than cytotoxin. Cytotoxin was inactivated by treatment at pH 2 for 24 hr, whereas interferon activity was well preserved after this treatment. These results suggest that both activities are the result of different substances.

View full abstract

The time course of the occurrence of hyperreactivity in interferon and cytotoxin responses to the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) and bacterial lipopolysaccharide (LPS) and of the hyperreactivity to their lethal effects was followed after infection with BCG in SMA and ICR strains of mice. The duration of these hyperreactivities of BCG-infected mice depended on the inoculum doses of BCG. The time patterns of the hyperreactivity to the lethal effects of neutral CPS-K and LPS were similar in both strains of mice, although the maximum toxicity of LPS by the intraperitoneal route in BCG-infected mice on a weight basis was stronger than that of neutral CPS-K. Irrespective of inducer and mouse strain, the time pattern of the hyperreactivity to produce cytotoxin was similar to that of the hyperreactivity to produce interferon. The patterns for these phenomena when neutral CPS-K was used as an inducer were also similar to those when LPS was used. In ICR mice the hyperreactivity in interferon and cytotoxin responses to either neutral CPS-K or LPS decayed significantly earlier than the hyperreactivity to their lethal effects, whereas in SMA mice the occurrence of both types of hyperreactivities seemed to be associated. Therefore, it is suggested that the mechanism for the hyperreactivity in interferon and cytotoxin responses to neutral CPS-K or LPS in BCG-infected mice is not necessarily the same as that for the hyperreactivity to their lethal effects.

View full abstract

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10⁸-10⁹) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10⁹ SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice or CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3-4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.

View full abstract