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This paper examines helper T-cell responses and antibody titres and isotypes following immunization with a peptide antigen in association with three different adjuvants. B10.A mice were primed with pigeon cytochrome c fragment 81-104 in association with the adjuvants complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum. Strong antibody responses, dominated by IgG1, were observed upon priming with CFA and IFA. In contrast, priming with alum induced a weak antibody response with little or no detectable antigen-specific IgG1. These differences did not correlate with differences in T-cell priming, as immunization with peptide in association with all three adjuvants induced comparable T-cell proliferative responses and frequencies of antigen-specific cells. In addition, no significant differences in iiiterleukin-2 (IL-2), interferon-gamma (IFN-γ) and IL-4 production could be found, suggesting that the adjuvants did not differentially affect Th1 and Th2 cells. [ABSTRACT FROM AUTHOR]

The regulation of secondary antibody production in mice is studied in adoptive transfer experiments. Mice immunized twice with chicken red blood cells (CRBC) in Freund's complete adjuvant (adjuvant) at 28 day intervals subsequently produce high levels of IgG anti-CRBC antibody for several weeks. Immunization with CRBC in saline also induces prolonged splenic production of IgG antiCRBC antibody but at a much lower level. It was not possible to augment the level of antibody production in mice immunized with CRBC in saline by transfer of very large numbers of spleen cells from mice immunized with CRBC in adjuvant. Transfer experiments in the opposite direction failed to suppress antibody responses in mice immunized with CRBC in adjuvant. Similar experiments were carried out with mixed transfers into third party non-immune irradiated recipients. The requirement for continued antigen supply to maintain secondary antibody production was shown in that IgG anti-CRBC antibody production in nonimmune recipients of immune spleen cells ceased within five days when the recipients were not given antigen. It is concluded that: (i) while strong regulation of secondary antibody production against CRBC exists, this is not a function of transferrable suppressors; (ii) increased antibody production in animals treated with adjuvant is not due to increases in the number of antigen-sensitive T or B lymphocytes; and (iii) the data are most easily explained by a hypothesis which implicates the rate of triggering of lymphocytes as the regulatory step in secondary antibody production. This could be a factor relating to the supply of antigen or the number of sites in secondary lymphoid tissue at which antigen can trigger. [ABSTRACT FROM AUTHOR]

The effect of synthetic complexes of polyriboinosinic and polyribocytidylic acids (poly-IC) on the production of homocytotropic antibodies by guinea-pigs was studied. It was observed that poly-IC ires a definite adjuvant effect on the production of both IgG1 and IgE. The adiuvant effect was present when poly-IC was injected 24 hours after the antigen, but was absent when the polynucleotide was injected 48 hours after the antigen. In comparison with lipopolysaccharide (LPS) the adjuvant effect of poly-IC was weaker, more than 0.5 mg/kg of this polynucleotide being required to show an adjuvant effect, whereas only 20 µg/kg of LPS was enough to show a positive adjuvant effect. Furthermore, considerably less antibody was detected in the animals stimulated with the polynucleotide. [ABSTRACT FROM AUTHOR]

Aluminium hydroxide (alum), the most widely used adjuvant in human and animal vaccines, has long been known to promote T helper type 2 (Th2) responses and Th2-associated humoral responses, but the mechanisms have remained poorly understood. In this study, we explored whether alum is able to directly modulate antigen-presenting cells to enhance their potency for Th2 polarization. We found that alum treatment of dendritic cells failed to show any Th2-promoting activities. In contrast, alum was able to enhance the capacity of basophils to induce Th2 cells. When basophils from interleukin-4 ( IL-4) knockout mice were examined, the intrinsic Th2-promoting activities by basophils were largely abrogated, but the alum-enhanced Th2-promoting activities on basophils were still detectable. More importantly, Th2-promoting adjuvant activities by alum found in IL-4 knockout mice were also largely reduced when basophils were depleted by antibody administration. Therefore, basophils can mediate Th2-promoting activities by alum both in vitro and in vivo through IL-4-independent mechanisms. Further studies revealed that secreted soluble molecules from alum-treated basophils were able to confer the Th2-promoting activities, and neutralization of thymic stromal lymphopoietin or IL-25 attenuated the IL-4-independent development of Th2 cells elicited by alum-treated basophils. Finally, alum was able to activate NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in murine basophils in the same way as alum in professional antigen-presenting cells, but NLRP3 was not required for Th2-promoting activities on basophils by alum in vitro. These results demonstrated that alum can enhance the capacities of basophils to polarize Th2 cells via IL-4- and NLRP3-independent pathways. [ABSTRACT FROM AUTHOR]

Understanding the nature of adjuvants and the immune priming events in autoimmune diseases, such as rheumatoid arthritis, is a key challenge to identify their aetiology. Adjuvants are, however, complex structures with inflammatory and immune priming properties. Synthetic polymers provide a possibility to separate these functions and allow studies of the priming mechanisms in vivo. A well-balanced polymer, poly-N-isopropyl acrylamide (PNiPAAm) mixed with collagen type II (CII) induced relatively stronger autoimmunity and arthritis compared with more hydrophilic (polyacrylamide) or hydrophobic (poly-N-isopropylacrylamide-co-poly-N-tertbutylacrylamide and poly-N-tertbutylacrylamide) polymers. Clearly, all the synthesized polymers except the more hydrophobic poly-N-tertbutylacrylamide induced arthritis, especially in Ncf1-deficient mice, which are deficient in reactive oxygen species (ROS) production. We identified macrophages as the major infiltrating cells present at PNiPAAm-CII injection sites and demonstrate that ROS produced by the macrophages attenuated the immune response and the development of arthritis. Our results reveal that thermo-responsive polymers with high immune priming capacity could trigger an autoimmune response to CII and the subsequent arthritis development, in particular in the absence of NOX2 derived ROS. Importantly, ROS from macrophages protected against the autoimmune priming, demonstrating a critical regulatory role of macrophages in immune priming events. [ABSTRACT FROM AUTHOR]

Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed bone marrowderived MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, bone marrow-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described as favouring phagocytosis of mast cells by professional antigen-presenting cells. [ABSTRACT FROM AUTHOR]

The IMi Ds® immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/ II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell ( DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 μ m pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMi D® immunomodulatory compounds increased expression of Class I ( H2- Kb), CD86, and pomalidomide also increased Class II (I- Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μ m pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8+ T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4+ T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies. [ABSTRACT FROM AUTHOR]

Susceptibility to experimental autoimmune encephalomyelitis (EAE), which is an autoimmune disease inducible by immunization with a brain-specific antigen in complete Freund's adjuvant (CFA), is different among strains. In an attempt to resolve the immune mechanisms by which the difference in susceptibility to EAE is regulated, we re-estimated susceptibility of several strains of rats, and the frequency of antigen-reactive T cells in each strain was determined by limiting-dilution analysis. EAE was induced in Lewis (LEW), PVG/c and BN rats using four different methods: (i) active immunization with guinea-pig myelin basic protein (GPBP) in CFA; (ii) immunization with GPBP in CFA that had been further supplemented with Mycobacterium tuberculosis H37Ra (supplemented CFA); (iii) adoptive transfer of GPBP-activated spleen cells into syngeneic rats; and (iv) transfer of a GPBP-specific T-cell line. The LEW strain was susceptible to all four methods. The PVG/c strain was resistant to immunization with GPBP in conventional CFA (GPBP/conv. CFA), but was susceptible to immunization with GPBP in supplemented CFA (GPBP/suppl. CFA) and to transfer of activated spleen cells. The BN strain was resistant to all methods. Limiting-dilution analysis using T cells from LEW, PVG/c or BN rats has revealed that each strain of rat displays a different pattern of frequencies of GPBP-reactive or the 68–88 sequence (GP68–88)-reactive T cells. LEW rats showed relatively high frequencies of GPBP-reactive and GP68–88-reactive T cells after immunization with either GPBP/conv. CFA or GPBP/suppl. CFA, symptomatic rats showing higher values than asymptomatic rats. In asymptomatic PVG/c rats, the frequency of GP68–88-reactive T cells was lower than that of GPBP-reactive T cells. In PVG/c rats with clinical EAE, however, GP68 88-reactive T cells increased in frequency and were almost the same as GPBP-reactive T cells. BN rats, on the other hand, responded very poorly not only to the GP68–88 sequence but also to the whole GPBP molecule, even after immunization with GPBP/suppl. CFA. These findings, obtained by limiting-dilution analysis, strongly suggest that the development of EAE in LEW, PVG/c and BN rats is closely related to the frequency of GPBP-reactive T cells. Furthermore, it is shown that resistance to EAE found in PVG/c and BN rats may be generated by different immune mechanisms. [ABSTRACT FROM AUTHOR]

Solutions of BeSO4 were added to soluble macromolecules and the mixtures brought to neutrality. Under appropriate conditions much of the beryllium did not precipitate out as insoluble hydroxides but became attached to naturally occurring sulphated proteoglycans and polysaccharides (e.g. heparin, chondroitin sulphate, fucoidan, etc.) and to synthetic, sulphonated aromatic dyestuffs (e.g. trypan, Evans and Coomasie blues, Suramin, etc.). At physiological conditions of pH and ionic strength, these addition compounds were relatively stable and did not dissociate during dialysis. When such materials were injected s.c. into sheep (in doses containing about 50 μg Be), rapid lympho- proliferative responses took place in the regional nodes so that immunoblasts appeared in the efferent lymph in numbers that exceeded those provoked by powerful conventional antigenic stimuli. Significant amounts of the injected materials passed through the nodes and, in intact animals, became systematized. A method was devised for attaching beryllium to particulate carriers, such as lymphocytes or red cells that had been fixed with glutaraldehyde. The injection of suspensions of such materials also provoked vigorous immunoblastic responses, but the particulate materials did not pass beyond the regional node. In the doses usually used, none of these materials was grossly toxic and they may be suitable for consideration for development into a new type of immunostimulator cum adjuvant. [ABSTRACT FROM AUTHOR]

The effects on lymphokine production of two agents known to potentiate delayed-type hypersensitivity (DTH), pertussigen (pertussis toxin) (PT) and cyclophosphamide (CY) have been investigated. These two agents were administered to immunized mice. Subsequently, lymph nodes and spleen cells were exposed to specific antigen in vitro. The resulting culture supernatants were assayed for the presence of lymphokines. Only supernatants of cells from the mice given PT contained appreciable quantities of interferon-γ (IFN-γ) and stimulated cells of the monocyte-like WEHI-265 cell line to produce procoagulant activator and plasminogen activator. On the other hand, CY was more effective than PT on the production of interleukin-3 (IL-3). Both adjuvants had small enhancing effects on the production of interleukin-2 (IL-2). With either adjuvant, the cell populations induced had a similarly enhanced capacity to transfer DTH. These results demonstrate that the capacity of cells to transfer DTH does not necessarily correlate with their release of particular lymphokines. The potentiation of DTH by cyclophosphamide did not depend on significantly enhanced generation of IFN-γ, procoagulant activator, or plasminogen activator. The amount of IFN-γ in the culture supernatants correlated with their capacity to produce procoagulant activator and plasminogen activator, whereas the amount of IL-2 and IL-3 did not. [ABSTRACT FROM AUTHOR]

Klebsiella 03 lipopolysaccharide (LPS) and Escherichia coli 09 LPS were previously shown to have potent adjuvant activities in augmenting antibody response and delayed-type hypersensitivity to protein antigens. The R form LPS extracted from the O-specific polysaccharide-less mutants derived from Klebsiella 03 and E. coli 09 strains were characterized by chemical and electrophoretic analyses. The adjuvant activities of the R-LPS in augmenting antibody response and DTH to protein antigens were much weaker than those of the parental LPS. The strength of the adjuvant activities of the R-LPS was similar to that of the activities of Salmonella minnesota LPS and Ra-LPS. By contrast, polyclonal B-cell activation effects of the R-LPS were stronger than those of the parental LPS. The common feature of the parental LPS is that their O-specific polysaccharides are mannose homopolymers (mannans). From these results, it is suggested that mannose homopolymers as the O side-chains of LPS contribute to the action of LPS in enhancing strongly the T-cell dependent immune responses to protein antigens. [ABSTRACT FROM AUTHOR]

Referring to the strong immunostimulating activity of combinations of lipophilic agents and dextran sulphate, conjugates with chemical determinants of both types of adjuvants were synthesized and then examined for immunostimulatory capabilities in mice. Saturated fatty acids with varying chain lengths and sulphate groups were coupled covalently at defined ratios to the polysaccharide Ficoll (MW 400,000). Chemical analysis of 60 of the sulpholipopolysaccharides synthesized revealed that the number of sulphate groups per monosaccharide unit varied from 0 to 1.6, and the number of lipid groups from 0 to 0.8. Adjuvanticity of these conjugates for the humoral immune response was determined using sheep red blood cells (SRBC) and dinitrophenylshaptenated bovine serum albumin (DNP-BSA) as antigens. Five days after intraperitoneal injection of adjuvant and antigen, the numbers of direct anti-SRBC plaque-forming cells (PFC) in the spleen were determined. Anti-DNP antibody titres were measured from 1 to 4 weeks after immunization. PFC responses to 2 × 106 SRBC were augmented up to a 100-fold by conjugates of Ficoll and sulphate (sulphopolysaccharides: SPs) or lipid groups (lipopolysaccharides: LPs). Introduction of low or moderate numbers of lipid groups in SPs reduced adjuvanticity. Adjuvant activity of sulpholipopolysaccharides (SLPs) with varying sulphate and high lipid content depended on the sulphate contents and the chain length of the lipids. Sulphate reduced adjuvanticity of the SLPs, and the number of sulphate groups required for complete annihilation increased with the chain length of the lipid. LPs and SLPs, including conjugates that did not enhanc anti-SRBC PFC responses, augmented serum antibody responses to DNP-BSA while SPs were hardly effective. [ABSTRACT FROM AUTHOR]

The effect of intravenous (i.v.) administration of bacterial x-amylase (BαA) on the IgO antibody response to a subsequent challenge with BαA in incomplete Freund's adjuvant (IFA) varied with the difference in responsiveness of the parental strains. High-responder C3H/He (C3) mice given injections of either 200 or 4 ,μg of BαA, which alone were unable to trigger a detectable IgG antibody response, generated an enhanced response to an immunogenic challenge given 25 days after the last i.v. injection. The response of low-responder CS7BL/6 (B6) mice previously exposed to BαA, following a different kinetic course depending on the exposing dose, reached a plateau lower than the levels of control responses (tentatively designated as high- and low-zone suppression). Prior exposure of (B6 × C3)F1 hybrids to 200 pg led to the enhanced response, whereas pretreatment with 4 pg rendered them partially tolerant to a subsequent challenge. These results suggest that the capacity to achieve low-zone suppression is inherited as a dominant trait. Isoelectric focusing (IEF) analysis revealed that these enhanced responses expanded antibody heterogeneity in a strictly restricted, strain-specific manner as observed during the normal antibody response, although the rate of expansion was accelerated. The specific antibodies produced by individual high-zone suppressed B6 mice were focused as a limited set of bands in a narrow pH range where the specific antibodies produced early in the normal response were focused. In contrast, the response of low-zone suppressed B6 and F1 hybrid mice was characterized by a unique process of heterogeneity expansion. [ABSTRACT FROM AUTHOR]

Experiments were carried out to determine the effect of a range of adjuvants on the antibody response of sheep to a subcutaneous injection of ovalbumin. Incomplete Freund's adjuvant (IFA) was the best of the repository type adjuvants. From a range of soluble immunopotentiating substances tested, the polyion DEAE-dextran, or dextran sulphate, produced the greatest enhancement of the response. These results were confirmed in a factorial experiment, but no interactions were found between various classes of adjuvants. Further experiments were carried out to determine the effect of superior adjuvants, prior immunization or intradermal injection on the rate and mode of uptake of antigen from the injection site to afferent lymph. These experiments showed that, with IFA, most antigen is retained at the injection site, less than 15% entering lymph by 3 days. There was some delay in antigen uptake with adjuvant 65, while for aluminium phosphate precipitated antigen, the rate of uptake was the same as following a saline injection. There was some delay in uptake of antigen following injection with DEAE-dextran or in primed sheep but, following intradermal injection, the rate of uptake into lymph was the same as aqueous antigen injected subcutaneously. Experiments in which the distribution of radioactive antigen between cells and plasma in lymph was examined showed that, in unprimed sheep, virtually all antigen is unassociated with cells. In primed sheep, however, about 0.5% of antigen in lymph was found to be cell-associated. Gel filtration of lymph plasma from primed sheep demonstrated that nearly all the antigen in lymph is carried to the node as high molecular weight material, presumably antigen-antibody complexes. [ABSTRACT FROM AUTHOR]

Immune complexes were formed in vitro with antibodies obtained from rabbits immunized with bovine serum albumin in Freund's complete (FCA) or incomplete (FIA) adjuvant. The antibodies were mixed with different amounts of antigen. Immune complexes formed at maximum precipitation proportions were efficient in complement activation, whereas immune complexes formed at antigen excess had weak complement-activating properties. When injected into rabbit knee joints, the immune complexes formed at maximum precipitation proportions with FCA or FIA antibodies caused a moderate leucocyte migration into the joints with maximum cell counts 6-8 hr after injection. Injection into the joints of immune complexes formed with FCA antibodies at antigen excess induced a pronounced leucocyte migration with maximum numbers 18 hr after injection. Immunofluorescent studies indicated that these immune complexes became associated with the leucocyte membrane, whereas immune complexes formed at maximum precipitation proportions were found in the leucocyte granulae, indicating that only the complement-activating immune complexes were efficiently eliminated. [ABSTRACT FROM AUTHOR]

The immunogenicity of different preparations of liposome-associated bovine serum albumin (LSM-BSA) was compared in order to examine the significance of the site of association between a protein antigen and the liposomal carrier. Animals immunized with liposomes containing both entrapped and surface-adsorbed BSA, (LSM-BSA)c + a, were found to give a BSA-specific plaque-forming cell (PFC) response comparable with that elicited by animals immunized with trypsinized (LSM-BSA)c + a which contained mainly entrapped BSA. In contrast, the PFC response elicited by animals immunized with liposomes containing BSA adsorbed on the membrane surface, (LSM-BSA)a, was significantly less than that of the above two groups. Finally, animals injected with trypsinized (LSM-BSA). only elicited a marginally detectable PFC response. Results of this study demonstrate that liposomes can enhance the antibody response of a protein antigen regardless of whether the antigen is entrapped within the vesicle or adsorbed on the outer surface. Entrapped antigen, however, proved to be more immunogenic than that which was surface-adsorbed. [ABSTRACT FROM AUTHOR]

Using a new adaptation of the 51Crk release assay, it was found that plasma membrane vesicles from the human placental microvilious surface were not only capable of eliciting both cellular and humoral immunity in mice, but were also susceptible to lysis by these immune components. The production of cytotoxic cells and circulating antibody in immunized animals was only observed in the presence of Freund's complete adjuvant. The cells exhibited reactivity only against vesicles from a limited range of placentae, while the antibodies showed reactions against vesicles from a wider range of placentae. These vesicles were also susceptible to lysis by antisera raised against placental alkaline phosphatase but were resistant to attack by antibodies raised against normal human serum and HLA (multispecific) determinants and by a cytotoxic monoclonal antibody against human β2-microglobulin. The cellular cytotoxicity in the spleens of immunized animals could be abrogated if the cells were pretreated with AKR anti-C3H antiserum and complement. Further, in the spleens of unimmunized animals, a population of cells was detected that exhibited a 'natural' cytotoxicity against several of the membrane preparations. This cytotoxicity was heat-labile, being abrogated if the cells were preincubated at 37° for 4 hr before the assay, but was resistant to attack by the anti-Thy 1 antiserum. The results indicate that the use of 51Crk-labelled placental microvillous vesicles is a useful way of searching for anti-trophoblast immunity which may now be applied to the search for immune responses in pregnancy. [ABSTRACT FROM AUTHOR]

Thymosin (TFX) stimulates polyclonal antibody synthesis of human peripheral blood, lymph node and spleen lymphocytes. This stimulation is observed only with T-cell-dependent polyclonal activators of immunoglobulin production and its mechanism appears to be associated with TFX-induced activation of T-helper cells. On the other hand, antibody production in vitro of lymphocytes already involved in high humoral responses is inhibited by thymosin. TFX has similar activity both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Separation of immunostimulating polysaccharide (ALP) from Angelica and its biological activities were investigated. AlP was separated as an acetone-insoluble and non-dialysable fraction from hot water extract obtained by heating the root of Angelica acutiloba in water at 95°-98° for 30 min. It is a water-soluble heteropolymer(s) consisting of uronic acid, hexose and peptide. The anti-turnout activity of AIP was observed in terms of prolongation of the survival period of mice beating Ehrlich ascites cells. The uptake of tritiated thymidine into murine and human spleen cells could be stimulated by AIP in a dose-dependent manner. Murine B cells were activated polyclonally by AIP and differentiated to antibody-forming cells even in the absence of either helper T cells or macrophages. The possibility that the biological activity of AIP might be due to contamination by bacterial lipopolysaccharides (LPS) or lipid A-associated protein (LAP) was ruled out for the following reasons: (i) polyclonal B-cell activation by AlP was shown in spleen cell cultures of C3H/HeJ mice, a low responder strain to LPS; (ii) the activity of AIP disappeared completely after a mild periodate oxidation whereas that of LPS containing LAP was not lost by similar treatment. In addition, the primary antibody response to sheep erythrocytes was markedly augmented by an intraperitoneal injection of AlP. This result show that AlP is a potent adjuvant. [ABSTRACT FROM AUTHOR]

The immunogenic potential of lipid A, isolated from Salmonella minnesota R595 and made soluble by mild alkaline hydrolysis, was investigated using ICR mice and a modification of the Jerne plaque assay. In the absence of carrier protein or Freund's adjuvant, a single intravenous injection of 100 µg lipid A induced the development of anti-lipid A antibody-producing cells in the spleen. At the doses used, no heterophile-antibody plaques specific for sheep red blood cell antigen were detectable in recipients of alkali-treated lipid A. Moreover, the specific (anti-lipid A) plaque-forming ability of mice injected with alkali-treated lipid A nearly equalled the ability of mice injected with free lipid A, suggesting that mild alkaline hydrolysis treatment left intact the immunodominance of free lipid A. We also found that alkali-treated lipid A induced an increase in [³H]-thymidine incorporation by splenic T and B lymphocytes. This suggests the possible collaboration of T and B cells in the in vivo production of anti-lipid A antibody. [ABSTRACT FROM AUTHOR]

Water-in-oil emulsions of metabolizable fatty acid esters, with the non-toxic surfactant Pluronic L122 as emulsifying agent, potentiated the humoral response to bovine serum albumin and staphylococcal toxoid in the mouse. Adjuvant activity was increased by changing the chemical nature of the esters as follows: (i) using a series of ethyl esters, adjuvant activity appeared when the acyl chain length of the fatty acid component was 16 or greater; (ii) isobutyl and isopropyl esters of palmitic acid (CI 6:0) were superior to ethyl; (iii) the ethyl esters of oleic (C18:1) and linoleic (C18:2) acids were better than stearic (C18:0). Since emulsions prepared with longer chain saturated esters are very viscous or solid at room temperature, and unsaturated esters are chemically reactive, emulsions were prepared with differing proportions of ethyl caprate (C10:0) and butyl stearate. At a ratio of 9:1 the emulsions possessed the low viscosity of ethyl caprate, but gained the adjuvant activity of butyl stearate. 125I-labelled BSA was retained in the footpad to a significantly greater extent than with a caprate emulsion, but reasons are given for believing that slow release of antigen is not the only mechanism of adjuvant activity. The ester emulsions caused more acute but less chronic local inflammation (footpad swelling) than Freund's incomplete adjuvant. [ABSTRACT FROM AUTHOR]

The output and immunoglobulin class of antibody-containing cells (ACC) in lymph from different regions of adult sheep has been compared following intraperitoneal or subcutaneous injection of ovalbumin in Freund's complete adjuvant (FCA). Intraperitoneal injection of antigen in FCA elicited no response at all in popliteal lymph, but there were substantial responses in lymph from the hepatic, coeliac (which carries lymph mainly from the abomasum and anterior duodenum) and intestinal lymphatic ducts. A significant increase in the number of blast cells accompanied the ACC response. ACC reached a peak 7-days after injection when ACC comprised 3.7%+0.7%, 2.3%±0.7% and 3.3%±0.6% of total lymphocytes in lymph from the hepatic, coeliac and intestinal ducts respectively. At this time ACC were distributed among three immunoglobulin classes IgM, IgG1 and IgA with the latter constituting 25%–40% of ACC in lymph from the three regions. ACC did not appear in intestinal lymph following subcutaneous injection of ovalbumin in FCA in the leg immediately below the tarsus. This contrasted with the vigorous response in popliteal lymph where 6 days after the injection ACC constituted 3.9% of cells in lymph. Most (83%) of the ACC were IgG1-specific at the peak of the response. The remaining ACC were distributed among the IgM, IgG2 and IgA classes with the latter comprising 3% of total ACC. [ABSTRACT FROM AUTHOR]

Experiments are described which demonstrate that a single intraperitoneal (i.p.) injection of antigen in Freund's complete adjuvant results in the appearance of IgA-specific antibody-containing cells (ACC) in the intestinal lamina propria of sheep and that these cells reach the intestine via the intestinal lymph and blood circulation. Following intraintestinal administration of antigen to sheep immunized i.p. 2 weeks previously, an enhanced ACC response occurs in the intestine but cannulation and drainage of the intestinal duct does not interfere with this enhancement. Evidence is presented which suggests that the enhanced ACC response may be accounted for by antigen-induced local proliferation of ACC in the lamina propria of the intestine. [ABSTRACT FROM AUTHOR]

Mice were injected in their hind footpads with peroxidase (P0) emulsified in Freund's complete adjuvant. The development of cells secreting anti-peroxidase antibody (Ab) and cells secreting immunoglobulins (Ig) were detected in the draining popliteal lymph nodes in the subsequent 35 days, using local haemolysis plaque assay with sheep red cell blood cells coated with either PO or anti-mouse lg antibody. Plaque-forming cells (PFC) were isolated from the centre of plaques by micromanipulation and after appropriate treatment, were examined by electron microscopy for their intracellular Ab content and incorporation of [³H]-thymidine. Four subpopulations of Ig secreting cells were distinguished: (1) cells secreting Ig without Ab function and not containing intracellular Ab detectable between days 5 and 20; (2) cells secreting Ig without Ab function but containing Ab appearing on day 6 and present throughout the immune response; (3) cells secreting Ab and containing Ab; (4) cells secreting Ab, but without detectable intracellular Ab. These last subpopulations appeared on day 7 and were found in all subsequent assays. The analysis of the kinetics of these subpopulations suggest that cells secreting Ig without Ab function might be precursors of Ab secreting cells. [ABSTRACT FROM AUTHOR]

The B 19S strain of Brucella abortus is found to act as an adjuvant to the anti-sheep red blood cell (SRBC) reaction in some congenic strains of mice but not in others. If the recipient is H-2b, there is no adjuvant effect (B 19S); neither is there (thymus-dependent) anti-B 19S reaction as measured by thymocyte activation and change of electrophoretic mobility. In contrast, there is a thymus-independent anti-B 19S reaction (production of haemagglutinins) as good in the H-2b mice as in the others. Experiments with T cell deprived mice show that the adjuvant action of B 19S is thymus-dependent. As the antiSRBC reaction without adjuvant is also thymusdependent, it is difficult to distinguish anti-SRBC and anti-B 19S reactions from the adjuvant action of B 19S on the anti-SRBC reaction. Several explanations are possible, all involving H-2 (Ir?) controlled thymus dependent mechanisms. [ABSTRACT FROM AUTHOR]

Genetic controls on the concentration and affinity of the anti-dinitrophenyl antibodies produced by different inbred strains of mice in response to immunization with dinitrophenylated bovine gamma globulin in Freund's complete adjuvant were demonstrated. Based upon data on F1 hybrids and backcrosses, it can be concluded that there are genetic controls on antibody affinity which are not linked to the major histocompatibility complex. In addition, the genetic controls on affinity which were studied here appear to be inherited independently of genetic controls on antibody concentration. [ABSTRACT FROM AUTHOR]

Mice were immunized with preformed complexes of dinitrophenylated haemocyanin (DNP- KLH) and anti-DNP or anti-KLH antibodies, and subsequently assayed for the generation of B memory (BM) cells. Complexes formed at equivalence or in slight antigen excess were far more effective than antigen alone in generating memory: as little as 100 ng DNP-KLH-anti-DNP produced substantial B cell priming. Anti-carrier and anti-hapten antibodies were equally effective. Complexes generated memory more rapidly than antigen alone, and the adjuvant effect was not simply due to aggregation of the antigen. Optimal priming by complexes required the integrity of the Fe portion of the antibody: F(ab')2 antibody fragments were less effective The capacity of complexes to prime BM cells was abrogated by depriving mice of C3. C3 was also required for localization of complexes within splenic lymphoid follicles; complexes made with F(ab')2 localized in follicles, but less efficiently than those made with intact antibody. These results extend earlier findings (Klaus & Humphrey, 1977) and strongly suggest that the generation of BM cells involves the C3-dependent localization of antigen-antibody complexes within lyraphoid follicles and strengthen the concept that germinal centres are the birthplace of BM cells. [ABSTRACT FROM AUTHOR]

The immunogenicity of smooth muscle actin is increased by 'ageing' at 4° for at least a week. Rabbits lacking natural smooth muscle antibodies were injured with 1 mg of aged purified actin in adjuvant. Fourteen out of thirty-six rabbits produced serum antibodies which precipitated with actin solution, but not with smooth muscle tropomyosin, myosin, light or heavy meromyosin or with other unidentified non-actin proteins in crude extracts. Analysis of crude actin extract before and after precipitation by antiserum (i) by Sephadex G-200 chromatography and (ii) for its stimulating effect on myosin ATPase activity showed that actin was selectively removed. The precipitate itself, analysed on SDS-polyacrylamide gel, showed one band in the actin position, and otherwise only bands representing immunoglobulins. The antiserum also inhibited the ability of actin to stimulate myosin ATPase activity, and prevented polymerization of G-actin to F-actin, as shown by viscosity and EM studies. On immunofluorescence with cryostat tissue sections or ceil cultures, anti-actin serum stained smooth muscle fibres and many non-muscle cells, in the latter staining the microfilaments. The staining was prevented by absorbing the antiserum with actin (16 µg per 5 µl serum), and was abolished by pretreatment of the cells with cytochalasin B. No species specificity was demonstrated for these anti-actin antibodies. [ABSTRACT FROM AUTHOR]

A water-soluble mycobacterial glycopeptide was obtained in large quantities from the culture supernatant fluid of M. tuberculosis strain DT. This glycopeptide was strongly adjuvant-active when injected, in a water-in-oil emulsion containing ovalbumin, into guinea-pigs. In addition, it was devoid of cord factor toxicity in mice, polyarthritogenic activity in rats and cavity stimulating activity in rabbit lungs. [ABSTRACT FROM AUTHOR]

Polymerized flagellin and E. coli lipopolysaccharide both express adjuvanticity in vivo and in vitro for responses to hapten conjugated to sheep crythrocytes, and hapten conjugated to soluble thymus-dependent antigens. In the case of erythrocyte-bound hapten, adjuvanticity is expressed in the absence of thymus-derived cells (T-cells). However, in the case of responses to soluble thymus-dependent conjugate, carrier-specific T cells would appear to be necessary for adjuvanticity to be expressed. On the basis of these observations on hypothesis for the mechanism of B-cell induction and tolerance is proposed. [ABSTRACT FROM AUTHOR]

By using T cell-deprived mice and appropriate controls it has been shown that the adjuvant activity of LPS on the immune response to different doses of SRBC, tends to be oriented more towards B cells than towards T cells. In contrast, lentinan seems to mediate its adjuvant activity largely through a stimulatory effect on helper T cells. [ABSTRACT FROM AUTHOR]

Precipitating antisera against the four subclasses of human IgG were prepared by immunizing different species of animals (monkeys, rabbits, chickens, guinea-pigs and sheep) with whole IgG or Fc fragments. Freund's complete and incomplete adjuvant and Al(OH)3 were used to enhance antibody formation. In most of the experiments with rabbits, immunization against a particular subclass was accompanied by induction of unresponsiveness to the other subclasses. Different animal species showed distinct preferences for production of certain IgG subclass antisera. Anti-IgG3 could quite easily be produced in each of the animal species tested (monkey, rabbit and guinea-pig). Anti-IgG2 and anti-IgG4 could be raised in monkeys and, with greater efforts, in rabbits. Anti-IgG1 could easily be raised in guinea-pigs and with difficulty in rabbits. In our hands monkeys did not react to IgG1, possibly because only Gm(f+) paraproteins were used; experiments with rabbits and guinea-pigs showed that antibody formation against IgG1 was promoted by the presence of the Gm(a) marker. The Gm(n) marker was found to influence anti-IgG2 formation to an even greater extent: no subclass-specific antibodies were obtained when Gm(n-) IgG2 paraproteins were used as antigen. Apart from IgG subclass-specific antibodies the following additional antibodies were often found: antibodies directed to a combination of a certain heavy chain and a kappa light chain in monkeys; antibodies specific for one or more antigenic determinants common to IgG2 and IgG3 in monkeys and rabbits immunized with IgG2; antibodies specific for one or more antigenic determinants common to IgG1 and IgG2 in guinea-pigs and rabbits immunized with Gm(f+) IgG1. [ABSTRACT FROM AUTHOR]

The chemoattractant effect of soluble protein antigens for B cells from immunized mice was examined. Mice were immunized either via the footpad with ovalbumin (OVA) in complete Freund's adjuvant (CFA) or with CFA alone; or intraperitoneally with OVA incorporated in immune-stimulating complexes (OVA-ISCOM) or with saline. After 8-14 days B cells were purified from the spleens or the draining popliteal nodes and tested in vitro for locomotor responses to antigen using polarization (shape-change) and filter assays. Cells obtained by both routes of immunization, but not cells from control mice, gave locomotor responses to OVA. Responses were seen in B cells directly after preparation (5-8% of cells responding) but were enhanced if the cells were cultured overnight in the presence of interleukin-4 (IL-4) before testing (10-12% of cells responding). Checkerboard filter assays suggested that the response to OVA was chemotactic. The response was antigen specific since cells from OVA-immunized mice did not respond to bovine serum albumin (BSA), and cells from BSA-immunized mice responded to BSA but not to OVA. The response to OVA was inhibited by preincubation of OVA with anti-OVA but not with anti-BSA. Many of the cells that polarized in response to antigen were larger than any B cells found in control populations suggesting that the responsive cells are those that had been stimulated to enter cell cycle following immunization. [ABSTRACT FROM AUTHOR]

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s-IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasaily to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome-mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facifitating local and systemic antibody responses. [ABSTRACT FROM AUTHOR]

We have investigated the possibility of a T-cell epitope peptide providing help for a B-cell epitope peptide when both peptides are co-entrapped in the same liposomes. Epitope models used were a 28 amino acid peptide from the S region of the hepatitis B surface antigen (HBsAg) (subtype adw) containing an H-2′. Th-cell epitope, and a 33 amino acid peptide from the pre-S1 region of the HBsAg (subtype adw) designed to exclude an adjacent H-2′ T-cell epitope, the latter (pre-S1) peptide being recognized by SJL (H-2′) mice as a B-cell epitope. SJL(H-2′) mice were immunized twice intramuscularly with S or pre-S1 peptide alone, co-entrapped in the same liposomes or entrapped in separate liposomes which were mixed before injection. Analysis of sera for anti-peptide IgG1 antibodies revealed that the Th-cell peptide provided help for the pre-S1 peptide only when the two peptides were co-entrapped in the same vesicles. This helper effect was found to correlate with the ability of S peptide (co-entrapped with the pre-S1) to stimulate T-cell proliferation in vitro. There was no IgGl response against pre-S1 peptide in mice immunized with a mixture of the free peptides or a mixture of separately entrapped peptides. A helper effect, albeit much weaker, was also observed in mice immunized with the two peptides emulsified in incomplete Freund's adjuvant. Antisera from mice immunized with both peptides co-entrapped in liposomes were found to bind to full length (pre-S1 containing) recombinant HBsAg. Moreover, binding values were much higher than those seen with antisera from animals immunized with the liposomal S peptide above, presumably because of full access of anti-pre-S1 antibodies to the pre-S1 region of the rHBsAg. It is concluded that liposomes could serve not only as an immunological adjuvant for peptides but also as a carrier for Th- and B-cell epitopes thus eliminating the need for covalent linkage to a carrier protein. [ABSTRACT FROM AUTHOR]

A model but poor immunogen, ovalbumin (OVA), was entrapped in a novel antigen delivery system comprising poly (D,L-lactide-co-glycolide) (PLGA) microparticles. Both the primary and the secondary lgG antibody responses obtained with OVA in microparticles were compared to those obtained with OVA emulsified in Freunds' adjuvants by two routes of immunization, intraperitoneal (i.p.) and subcutaneous(s.c.) injection. Following single i.p. or s.c. injections, the lgG serum antibody responses to OVA in microparticles were significantly greater than the responses to OVA in Freunds' complete adjuvant (FCA) for up to 10 weeks. After s.c. booster doses of OVA, the secondary lgG antibody responses to OVA in microparticles remained greater than the secondary responses to OVA in Freunds', but not significantly so. Furthermore, the primary IgG responses to OVA in microparticles obtained 8-12 weeks after a single i.p. injection were greater than the secondary responses to OVA in Freunds' obtained by repeat s.c. injections at Weeks 0 and 6. These results demonstrate that microparticles can function as potent antigen delivery systems for an entrapped antigen. Due to their ability to degrade slowly in vivo and to release entrapped antigens, microparticles have considerable potential as controlled release antigen delivery systems for the induction of long-term immune responses. [ABSTRACT FROM AUTHOR]

Cholera toxin B subunit (CTB) has been shown to augment the antibody responses to influenza virus haemagglutinin (HA) in BALB/c mice immunized with HA vaccine together with CTB. In this study, mouse strain differences in the adjuvant effect of CTB on anti-HA antibody responses were investigated along with those in the antibody responses to CTB or HA, using various inbred and H-2 congenic strains. The antibody responsiveness to CTB depended on the H-2 haplotype of the strain: strains with the H-2b haplotype were high responders, those with H-2a, H-2k and H-2s were low responders, and those with H-2d were intermediate. The responsiveness to HA was also related to the H-2 haplotype: H-2a and H-2k strains were high responders, H-2b and H-2s strains were low responders, and H-2d stratus were intermediate. However, the degree of the adjuvant effect of CTB on anti-HA antibody responses was almost constant, regardless of the H-2 haplotype or other genetic backgrounds of the strain. The lack of genetic restriction of the adjuvant effect would be favourable for application of CTB-combined HA vaccine to humans, who are genetically diverse. Moreover, these results suggest that the immunogenicity and adjuvanticity of CTB differ essentially in their mechanisms. [ABSTRACT FROM AUTHOR]

Cholera toxin is widely recognized as a potent stimulator of mucosal IgA responses after oral feeding. However, comparatively little is known of its ability to stimulate cellular responses. This is due in part to the direct inhibitory effects of cholera toxin on T lymphocytes, which can confound attempts to study primed T-cell responses in vitro. We have avoided this problem by using cholera toxin as an adjuvant to enhance the response to an unrelated protein fed simultaneously. Thus the simultaneous feeding of 10 µg of cholera toxin and 5 mg of keyhole limpet haemocyanin (KLH) results in the priming of T cells in the spleen, mesenteric lymph nodes, Peyer's patch and lamina propria that proliferate when restimulated with KLH in vitro. The feeding of KLH alone does not result in such responses. Both CD4- and CD8-positive antigen-specific T cells were involved in the response and the cells produced both interleukins 2 and 4 on antigen restimulation in vitro. [ABSTRACT FROM AUTHOR]

Certain strains of rats infested with the nematode parasite Nippostrongulus brasiliensis mount vigorous, persistent immunoglobulin E (IgE) responses. In the absence of parasites, adjuvants such as Bordatella pertussis or Al(OH)3 are needed to produce IgE responses to soluble antigens. These are short-lived, even in high IgE responder strains. In this study we have produced long-lived IgE responses in both low (Wistar) and high (Brown Norway) IgE responder strains of rats by repeated injections of ricin, a toxic lectin from castor beans, and phospholipase A2 (PLA2), a bee venom protein. Total IgE levels rose from 30 ± 20 ng/ml to 39,000 ± 7500 ng/ml in the Wistar rats compared with an increase from 120 ± 100 ng/ml to 47,000 ± 8000 ng/ml in the Brown Norway rats. An even greater (104-fold) increase was seen in PLA2-specific IgE antibody levels. Total and PLA2-specific IgE started to fall 6 weeks after treatment was stopped in the Wistar and after 12 weeks in the Brown Norway rats. The duration of the response was 204 and 248 days, respectively. The IgE-enhancing properties of ricin were compared in low, mid (Hooded Lister) and high IgE responder rats, Total IgE and PLA2-specific IgE but not IgG antibody (Ab) responses were enhanced in all animals given ricin and PLA2 but not in animals given ricin or PLA2 alone. The increase was greater in Wistar rats (48-fold) than in Brown Norway rats (eightfold) and by Day 24 the levels of both total and PLA2specific IgE in the three different strains were indistinguishable. PLA2-specific IgE antibody-secreting cells were detected in the spleen at a frequency of 1:5000. These results show: (i) that repeated immunization of rats with antigen and ricin produce a very large IgE response which was long-lived; (ii) that this response was indistinguishable in different IgE responder strains of rat; and (iii) that the IgE response declines earlier in low IgE responder strains of rats. [ABSTRACT FROM AUTHOR]

Generation of interleukin-1 (IL-1) in the draining lymph nodes after injection of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum was studied in line with IL-1 mRNA expression (cytoplasmic slot blot analysis) and IL-1β antigen detection (ELISA and immunohistochemistry) in rabbits. The expression of IL-1β mRNA was marked from 6 to 96 hr, with a maximum at around 24 hr post-injection of CFA, while injection of the other two adjuvants elicited only a moderate or negligible response. On the other hand, IL-1α mRNA expression was almost negligible during the entire 8-week observation period after injection of the above three adjuvants. Generation of IL-1β antigen in the draining lymph nodes after CFA injection paralleled the expression of IL-1β mRNA. immunohistochemistry revealed that cells containing IL-1β resided in the medullary sinuses, marginal sinuses and para-cortical area, but not in the follicles. Despite marked generation of IL-1β in CFA-treated draining lymph nodes, the primary antibody response (IgG) to ovalbumin differed only slightly between the three animal groups that were immunized with the antigen incorporated in CFA, IFA and alum. Further, rIL-1β did not significantly enhance the immune response when it was entrapped together with the antigen in IFA and alum. IL-1β enhanced the immune response only when it was injected with antigen without adjuvant. Thus, IL-1 seemed to play only a limited role, if any, in the augmentation of the primary immune response by the above-mentioned adjuvants. [ABSTRACT FROM AUTHOR]