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.     

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.     

A possible correlation between histological changes in regional subcutaneous tissue induced by bacterial lipopolysaccharides and their adjuvant activities

Previously it was demonstrated that Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) exhibited much stronger adjuvant action on antibody response to subcutaneously (s.c.) injected sheep red blood cells or deaggregated bovine serum albumin than did other kinds of LPS, the R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS (R-LPS), and the lipid A fractionated from KO3 LPS. We compared histological changes in the regional subcutaneous tissues of mice injected subcutaneously (s.c.) with KO3 LPS, the lipid A, and R-LPS. At the early stage after injection, KO3 LPS induced the infiltration of a large number of inflammatory cells, mainly polymorphonuclear leukocytes (PMN), at the site of injection. Neither R-LPS nor the lipid A induced the accumulation of PMN so much as KO3 LPS did. When injected s.c. with LPS from Escherichia coli O111 (EO111 LPS) and O55 (EO55 LPS), and Salmonella enteritidis (Sent LPS), the appearance of PMN at the regional site was much less than KO3 LPS. KO3 LPS could accumulate more 51Cr-labeled leukocytes at the injection site than EO111 LPS and Sent LPS. Administration of acetylsalicylic acid, which can inhibit leukocyte migration in inflammatory lesions, suppressed its adjuvant action. It was therefore suggested that the strong adjuvant action of KO3 LPS in s.c. injection might be dependent on its potent capability of accumulating PMN at the regional subcutaneous tissue. Furthermore, at the late stage after injection, the formation of several lymphoid follicles at the regional site was seen only in mice injected with KO3 LPS. It might be also related to the strong adjuvant action of KO3 LPS.     

Microbial adjuvant and autoimmunity. III. Histological studies of development of experimental autoimmune thyroiditis in mice immunized with syngeneic thyroid extract together with the capsular polysaccharide of Klebsiella pneumoniae.

Experimental autoimmune thyroiditis could be produced in SMA, C3H/He and C57BL mice by repeated injection at intervals of 30 days of syngeneic thyroid extract mixed with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a powerful adjuvant. The sequence of the development of autoimmune thyroiditis was histologically followed using SMA strain of mice, in which the thyroid lesions were most marked. A single injection of the thyroid extract mixed with CPS-K induced interstitial infiltration of small lymphocytes and other mononuclear cells, and follicular architecture was partially damaged. At early times (5 to 12 days) after the secondary injection of the material, there were aggregation of polymorphonuclear neutrophilic leukocytes with formation of microabscesses in the follicles and hyaline degeneration of small vessels in the thyroid glands, suggesting that this stage of the thyroid lesions was expression of an Arthus reaction. The maximal severity of the thyroid lesions was reached at 30 days after the secondary injection. At this time, the thyroid lesions consisted of extensive infiltration of small lymphocytes, plasma cells and other mononuclear cells with complete loss of follicular architecture and mild proliferation of fibrous connective tissues. Even at 200 days after the secondary injection, there was no evidence of spontaneous regression and resolution of the thyroid lesions. Based on the histological findings, it seems likely that in our system cell-mediated immunity plays a dominant role in initiation and maintenance of thyroiditis and humoral antibodies do so in its aggravation.     

Microbial adjuvant and autoimmunity. II. Production of lesions in mice immunized with syngeneic tissue extracts together with the capsular polysaccharide of Klebsiella pneumoniae.

The target organs of mice immunized with the respective syngeneic tissue extracts together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a powerful adjuvant were examined for production of lesions. In 15 out of 24 mice injected three times or more with syngeneic eyeball extracts and CPS-K adjuvant at intervals approximately 30 days, severe eyeball lesions developed in which the normal structure was almost completely lost. A large part of the eyeball tissue of these mice was replaced by infiltration with cells such as lymphocytes, plasma cells and other mononuclear cells and by connective tissue. No definite eye lesions developed in mice injected with CPS-K alone, eyeball extracts alone or eyeball extracts emulsified in complete Freund's adjuvant (CFA). In all of mice injected four times with thyroid gland extracts and CPS-K at intervals of approximately 30 days, definite thyroid gland lesions were produced. In three out of five mice of this group, the thyroid lesions were so severe that the normal thyroid follicular structure was almost completely lost, and a large part of the thyroid gland was replaced by infiltration with lymphocytes, plasma cells and other mononuclear cells and in part by connective tissue. In only one out of five mice injected with thyroid gland extracts emulsified in CFA, definite but milder thyroid gland lesions developed. No definite thyroid lesions developed in the remaining four mice of this group and also in any of the mice injected with thyroid gland extracts alone or CPS-K alone. Repeated injections of lymphoid tissue extracts and CPS-K also induced pathological changes in the spleen and lymph nodes, although less marked than those in the cases of the eyes and thyroid gland. The most remarkable change was a decrease in numbers of small lymphocytes at the areas surrounding the central arterioles in the white pulp of the spleen and the post-capillary venules in the cortex of the lymph nodes. From these results it has been concluded that our system can provide new and useful models for autoimmune diseases in man.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.     

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.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: III. Augmentation of the Antibody Response to Subcutaneously Injected Sheep Red Blood Cells by the Adjuvant Polysaccharide

The adjuvant action of the O3 antigen of Klebsiella (KO3) on the antibody response to sheep red blood cells (SRBC) was elucidated by injecting both KO3 and SRBC subcutaneously at the right inguinal region of SMA mice. We demonstrated that KO3 exhibits a novel ability to augment anti-SRBC plaque-forming cell responses in both the local lymph node and the spleen at a relatively late stage of immunization. Escherichia coli lipopolysaccharide, dextran sulfate and concanavalin A showed such an action only minimally. In parallel with the development of the adjuvant action, KO3 definitely activated B cells in the local lymph node polyclonally for either IgM or IgG synthesis, suggesting that the mechanism of the adjuvant action includes direct stimulation of B cells by KO3 at the late stage. Neither increase in trapping of lymphocytes in the local lymph node nor change in tissue distribution of antigen was shown to be primarily involved in the mechanism of the adjuvant action.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on the differentiation and functional capacity of macrophages cultured in vitro.

The effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) on the differentiation and functional capacity of macrophages cultured in vitro from various lymphoid tissues was investigated. In cultures of peritoneal cells, the number of macrophages did not change throughout incubation periods of from 1 hr to 3 days, and the addition of CPS-K had no affect. It appears therefore that CPS-K does not exhibit cytotoxic effects on macrophages. In cultures of spleen cells, only a small number of macrophages appeared within 1 hr, but the number of macrophages increased during further incubation. The addition of CPS-K to cultures of spleen cells at the start of incubation suppressed markedly the increase in the numbers of macrophage. This finding indicates that CPS-K blocks the process of the generation of macrophages, probably from their precursor cells in cultures of spleen cells. Only a small number of macrophages appeared in cultures of thymocytes or lymph node cells either with or without CPS-K. The phagocytic capacity of either peritoneal macrophages or macrophages generated in cultures of spleen cells was activated during incubation in vitro. Macrophages cultured in the presence of CPS-K for 24 hr or longer appeared to have an enhanced phagocytic activity, although the enhancement of their phagocytic activity by the addition of CPS-K was less marked in cultures of spleen cells than in those of peritoneal macrophages. Morphologically, macrophages in both cultures of peritoneal cells and spleen cells incubated in the presence of CPS-K for 4 days possessed much longer cytoplasmic processes than those incubated in the absence of CPS-K. From the present study, it appears that CPS-K exhibits dual effects on macrophage precursor cells and macrophages, a blocking effect on the differentiation from the former to the latter and an enhancing effect on the functional capacity of the latter.     

Differential response of B cells in the lymph node and the spleen to bacterial lipopolysaccharide

In vivo polyclonal activation of B cells in the lymph nodes and the spleens of mice injected with bacterial lipopolysaccharide (LPS) was compared. The peak of anti-trinitrophenylated sheep red blood cells plaque-forming cell (PFC) response in the lymph node was reached 6-8 days after the injection of LPS while that in the spleen was reached at 2 days. The maximal increase in the total number of Ig-producing cells in the lymph node also occurred at the later stage. These differences in time courses of polyclonal activation of B cells between the lymph node and the spleen were not due to the absence of B cells in the lymph node, migration of PFC from the spleen to the lymph node, or qualitative differences of B cells. This phenomenon was dependent on the environmental difference between the lymph node and the spleen, because B cells from the lymph node could respond to LPS rapidly in the spleen. Further, the polyclonal activation of B cells was accelerated in the lymph nodes of mice receiving prior injection of LPS. In in vitro cultures of lymph node cells of those mice, a significant amount of interleukin-1 could be detected by stimulation of LPS. It was possible that the delayed activation of B cells in the lymph node was due to the time lag necessary for construction of the environmental condition suitable for activation of B cells, whereas in the spleen this condition can be provided without delay.     

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.     

Systemic IL-1 and adjuvant treatment of an experimental tumor

In this investigation, systemic administration of interleukin-1 (IL-1) and local adjuvant therapy were shown to modify immunological parameters associated with the lymphatics draining the site of experimental tumor inoculation. These immunological parameters were shown to be modified early (within 7 days) following tumor inoculation and within the time period of IL-1 administration. IL-1 induced a marked increase in the number of lymphocytes within the brachial and axillary lymph nodes associated with the tumor inoculation site. This increase was characterized by an overall augmentation in the number of CD8⁺ and CD4⁺ lymphocytes.In vitro, these lymph node cells showed enhanced proliferation in response to interleukin-2 (IL-2) when compared to non-IL-1 treated animals, and were capable of mounting a potentially greater cytotoxic response for both NK sensitive and NK resistant tumor targets. Without IL-1 administration, temporal and sequential lymph node cellular changes were observed, but were diminished and delayed when compared to the IL-1 treated animals. By adoptive transfer of tumor resistance, lymph node cells from IL-1 treated animals were demonstrated to be tumor-protectivein vivo. These results demonstrate that systemic IL-1 induces regional changes in the lymphatics of mice undergoing primary tumor challenge with adjuvant therapy and that these changes result in tumor protection for the host.     

Adjuvant actions of polyclonal lymphocyte activators. II. Comparison and characterization of their actions in initiation and potentiation of immune responses to T-dependent and T-independent soluble antigens

Various polyclonal lymphocyte activators (PLA) such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide of Escherichia coli (LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and polyadenylic-polycytidylic acid (poly A:U) were compared in their effects on antibody response to T-dependent antigen (bovine gamma globulin (BGG) and dinitrophenylated (DNP)-BGG) and T-independent antigen (DNP-Ficoll) and on induction of tolerance to T-dependent antigen. All of these PLA acted more or less to trigger the initiation of the antibody-forming mechanism for deaggregated BGG (DBGG) or DNP-BGG through their actions on the carrier-specific T-cell function. All of these PLA tested also acted more or less to inhibit the induction of the carrier-specific T-cell tolerance to DBGG. Moreover, some of these PLA could act to augment antibody response to DNP-Ficoll. The adjuvant action of PLA in the response to DNP-Ficoll worked as well in athymic nu/nu mice as in nu/+ mice, whereas that in the response to DNP-BGG did not occur in athymic nu/nu mice. The order of the strength of the action of PLA to trigger the initiation of the whole immune response to DBGG, that to trigger the carrier-specific T-cell function to DNP-BGG, and that to inhibit the induction of the whole tolerance to DBGG was very similar to each other: i.e., CPS-K ? Con A > LPS, DS, poly A:U, PWM and PHA. By contrast, the order of the strength of the action to inhibit the induction of T-cell tolerance to DBGG was ≧ = LPS > Con A, PWM and poly A:U > DS and PHA, and that of the action to augment the antibody response to DNP-Ficoll was CPS-K > LPS > Con A. CPS-K was the most potent in all of these immunological activities. It was concluded that PLA act generally to stimulate the immune response at its initiation step in which T cells in the case of T-dependent antigen and B cells in the case of T-independent antigen play a predominant role, but that individual PLA share this adjuvant activity in different fashions.     

Effect of Capsular Polysaccharide of Klebsiella pneumoniae on Host Resistance to Bacterial Infections

When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 μg per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 μg per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.     

Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture

The effect of the capsular polysaccharide of Klebsiella pneumoniae type 1 Kasuya strain (CPS-K) on the formation of macrophage colonies in cultures of mouse spleen cells was investigated by the liquid culture technique during an incubation period of 7–8 days. CPS-K markedly inhibited further generation of macrophage colonies when added at any time after the beginning of culture, whereas it showed no destructive effect on macrophage colonies which were already formed before its addition. When CPS-K was present throughout the incubation period, such a low concentration as 0.05 μg/ml significantly inhibited colony formation, and the intensity of its inhibitory effect depended on its dose in the range of 0.005–50 μg/ml. The inhibitory effect persisted even if CPS-K was washed out after spleen cells were kept in contact with 20 μg of CPS-K per ml at 37 C for 6 hr. It was found that the inhibitory effect of CPS-K on colony formation was not mediated through its action on T cells, B cells or macrophages, and that it was not due to the generation of suppressor cells capable of inhibiting colony formation. It is concluded therefore that CPS-K directly inhibits the proliferation of macrophage colony-forming cells. The active substance responsible for the inhibitory effect of CPS-K on colony formation is the neutral polysaccharide fraction of CPS-K.     

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 actions of polyclonal lymphocyte activators: III. Two distinct types of T-initiating adjuvant action demonstrated under different experimental conditions

We demonstrated that each of various polyclonal lymphocyte activators (PLA) exhibits two types of adjuvant action to initiate the carrier-specific helper T-cell response to otherwise nonimmunogenic antigen. Type 1 action was characterized as that to initiate the T-cell response to subcutaneous injection of soluble bovine γ-globulin (BGG), and type 2 as that to initiate the response to intravenous injection of aggregated BGG. Each of various PLA showed these two types of adjuvant action in a dissociated fashion. The capsular polysaccharide of Klebsiella pneumoniae (CPS-K) showed both types of action to the highest degrees. Lipopolysaccharide of Escherichia coli exhibited type 2 action as markedly as CPS-K, but failed to show type 1 action. Concanavalin A showed definite type 1 action, but not type 2 action. Polyadenylic-uridylic acid showed definite type 2 action, but not type 1 action. Type 1 and type 2 actions of dextran sulfate were minimal. A hypothetical view is presented to consider that type 1 adjuvant action is directed to two mutually independent sites whereas type 2 action is directed to one site.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. I. Enhanced production of interferon and appearance of cytotoxin stimulated by capsular polysaccharide of Klebsiella pneumoniae or bacterial lipopolysaccharide.

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.     

Microbial Adjuvant and Autoimmunity

Definite lesions in the exocrine pancreas were produced when SMA mice were immunized eight times at intervals of 30 days with a mixture of extract of pooled pancreas from syngeneic mice and the capsular polysaccharide of Klebsiella pneumoniae type 1 Kasuya strain (CPS-K), whereas no pancreatic lesions were produced in mice given CPS-K alone or pancreatic extract alone. The typical histological changes were characterized by infiltration with lymphocytes, plasma cells, and other mononuclear cells, degeneration and lysis of the acinar cells, destruction of the lobular architecture, and replacement by fatty tissue and fibrous connective tissue. The endocrine islets were well preserved. No specific histological changes were produced in the organs other than the pancreas in these mice. Most of mice immunized with pancreatic extract mixed with CPS-K produced serum precipitins to syngeneic pancreatic antigens. However, severe pancreatic lesions were also produced in mice showing no definite precipitin production.