Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was performed to clarify the roles of primary and secondary injections of antigen and adjuvant (capsular polysaccharide of Klebsiella pneumoniae, CPS-K) in induction of antibody responses and in development of immunological memory in mice to bovine serum albumin (BSA). A primary injecion of BSA alone neither induced significant primary antibody response nor increased immunological memory for a secondary antibody response but, if primary injections of BSA and CPS-K were performed simultaneously, high antibody responses were induced. Moreover, a prior injection of BSA alone or CPS-K alone decreased the level of primary antibody response and the degree of increase in memory following the subsequent injection of BSA mixed with CPS-K. In contrast, a secondary injection of BSA alone into mice once primed with a mixture of BSA and CPS-K elicited very high secondary type antibody response and increased secondarily the memory for a tertiary antibody response. Injection of CPS-K simultaneously with or shortly before or after the secondary injection of BSA did not increase the level of the secondary antibody response and the degree of the secondary increase in memory. Augmentation of the secondary antibody response was elicited by simultaneous injection of CPS-K only when the secondary response was induced inadequately by a suboptimum or supraoptimum dose of antigen.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Study was made to clarify the experimental conditions for the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to exhibit maximum adjuvant effect on antibody production to bovine serum albumin (BSA) in mice. To obtain the maximum primary antibody response and also the strongest priming for a secondary response to BSA, 1000 μg of CPS-K had to be injected intramuscularly into the same or adjacent site of BSA injection within the period of 1 hr before to 6 hr after the BSA injection. The optimum amount of BSA giving the maximum antibody response and also the strongest priming under these experimental conditions was 15 mg. In mice thus primed, an extremely high secondary response was induced by injecting 0.5 mg of BSA 30 days after the initial injection. The minimum amount of CPS-K, to exhibit a strong adjuvant action, was 100 μg, which was equal to the minimum amount to induce immunologic paralysis to a homologous antigen. Extremely large amounts, such as 100 to 300 mg per mouse of BSA, were also strongly immunogenic when injected together with paralyzing doses of CPS-K. In vitro admixture of BSA and CPS-K before injection did not strengthen adjuvant action of CPS-K. Alum-precipitated BSA mixed with CPS-K was not more immunogenic than native BSA mixed with CPS-K. Addition of Freund's complete adjuvant to an injection of BSA and CPS-K mixture did not enhance the adjuvant effect of CPS-K.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was made to characterize the active substance for the extraordinarily strong adjuvant effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain. CPS-K was fractionated into acidic and neutral CPS-K by the addition of cetyl-pyridinium chloride. Neutral CPS-K exhibited an extremely strong adjuvant effect. The active substance in neutral CPS-K was precipitable when mixed with a rabbit homologous antiserum. The neutral CPS-K antigen was serologically distinct from the O antigen and from the acidic CPS-K which was the type-specific capsular antigen. Among preparations of neutral CPS-K from eight different strains of K. pneumoniae tested, the preparation from only one strain (MH-2) exhibited a strong adjuvant effect comparable to that of the neutral CPS-K from the Kasuya strain. The neutral CPS-Ks from Kasuya and MH-2 strains were antigenically identical. This antigen was not found in all preparations of neutral CPS-Ks obtained from seven different strains. Preparations of acidic CPS-Ks from all strains of K. pneumoniae tested with various serologic types including Kasuya and MH-2 strains were found to exhibit only weak adjuvant effects. The active substance (neutral CPS-K antigen from Kasuya strain) was shown to form a single peak upon analyses by gel filtration (Sephadex G-100) and ultracentrifugation. Sedimentation coefficient of the substance was approximately 20 S at a concentration of 5 mg per ml in 0.1 M NaCl. The active substance finally purified by gel filtration contained 65% sugars (as glucose equivalents), 6.8% hexuronic acids, 2.6% hexosamine, 2.3% proteins, and very small amounts of lipids.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.     

Initiation of the Primary Immune Response to Sheep Red Blood Cells in the Dissociated Mouse Spleen Cell Culture

From a suspension of mouse spleen cells were separated two functionally different cell types on the basis of their ability or inability to adhere to plastic dishes during a short period of incubation. Morphological observations of cells of these two fractions were made with the aid of histochemical methods. The majority of cells in the adherent fraction possessed β-glucuronidase, which is one of the lysosomal enzymes rich in macrophages or phagocytic cells. In contrast, almost all cells in the non-adherent fraction were devoid of this enzyme activity and identified morphologically as small lymphocytes. The adherent and non-adherent cells were found to associate in cell clusters during the cultivation. In the cultures stimulated with sheep red blood cells, some of the non-adherent cells which were located peripherically in the cell clusters began to show alkaline phosphatase activity in their cytoplasm. This may perhaps indicate that antibody synthesis is going on in these alkaline phosphatase-positive cells, since in an in vivo study such cells were found to arise in the lymph nodes of immunized animals concomitantly with the appearance of specific serum antibody.     

Electrical Sizing of Particles in Suspensions: III. Rigid Spheroids and Red Blood Cells

The processes involved during the passage of a suspended particle through a small cylindrical orifice across which exists an electric field are investigated experimentally for an approximate prolate spheroid in the form of two tangent, rigid spheres (ragweed pollen particles) and for fresh, human red blood cells. Oscillograms of current pulses produced by both types of particles are presented and discussed in terms of particle shape and orientation and the effects of the hydrodynamic field. It is concluded that all the particles enter the orifice with their major axes aligned parallel to the orifice axis (electric field), but that during their passage some are rotated by the hydrodynamic field. Cells with their equatorial plane perpendicular to a radius of the orifice change their orientation with respect to the electric field as they are rotated, the others do not; only in the former case is there any deformation. It is shown that the bimodal or skewed size distributions can be explained on this basis, and that size (shape factor × volume) is actually a normally distributed variable (P > 95%). The average size of samples from 10 healthy adults was found to be 102.7 μ³ with a coefficient of variation of 1.8%. For a volume of 87 μ³, this corresponds to a shape factor of 1.18, an axial ratio (assuming a perfect oblate spheroid) of 0.26, and an equivalent major axis of 8.6 μ. The effect of high electric fields on red cell size distributions is mentioned.     

Polysaccharide Produced by the Genus Pullularia: I. Production of Polysaccharide by Growing Cells

A dextranlike polysaccharide was found to be produced on substrates of sucrose, maltose, glucose, and fructose by growing cells of various strains of the genus Pullularia. The polysaccharide, obtainable in amounts as large as 2 to 3 g per 100 ml of culture medium using various carbohydrates as the carbon source, was soluble in cold water but not in 50% alcohol. The polysaccharide obtained had a α_d = +197.5° (c = 0.2 in water), and its molecular weight, determined by the light-scattering method, was found to be approximately 250,000.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on host resistance to bacterial infections. III. Further study of its effects on interactions between peritoneal leukocytes and virulent Salmonella enteritidis.

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 the 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.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane.

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.     

Studies of the Biochemical Background to Differences in Glucosinolate Content in Brassica napus L. III. Further Studies to Localize Metabolic Blocks

To localize the metabolic block(s) in the biosynthesis of glucosinolates in the Brassica napus L. cv. Bronowski, 5-methylthiopentanal oxime-1-¹⁴C was synthesized and fed to this cultivar and cv. Regina II, which has average levels of glucosinolates. The aldoxime was as efficient as a precursor of 3-butenylglucosinolate in both cultivars, but less efficient as a precursor of 2-hydroxy-3-butenylglucosinolate in Bronowski. These results suggest that there is a block in the biosynthesis of 3-butenylglucosinolate in Bronowski that is situated before the synthesis of the intermediate 5-methylthiopentanal oxime, as well as a block in the hydroxylation step. The silique walls of Bronowski contained increased amounts of total sulphur and inorganic sulphate.