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Intracutaneous immunization of mice with 105 or 106 viable listeria resulted in acquired cellular resistance (ACR) of short duration (7 days) and in delayed-type hypersensitivity (DH) lasting at least 27 days. The ACR was partially non-specific, as 50% of the mice were also protected against a lethal challenge with Salmonella enteritidis. The specific element of the ACR could be transferred by non-adherent spleen cells from immune mice to normal recipient mice. Such transfer was not possible with adherent spleen cells from immune mice or with spleen cells from normal mice. Two systems of multiple immunizations to extend the period during which mice were protected against a challenge with 50 LD50 listeria were used. In the first system, mice were immunized with 106 viable listeria and subsequently challenged with 50 LD50 (=107) viable listeria. Mice surviving the challenge were actually boosted at the challenge injection for ACR. In the second system mice were immunized and boosted with 108 killed listeria mixed with the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). In the former system after each booster injection with viable listeria the interval during which the mice were protected doubled and reached a maximum of 31 days. In the latter system all intervals between two booster injections were equally long and never exceeded 28 days. In both systems the existence of immunological memory was suggested. The difference in results obtained after immunization with viable listeria and killed listeria mixed with DDA are discussed. [ABSTRACT FROM AUTHOR]

Mice were immunized i.e. with various enlarged haptens conjugated to bovine serum albumin and mixed with the cationic, surface active lipid, dimethyl dioctadecyl ammonium bromide (DDA). This immunization generated delayed-type hypersensitivity (DH) in these mice without detectable concomitant antibody formation. The DH was measured as footpad swelling. However, both direct and indirect hapten-specific PFC could be detected in peripheral lymph nodes and in spleens 4 days after a challenge injection. Although the adjuvant, DDA, promotes a strong cross-reactivity in DH between heterologous hapten--carrier complexes, the antibody-forming cells produced 4 days after challenge showed relatively high specificity for the immunizing hapten. This indicates only weak cross-reactivity at the anti-body-forming cell level co-existent with high cross-reactivity in DH expression to the same hapten-carrier complexes. These results are consistent with the possibility that B-cell receptors may be capable of expressing a greater degree of hapten specificity than T-cell receptors. It is tentatively concluded that Thelper cells participating in antibody formation may represent a subset of the T cells involved in DH. [ABSTRACT FROM AUTHOR]

Tolazoline, a drug used in the treatment of hypertension, has been described as a typical H2 agonist. In this study possible immunomodulating properties of tolazoline were investigated. A single injection of tolazoline 1 day before immunization caused an effect on delayed hypersensitivity that depended on the antigen dose. The response to 105 and 106 sheep red blood cells (SRIK) was decreased, whereas the response to 109 SRBC was enhanced. Administration of tolazoline 4 days after immunization predominantly affected the humoral response. The IgM response was inhibited in favour of the IgG response. Low doses of tolazoline, given to animals simultaneously with the elicitation for delayed hypersensitivity, lead to a more severe inflammation. The possible involvement of suppressor cells and vessels in tolazoline action is discussed. The application of tolazoline in the immunotherapy of human cancer is suggested. [ABSTRACT FROM AUTHOR]

Non-ionic block polymers (NBPs) have proved to be potent adjuvants for the humoral immune response against liposomes haptenated with tripeptide-enlarged dinitrophenyl groups (hapten J). Since both reversed triblocks and normal octablocks displayed adjuvant activity, reversed octablocks, in which structural properties of both groups are combined, were also tested for their adjuvant activity. The latter compounds displayed very strong adjuvant activity for J-haptenated liposomes, not only in normal BALD/c but also in (CBA/N × BALB/c)F1 progeny. To test the applicability of NBPs as adjuvants in semi-synthetic vaccines, the capacity of NBPs to stimulate the immune response against liposomes haptenated with Streptococcus pnewnoniae type 3 capsular polysaccharide-derived oligosaccharides was analysed. In these studies, again NBPs proved potent adjuvants, stimulating antibody production to a large extent. In male (CBA/N × BALB/c)F1 mice, which carry a X-cbromosome-linked iminunodeficiency (Xid), antibody levels were stimulated to the largest extent by a normal octablock. Stimulation of antibody titres, however, did not result in increased protection in these Xid mice. [ABSTRACT FROM AUTHOR]

CBA/N mice have an X-linked S-cell defect which prevents them from responding to non- mitogenic thymus-independent (T1-2) antigens such as haptenated Ficoll. The CBA/N mice do, however, respond to another group of thymus-independent (TI-2) antigens among which are haptenated liposomes. The F1 male progeny of CBA/N female mice express the same characteristics. Hapten-specific plaque-forming cell (PFC) responses to haptenated proteins (thymus-dependent, TD, antigens) by CSA/N mice and CBA/N × C3H/HeN F1 male mice can be blocked by concomitant exposure to TI-2 antigens bearing the same hapten. Simultaneous exposure to haptenated liposomes (TI- I antigen) and TD antigens, however, results in a synergistic response to the hapten if the antigens share common epitopes. The tripeptide-enlarged hapten dinitrophenyl-β-alanylgly- cylglycine (J), conjugated to phosphatidyl-ethanol- amine (PE) and incorporated into liposomal model membranes, was used throughout the experiments. In F1 male mice the moderate responses to TD antigens could be restored to values equivalent to those seen in F1 female mice. This adjuvant effect of haptenated liposomes is hapten-specific, time-dependent, and restricted to certain structural forms of the liposomes. [ABSTRACT FROM AUTHOR]

This paper describes the effect of altering liposomal membrane composition on humoral and cellular immunogenicity of haptenated liposomes in mice. Antibody formation was determined by enumeration of direct, plaque-forming cells in the spleen and delayed-type hypersensitivity (DH) was measured with a footpad swelling test. Humoral immunogenicity of haptenated liposomes was strongly influenced by membrane phospholipid, cholesterol and charged amphiphile composition. Haptenated liposomes prepared from phospholipids with a low (diolcoyl- and dilauroyl-phosphatidylcho- line) or high (distearoyl phosphatidylcholine) phase- transition temperature were less immunogenic than those prepared from phospholipids with an intermediate phase-transition temperature (dipalmitoyl phosphatidylcholine and sphingomyelin). In general, increasing the amount of liposomal membrane cholesterol induced a higher humoral response. These results are discussed in relation to liposomal membrane fluidity. Induction of an optimal DH with haptenated liposomes did not occur in the absence of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). When DDA was used, alterations in membrane composition did not influence cellular immunogenicity. From these results it was concluded that ‘intermediate’ liposomal membrane fluidity is the most important requirement for induction of optimal antibody formation with haptenated liposomes and that a certain physicochemical configuration of the antigen, provided by the adjuvant DDA, is a prerequisite for induction of DH. [ABSTRACT FROM AUTHOR]

This paper describes the induction of IgM immune memory to haptenated liposomes in mice. The tripeptide-enlarged haptens 3-(p-azo-benzene-arsonate)-N-acetyi-L-tyrosylglycyiglycine (A) and N-(2,4-dinitrophenyl)-β-alanylglycylglycine (J) were coupled to phosphatidylethanola mine (PE) and the conjugates A-PE and J-PE were incorporated into separate liposomal membranes (monofunctional A-PE-liposomes or J-PE-Liposomes) or into the same liposomal membranes [bifunctional (A,J)PE-liposomes]. The magnitude of the humoral response was measured by the appearance of direct and indirect plaque-forming cells in the spleens of immunized mice. Intracutaneous priming of mice with A-PE-liposomes mixed with the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) and secondary immunization with bifunctional (A.J)-PE-liposomes resulted in enhanced hapten A- and J-specific IgM responses. However, no switch from IgM to IgG antibodies was observed in these mice. The J-specific IgM response was enhanced only when hapten I was incorporated into a bifunctional liposome which also contained A-epitopes. in mice primed with A-PE-liposomes in the absence of DDA, a greatly diminished memory to both hapten A and J was observed. This finding indicates a crucial role for the adjuvant in the induction of memory. J-PE-liposomes and DDA were not able to induce memory to monofunctional J-PE-liposomes or bifunctional (A,J)-PE-liposomes. The possibility that hapten A-specific B lymphocytes were responsible for the induction of memory was excluded by hapten-specific blockade of these cells with a B-cell tolerogen. These data, in addition to the observation that no IgM memory could be induced in congenitally athymic nude mice, suggest that the observed memory can be ascribed to priming of hapten A-specific T lymphocytes. [ABSTRACT FROM AUTHOR]

Lisseria monocytogenes was labelled with [³H]-thymidine and phagocytosis in vivo, measured after the intraperitoneal injection of killed or viable listeria. The loss of intracellular radioactivity after incubation in vitro was used as a measure for digestion. Killing was assessed by counting the numbers of viable listeria before and after in vitro incubation. Peritoneal macrophages from mice immunized with viable listeria showed better phagocytosis and digestion of both killed and viable listeria than macrophages from normal mice. Viable listeria were digested to a lesser degree than killed ones by both normal and immune macrophages. The simultaneous injection of the surfactant dimethyldioctadecylammonium bromide (DDA) with killed or viable listeria greatly depressed the digestion of listeria. Phagocytosis of viable listeria was not affected whereas that of killed listeria was slightly enhanced. The injection of 108 killed listeria mixed with DDA greatly impaired the digestion of viable listeria 7 days but not 1 day after injection. The impairment of digestion was accompanied by an increase in the number of intracellular viable listeria. [ABSTRACT FROM AUTHOR]

This paper describes the specificity of delayed-type hypersensitivity (DH) and antibody formation in the mouse to the tripeptide-enlarged hapten, 3-(p-azobenzenearsonate)-N-acetyl-L-tyrosylglycylglycine (A). Hapten A was coupled to phosphatidylethanolamine (PE) and incorporated into liposomal membranes (A PE-liposomes). DH was measured as footpad swelling and antibody formation by the enumeration of direct plaque-forming cells in the spleen. A-PE-liposomes mixed with the cationic, surface-active lipid, dimethyl dioctadecyl ammonium bromide (DDA) induce, on intracutaneous injection in mice, hapten-specific DH without a contribution by the carrier. With other haptenated liposomes it was not possible to induce DH to those haptens, including the closely related hapten 3-(p-azobenzenesulphonate)-N-acetyl-L-tyrosylglycylglycine (S). A-PE- and S-PE-liposomes evoke, after intravenous injection in mice, a humoral response. The antibody formation to A-PE-liposomes was thymus-independent. In this response a considerable cross reaction between haptens A and S was observed. [ABSTRACT FROM AUTHOR]

This paper describes the induction of delayed-type hypersensitivity (DH) in the mouse and guinea-pig to haptenated liposomes. The tripeptide- enlarged hapten 3-(p-azobenzenearsonate)-N-acetyl- L-tyrosylglycylglycine (A) was coupled to phosphatidylethanolamine (PE) and incorporated into liposomal membranes (A PE-liposomes). In mice DH was measured as footpad swelling and in guinea pigs by skin testing. To induce hapten A-specific DH in mice with A-PE- liposomes the application of the cationic, surface- active lipid, dimethyl dioctadecyl ammonium bromide (DDA) was necessary. The use of Freund's complete adjuvant (FCA) did not result in the induction of DH to hapten A. In guinea-pigs, however, FCA and DDA had equally good adjuvant properties in the induction of DH. The time course of the DH and the optimal time interval between immunization and elicitation were determined for the mouse system. Also, the effect of dose and epitope density was studied in that system. Cyclophosphamide treatment, before immunizing mice with A-PE-liposomes and DDA, resulted in greatly impaired DH, probably caused by the short lifetime of the integrity of liposomes after intracutaneous administration to mice. The results make it very likely that presentation of hapten A in a liposomal or micellar structure is required to induce a cellular immune response to this hapten in mice. [ABSTRACT FROM AUTHOR]

This paper describes a rather simple coupling method for tripeptide enlarged haptens to phosphatidylethanolamine (PE) and the incorporation of these conjugates into liposomal model membranes (haptenated liposomes). These haptenated liposomes evoke a hapten-specific humoral immune response in mice. The magnitude of the response as measured by the appearance of direct plaque forming cells in the spleen is dependent on the route of immunization and the dose and epitope density of the hapten-PE derivatives. It was not possible to evoke an IgG response after either primary or secondary immunization with haptenated liposomes (as measured by the production of indirect plaques or mercaptoethanol-resistant antibody). These data, in addition to the observations that mice depleted of, or deficient in thymus-derived (T) lymphocytes respond to haptenated liposomes, indicate that these haptenated liposomes are T-cell independent antigens. [ABSTRACT FROM AUTHOR]

Delayed-type hypersensitivity (DH) and acquired cellular resistance (ARC) to Listeria monocytogenes in mice was studied following immunization with killed bacteria in combination with Freund's complete adjuvant or the adjuvant dimethyldioctadecylammonium bromide (DDA). Intracutaneous or intraperitoneal injections of killed listeria mixed with Freund's complete adjuvant did neither result in DH nor in ACR. Intracutaneous injections of killed listeria and DDA resulted in an antigen-dose dependent DH but not in ACR. Intraperitoneal injection of listeria and DDA, however, induced ACR but no DH. Optimal conditions for the induction of ACR were simultaneous intraperitoneal injection of 15 mg DDA/kg body weight and 107 or 108 listeria. The optimal interval between immunization and challenge was 7 days. No protection was found against challenge with a lethal dose of Salmonella enteritidis, suggesting that the protection is specific. Intraperitoneal injection of mice with DDA resulted in inhibition of phagosomelysosome fusion in macrophages harvested 24 h later. Interference with macrophage activity is discussed as one of the possible mechanisms for the adjuvant effect of DDA. [ABSTRACT FROM AUTHOR]

Immunization of mice with antigen mixed with the cationic surface active lipid dimethyl dioctadccyl ammonium bromide (DDA) produced delayed type hypersensitivity (DH), measured as a footpad swelling. The DH to sheep red blood cells or dinitrophenyl conjugated with bovine serum albumin (DNP28-BSA) in DDA exceeded the response or the same antigens in Freund's Complete Adjuvant (FCA) significantly. Treatment of mice with CY 8 hr prior to the injection of antigen in FCA or DDA resulted in delay of the onset of footpad swelling past day 5 and in elimination of the differences in the response due to the adjuvants. Immunization with carrier or hapten-carrier complexes with different epitope density in DDA and elicitation with the homologous and heterologous antigens revealed that the DH was DNP-specific. In viva priming with DNP28-BSA in DDA and in vitro stimulation with the same antigen resulted in peak responses which were twice as high and were reached almost twice as fast as the earlier found response following immunization in FCA. The advantages of DDA as adjuvant over covalently linked fatty acid chains and over FCA are discussed. [ABSTRACT FROM AUTHOR]

Anti-thymus serum (ATS) absorbed with Peyer's patch cells and anti-Peyer's patch serum (APS) absorbed with thymus cells were prepared. ATS treatment of mice resulted in a suppression of the immune response, measured with Vibrio cholerae as an antigen. APS did not have this effect. ATS and APS were used in the indirect fluorescence test to detect T and Bp cells. The cells staining with APS were called Bp cells to indicate that APS might only detect a special fraction of B cells (mature B cells). In Peyer's patches 66 per cent of the cells reacted with APS (Bp cells), whereas only 19 per cent cells reacted with ATS (T cells). The percentage of T cells in the lymph nodes was high (73 per cent), whereas only 14 per cent Bp cells were found in this organ. In the spleen almost equal numbers of Bp and T lymphocytes (32 per cent and 33 per cent) were detected. However, 35 per cent of the lymphocytes were non-reactive with either anti-serum. Boric marrow contained only small numbers of reactive cells (1 per cent T and 2 per cent Bp cells). Treatment of mice with dimethylbenzanthracene (DMBA) caused an increase in the number of non-reactive cells in Peyer's patches and a dramatic decrease in the number of Bp cells. The relative increase of the number of T cells in spleen and lymph nodes was not found in Peyer's patches. Cortisone acetate seems also to act primarily on B cells, especially on those of Peyer's patches. In this case also an increase in the proportion of T cells in the spleen was detected. [ABSTRACT FROM AUTHOR]

The role of Peyer's patches in primary and secondary immune response was studied with Vibrio cholerae as the antigen. V. cholerae E1 Tor ME 7 is a typical enteric antigen which was shown to be suitable for the demonstration of antibody forming cells by localized lysis in agar. No plaque-forming cells (PFC) against this antigen were found in the organs of non-immunized mice. Consequently a primary immune response could be studied. In vitro an immune response against this antigen could only be obtained with spleen cells when the donor animal had been primed. After primary intraperitoneal or enteral immunization many PFC were found in the spleen but none in Peyer's patches. this cannot be explained by insufficient penetration of the antigen into Peyer's patches as secondary responses were obtained with both routes of immunization. After local injection of antigen directly into Peyer's patches, PFC only appeared after 5 days. This is the indication that an essential cell type for the unable to restore the immune response in lethally irradiated mice, unless they were injected together with thymus cells or cells from peripheral lymph nodes. This suggests the absence of active T cells and indicates the presence of B cells in Peyer's patches. The combination of thymus cells and bone marrow cells was inactive in restoring the immune response. This is an indication that the B cells in Peyer's patches are immunologically more mature than bone marrow cells. [ABSTRACT FROM AUTHOR]

The cell types involved in the cellular immune response were studied with the GVH in vitro as a test system. Comparison of the activities of cells of different lymphoid organs in the GVH in vitro showed the cells of peripheral lymph glands and thoracic duct to be the most active. Second in activity were the cells from peritoneal exudate, mesenterial lymph nodes and spleen. Weak activity was found in thymus and bone marrow cells, whereas cells from Peyer's patches showed no activity at all. Treatment of donor animals with ATS, cortisone or cyclophosphamide simultaneously with (acceptor-) transplantation antigen, resulted in spleen cells which were less active in the GVH in vitro. No such results were obtained after treatment of donor animals with dimethylbenzanthracene or cyclo- phosphamide without antigen. Fractionation of donor spleen cells on a bovine serum albumin density gradient resulted in an accumulation of activity in one of the high density fractions (D), whereas this was reduced in the low density fractions (C, B and A) and absent in the highest density fraction (E). Separation of thymus cells on a glass bead column coated with transplantation antigen resulted in augmented activity of the adherent cell fractions, while the effluent cells were less active than the original cells. Fractionation of cells on an uncoated column resulted in adherent and non-adherent cells which were both inactive in the GVH in vitro. [ABSTRACT FROM AUTHOR]