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Salmonella bacteria are a major cause of food-borne infectious diarrhoea and there is great interest in understanding the pathogenesis of Salmonella infection and in vaccine development. Potential vaccines include the aromatic mutants of S. typhimurium. Such non-lethal Aro mutants have also been useful for studying Salmonella infections in mouse models. Studies of systemic infection, using these Aro mutants, in both normal and cytokine gene knockout mice, indicate that interferon-gamma (IFN-gamma) plays a key role in the resolution of Salmonella infection. The present studies have investigated the outcome of oral infection in mice with attenuated Salmonella because this infection route mimics natural infection in humans. In IFN-gamma gene knockout (IFN-gamma-/-) mice, intestinal immunity was impaired and oral challenge resulted in disseminated septicaemia 2 weeks later. No dissemination of infection was seen in wild-type mice. In wild-type mice, both CD4 and CD8 cell numbers increased in the gut following Salmonella challenge, together with increased expression of major histocompatibility complex (MHC) II and vascular cell adhesion molecule-1 (VCAM-1). No such changes were seen in IFNgamma-/- mice. Following oral challenge, antilipopolysaccharide (LPS) and antiphosphoryl choline antibodies increased by more than 100-fold in both serum and faecal pellet extracts of IFNgamma-/- mice compared with wild-type mice. Our data show that IFN-gamma production is essential for resolution of enteric Salmonella infection and that antibody has little effect on this process.

Recent studies have demonstrated that systemic and mucosal administration of soluble antigens in biodegradable microparticles can potentiate antigen-specific humoral and cellular immune responses. However, current microparticle formulations are not adequate for all vaccine antigens, necessitating the further development of microparticle carrier systems. In this study, we developed a novel microparticle fabrication technique in which human serum albumin (HSA) was entrapped in starch microparticles grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS), a biocompatible silicone polymer. The immunogenicity of HSA was preserved during the microparticle fabrication process. Following intraperitoneal immunization of mice, TS-PDMS-grafted microparticles (MP) dramatically enhanced serum IgG responses compared with ungrafted MP and soluble HSA alone (P < 0.001). When delivered orally, both TS-PDMS-grafted and ungrafted microparticles elicited HSA-specific IgA responses in gut secretions, in contrast to orally administered soluble antigen. Indeed, TS-PDMS-grafted microparticles stimulated significantly stronger serum IgG (P < 0.005) and IgA (P < 0.001) responses compared with those elicited by ungrafted microparticles. These findings indicate that TS-PDMS-grafted starch microparticles have potential as systemic and mucosal vaccine delivery vehicles.

Lewis rats immunized with myelin basic protein (MBP) in Freund's complete adjuvant (FCA) suffer from a single episode of paralysis from which they recover spontaneously. Subsequent to recovery, further episodes of paralysis cannot normally be induced by reimmunization with MBP in FCA. It is well established that serum, obtained from rats in the refractory state, can suppress the induction of experimental allergic encephalomyelitis (EAE) when given to animals from the time of immunization with MBP in FCA. Here it is shown that treatment with some such sera from Day 7 after immunization also suppressed the disease. However, not all convalescent sera were suppressive, indicating that rats immunized with MBP in FCA could become refractory to EAE without assayable levels of suppressive activity in their sera. In the context of this result it was notable that a correlation was found between the level of antibody specific for the encephalitogenic peptide in sera and the ability to suppress EAE. An inverse relationship was also shown between the amount of anti-encephalitogenic peptide antibody produced after immunization and the severity of EAE induced. Spleen cells from animals treated with Lewis anti-MBP serum after immunization with MBP in FCA could be activated to transfer EAE by in vitro culture with MBP despite the absence of any clinical signs in the donor animals, i.e. the serum inhibited the expansion or differentiation of these cells rather than preventing their priming or bringing about clonal deletion.

Three monoclonal antibodies to human C3 have been obtained from a fusion of the rat myeloma line Y3 Ag 1.2.3. with spleen cells from rats immunized against C3. One, from clone 4, reacts with an antigenic determinant in C3c showing the expected reactivity of the 'C' antigen of C3. The specificity of the other two monoclonal antibodies correspond less clearly with known C3 antigens. By agglutination analysis of complement coated cells the determinant reacting with clone 3 is present in C3d while that for clone 9 appears as a neoantigen on C3bi. In both cases the co-precipitation results are anomalous and more direct studies are needed to define the exact specificity. The possibility that internal sequence duplications in C3 may explain some anomalies is discussed. None of the monoclonal antibodies significantly inhibit C3 functions. The monoclonal antibodies have been found to have unusual properties in co-precipitation assays being able to diffuse through a precipitation line with which they react to react with a further line. One antibody is also able to react strongly with the anodal half of what appears as a single line with a polyclonal antiserum.

The in vivo effects of Lipid A and dextran sulphate on the antibody response to the thymus-dependent antigen SRBC in piglets are reported. No alteration of the low primary antibody response was observed with any of the B-cell activators. However, both substances induced an increase in antibody titre after secondary challenge with the antigen, which was shown to be of the IgG class. An activation of immunological memory in the absence of primary antibody response is suggested.

Thoracic duct lymphocytes (TDL) from (AS2 x AS)F1 rats previously injected with human serum albumin (HSA) were transferred to 900 r irradiated syngeneic recipients which were challenged with various doses of soluble HSA (s-HSA). TDL from partially tolerant rats, which were deficient in high avidity B cells, produced a maximum PFC response to the largest challenge dose (1 g s-HSA). In contrast, high avidity B cells from primed donors were optimally stimulated by 1 microgram and maximally inhibited by 1 mg s-HSA (day 7 PFC). An additional increase in antigen concentration by 1000 fold failed to diminish the PFC numbers further. Plaque inhibition profiles indicated that these antibody forming cells resisting inhibition were of the same high avidity as those triggered by low doses of antigen. The inability of s-HSA to completely inhibit antibody synthesis is discussed with regard to current views on B cell inactivation. Evidence is also presented which indicates that the standard haemolysis-in-gel test may fail to detect many low avidity antibody forming cells to proteins.

A procedure is described for detection of antibodies and antigens in 'immune complex' nephritis. Kidney tissue is minced, washed and sealed in a well of an agarose or agar plate. Separation of immune complexes is achieved by electrophoresis at 56 degrees. Afterwards, antibody is readily detected in reaction with a proper anti-IgG serum. Antigen is identified by its corresponding antibody, the reaction with which is, in some instances, strengthened by counterimmunoelectrophoresis.

Intradermal skin tests performed in the pinna of the rat ear appeared to be 100 times more sensitive than classical flank skin tests in measuring Arthus and delayed-type hypersensitivity (DTH) reactions. One of these tests was antigen-induced thickening of the pinna of the ear. It was found to be a sensitive measure of Arthus reactivity at 4 h after irritation with antigen in both actively immunized rats and recipients of precipitating immune serum. The other test, radiometric ear index determination, exploits the fact that monocytes and monocyte derived macrophages accumulate at DTH reaction sites. The test was performed by labelling the precursors of these cells with a pulse of [3H]-thymidine and by determining radioactivity in biopsy specimens taken from test sites in the pinna of the ear. At a certain antigen dose range this objective and highly sensitive method was shown to measure a purely cell mediated reaction which could be transferred to normal recipients with thoracic duct lymphocytes but not with immune serum. It also behaved as a typical DTH reacttion in response to desensitizing injections of the specific antigen. Testing with unnecessarily high antigen doses, however, should be avoided since the strong early inflammation induced by them may interfere with the determination of DTH while using this sensitive assay.

The solid phase enzyme linked immunosorbent assay (ELISA) has been used to quantify anti-keyhole limpet haemocyanin (anti-KLH) antibody in the serum of KLH-immune C57Bl/6 mice. When spleen cells from immune mice were cultured overnight in ELISA microtitre wells to which KLH had been adsorbed it was found that easily quantifiable amounts of anti-KLH antibody were synthesized and were detectable. It was found further that spleen cells from KLH-primed mice, when cultured in vitro in the presence of KLH, transferred to KLH-labelled ELISA plates, and cultured overnight, also produced detectable levels of antibody. Levels of antibody were detectable only after 4 and 5 days of in vitro stimulation. A comparison was made between detectable numbers of plaque forming cells to sheep red blood cells (SRBC) in SRBC primed CBA mice and levels of antibody detected by the ELISA procedure. It was found that the sensitivities of the two tests were comparable. The applications of this technique to the study of in vitro antibody synthesis using soluble antigens are discussed.

Lymphocytes are heterogeneous with respect to their life-span. Typical B cells, bearing on their membranes immunoglobulin receptors, easily detectable by immunofluorescence, belong mainly to the long-lived population: this can be observed using combined autoradiography and immunofluorescence. However, when primed mice receive (-3H) thymidine before a boosting injection of tobacco mosaic virus (TMV), many plasma cells appearing in the spleen during the secondary response are labelled. In irradiated recipients repopulated with spleen cells from donors primed with TMV and injected with tritiated thymidine 2 hours before killing, the majority of plasms cells appearing in the spleen after antigen injection were labelled. If irradiated mice were repopulated simultaneously with spleen cells from donors primed with TMV and injected with (-3H) thymidine, and from donors primed with haemocyanin, most of the anti-TMV plasms cells were labelled, while most of the anti-haemocyanin plasma cells were unlabelled. These results allowed us to exclude non-specific reutilization of labelled thymidine as the main reason of our observations. It is concluded that either plasma cells derive from shortlived precursors or they receive material from a labelled cell able to co-operate specifically with plasma cell precursors.

Follicular dendritic cells (FDC), isolated from human tonsils or adenoids, were tested for their capacity to retain monomeric, aggregated or antigen-bound human antibodies in the absence of serum. FDC retain fluorescein-labelled heat-aggregated human immunoglobulins, but not monomeric ones nor fluorescein-labelled F(ab')2 in monomeric or aggregated form. Ultrastructural observations showed that colloidal gold-labelled monomeric, or antigen-bound, antibodies directed against tetanus toxoid are retained by dendrites and membrane infoldings of FDC but are never located in cytoplasmic vesicles. This retention was inhibited by incubating FDC with unlabelled aggregated or antigen-bound antibodies. When gold-labelled anti-tetanus toxoid antibodies were incubated in the presence of protein-A before the contact with FDC, a strong reduction of their retention occurred. This further suggested the presence of Fc receptors on isolated tonsillar FDC. Endocytosis was not observed in isolated FDC, even after prolonged incubation in presence of labelled immune complexes: their Fc receptors are, thus, not related to a phagocytic activity as they are in macrophages. Simultaneous ultrastructural labelling of Fc and C3b receptors with colloidal gold particles of different sizes did not reveal any clear relations between these two receptors on the surface of FDC.

The serological and secretory immune responses of the chicken to infection with Eimeria tenella were evaluated in terms of various anti-coccidial activities. Serological responses were detected in the forms of precipitating, sporozoite neutralizing, anti-merozoite and anti-schizont antibodies. Similarly, anti-schizont and sporozoite neutralizing activities were found in caecal contents (containing mainly IgA) from infected birds and these also had the capacity to damage second generation merozoites. Moreover, the functional importance of IgA could be implied from the substantial predominance of IgA synthesizing cells in the intestinal immunocyte response as revealed by immunohistology. This was reflected in the immunoglobulin profile of caecal contents, for primary and secondary infection resulted in elevated levels of IgA whilst IgG and IgM generally remained extremely low or were usually undetectable. Taken with the well established lack of correlation between serum antibody and protection, these results suggest that the intestinal secretory IgA system plays an essential role in the protective immune response to E. tenella.

Anti-horse erythrocyte (anti-HRBC) antibody synthesis was studied in normal, early thymectomized and adult thymectomized axolotls. The kinetics of the responses were similar to those described in the same species for antibody synthesis against bacterial or viral antigens. Booster injections did not induce any characteristic anamnestic responses. Early and adult thymectomized axoltls gave in three experimental groups higher anti-HRBC responses than controls. It is concluded that HRBC acts in the axolotl as a thymus-independent antigen. The enhanced response in early as well as in adult thymectomized animals can be interpreted by the presence of a suppressor T-cell activity on anti-HRBC synthesis. These results do not exclude possible thymus-dependent responses for antibody synthesis in the axolotl, although such responses were not demonstrated in urodele. The questionable lacking of some functional T-cell subsets in urodele is discussed as a working hypothesis.

Rabbit dams fed 0.1% BSA for various periods before and during lactation produced anti-BSA of low avidity and IgG isotype in serum and milk. Milk anti-BSA and IgG concentrations were one-third to one-half of those in the serum. At birth, kits had IgG and anti-BSA serum concentrations approximately equal to their dams. Both fell rapidly for the first 10-20 days, levelling off at about 1 mg IgG/ml. Kits born to unimmunized dams and suckled by dams with anti-BSA in the milk showed increasing anti-BSA in serum for the first 12-16 days, falling by 20 days. Foster-suckling on immunized dams beginning at various times after birth showed antibody uptake from birth through 12 days of age. Thus immunoglobulins are among factors absorbed from milk that have potential for regulating the immune responses of rabbit neonates.

Rhesus monkeys vaccinated with merozoites in FCA are protected against challenge with several strains and variants of Plasmodium knowlesi. Vaccination induces sterilizing immunity which is species specific. Merozoite-blocking (inhibitory) antibody usually correlates with clinical immunity and protection can be passively transferred with immune sera provided these contain high levels of inhibitory antibody. However, vaccination using adjuvants other than FCA may induce inhibitory antibody without clinical protection. In addition, vaccinated animals may become susceptible to challenge 4-5 weeks after splenectomy, although inhibitory antibody levels are not reduced. These observations indicate that immunity induced by merozoite vaccination involves: (i) merozoite blocking (inhibitory) antibody, (ii) specific antibody or immune complexes acting synergistically with cytotoxic splenic cells stimulated by FCA.

Mice mated with H-2 incompatible males were shown to have an increased frequency of specific allocluster-forming spleen cells to paternal strain erythrocytes. The time course of the response was similar to that expected following a skin allograft. The graft-versus-host responsiveness of the maternal lymphocytes was generally elevated in the post-partum period in matings with males incompatible at either the H2 locus or non-H2 loci. The implications of these responses with respect to their different lymphocyte mediators and possible interactions are discussed with the view that a B-cell mediated immune response to paternal antigens may be contributory to foetal survival.

Mice, previously selected for the production of low affinity antibody after four injections of protein antigens in saline, fell into two groups on the basis of their antibody response after injection of adjuvantized antigen. One group produced antibody of sequentially rising affinity but the other produced only low affinity antibody. Mice from the latter group were interbred to produce low affinity non-maturing mice (low N/M mice). Daily injections in these mice produced a more rapid and severe glomerulonephritis than that observed in mice of the original low affinity line. Male low N/M mice were more severely affected than female low N/M mice. Susceptibility to the disease were associated not only with an inability to produce the maturational transition from low affinity to high affinity antibody with time but also with the production of low levels of antibody. It is suggested that these quantitative and qualitative defects in the antibody response may lead to increased susceptibility to immune complex disease.

The contribution of gut-associated lymphoid tissue (GALT) to the local response in the mammary gland is well documented in laboratory animals and has been evaluated in this study in ruminants. Ewes were immunized intraperitoneally (IP) with antigen in Freund's complete adjuvant (FCA), a procedure which stimulates the production of antibodies of the IgA class in the intestine, and challenged intramammarily (IMam) either during colostrum formation or mammary gland involution. Despite a substantial IgA antibody-containing cell (ACC) response in the intestine in IP immunized sheep, there was no evidence to suggest a relocation of IgA-specific ACC to the mammary gland. There was, however, an IgA antibody response in mammary secretion of IP immunized animals, regardless of whether the mammary gland was locally immunized, but the origin of this antibody is unclear. IP/IMam immunized sheep did have an enhanced antigen-specific ACC response of the IgG1 isotype in locally immunized glands, but whether these cells were of GALT or systemic origin is also unclear.

The immune response to the antigen horse spleen ferritin, has been investigated in ten inbred parental strains and seven different F1 hybrid strains of mice, using an antigen excess technique. The degree of dominance in an F1 hybrid system can be estimated by using the Fisher dominance index. The responses in F1 hybrid animals, obtained from crosses of high and low responder parents, varied from dominant to recessive but the overall mean dominance index for the ferritin immunogenetic system was found to be -0.0467 +/- 0.0083 (mean +/- s.e.), a value close to zero, which suggests a codominant mode of inheritance of IR-genes to ferritin and this is consistent with most published data in other F1 IR-gene systems.

Garter snakes (Thamnophis ordinoides) were immunized with hen egg albumin, human gamma-globulin and Keyhole limpet haemocyanin in Freund's adjuvant. Antibody was consistently detected by radioimmunoelectrophoresis and in three different gamma- and beta-globulin precipitin lines called IgM (approximately or equal to 20S), Ig-1 (approximately or equal to 9S) and Ig-2 (approximately or equal to 8-5S). Early antibody (day 31 after immunization) was frequently Ig-M whereas Ig-2 and especially Ig-1 were detectable for the longest duration (992 days). After immunization with antigen in Freund's adjuvant, Ig-1 serum concentration showed the greatest increase, from almost undetectable levels to the most prominent immunoglobulin in immune serum.

The margate, a marine teleost fish, was found to contain both high (16S) and low (7S) molecular weight antibodies 17 days after initial immunization. The 16S antibodies were detectable with both haemagglutination and antigen-binding assays, whereas the 7S antibodies were only detected by the latter technique. Margate 16S (molecular weight approximately 700,000) and 7S (molecular weight approximately 175,000) immunoglobulins were isolated and shown to be antigenically indistinguishable. They therefore appear to belong to the same immunoglobulin class and to have a tetramer--monomer relationship. Experiments with stored sera indicated the 7S protein is probably not an in vitro degradation product of the 16S molecule.

'Gelbsilber' (GS) rabbits which had been maintained for an unknown time as a closed colony, were found to respond uniformly well to the pig lactic dehydrogenase isoenzyme of the H4 type (P-LDH-H4), to which most New Zealand white (NZW) rabbits produced no detectable antibody. Two to three per cent of the splenic lymphoid cells of GS rabbits after secondary immunization were found to produce antibody to P-LDH-H4, while no such cells were detected in NZW rabbits. No differences were detected between the electrophoretic mobility of endogenous LDH of GS and NZE rabbits. The immunoglobulins of GS rabbits were heterogeneous with respect to immunoglobulin light and heavy chain allotypes. It was concluded that in GS rabbits the response to LDH-H4 is controlled by and Ir gene and that these animals might be useful in studying the genetic control of the immune response in rabbits.

Purified soluble C3d has been employed to measure the concentration of anti-C3d antibodies in immune rabbit sera. Multiple batches of C3d, prepared from C3-C3b substrate by treatment with C3b-inactivator (KAF), after labelling with 125I, retained 80% immunoreactivity, and were stable on storage at -50 degrees and +4 degrees. Concentrations of anti-C3d were determined by Scatchard analysis of equilibrium concentrations of bound and free C3d in a mixture of 125I-labelled C3d and anti-C3d. Separation of bound from free C3d was by G-75 Sephadex filtration. Assuming a 1:1 molar ratio in the antibody-C3d complex, anti-C3d antibody concentrations for four rabbit whole antisera and four IgG preparations fell in the range 288-2433 microgram/ml, with Ko values of 6-2 X 10(8)-2-9 X 10(9) litres/mol. One commercial antiglobulin-serum contained 3-6 microgram anti C3d/ml and had a Ko value of 1-7 X 10(8) litres/mol. Values for anti-C3d concentrations measured independently by an indirect method employing 125I-labelled sheep anti-rabbit IgG averaged 20% lower than those obtained with 125I-labelled C3d. Antibody concentrations were correlated with antiglobulin agglutination titres against C3d-coated red cells; a titre of 1 was given by an anti-C3d concentration of 0-5 microgram/ml.

The defined antigen substrate spheres (DASS) system was employed for analysing reactions between solid state antibody or antigen and soluble immune complexes. Sepharose beads covalently coupled with ovalbumin were used to represent tumour cells and beads coupled with antibody against ovalbumin were used to represent anti-tumour lymphocytes; the ovalbumin and corresponding antibody simulated tumour-derived antigen and antibody to tumour respectively. Binding of soluble complexes to the beads was measured by fluorimetry and/or radiometry of fluorescein or 125-I-labelled ovalbumin or antibody. Antigen-antibody complexes in antibody excess bound less effectively to the antibody beads than antigen alone, but complexes in slight or moderate antigen excess bound more effectively. Complexes in antibody excess were most effective in the complex before levelling off and then decreased in extreme antibody excess. The model demonstration of augmentation by antibody of antigen binding to solid state antibody might by analogy reflect a mechanism of inhibition of lymphocyte cytotoxicity. Complexes in a wide range of antibody excess should also be effective in blocking lymphocytotoxicity at the target cell level.

Studies of the effects of cyclophosphamide on a non-lethal primary Sendai virus infection of mice are reported. Treatment with cyclophosphamide resulted in failure to limit or eradicate virus, diminished and delayed the appearance of immunoglobulin-secreting cells in the lungs and the production of local and serum antibody, reduced and delayed the appearance of bronchial basement membrane damage and the desquamation of infected mucosal cells, and reduced the incidence of immune complex deposition in the kidneys. Evidence is presented which indicated that some escape from immunosuppression occured by day 6, resulting in local antibody production. The appearance of the local antibody response was associated with increased tissue damage in the lungs and the deposition of immune complexes of viral antigen and antibody in the kidney. Nine further experiments were performed in mice to investigate this renal manifestation and preliminary results are presented. In four of seven Sendai and one of two avirulent influenza A (Kunz) virus infections glomerular immune complexes were found. Studies in C3H and C57 B1 mice and their F1 hybrid suggested that genetic factors play some part in the renal findings. The results are discussed with respect to the possible beneficial and harmful effects of the immune response to trivial respiratory virus infections.

The effect of high mol. wt dextran sulphate (DS) on cell-mediated immune responses was studied. Three criteria were used to assess cell-mediated delayed-type hypersensitivity: footpad swelling, i.d. skin tests and macrophage migration inhibitory factor (MIF) production. Guinea-pigs sensitized with egg albumin (EA) and treated with DS showed strong positive delayed skin tests. Control animals given only EA showed negative skin tests. Lymphocytes from mice sensitized s.c. with EA and treated with DS showed an increase in MIF production. Delayed footpad swelling responses in mice sensitized s.c. with sheep red blood cells (SRBC) and treated with DS were increased when the mice were challenged 8 days after sensitization. Doses of DS which were effective in increasing delayed footpad swelling ranged from 50-200 mg DS/kg body weight. DS was only capable of increasing delayed footpad swelling responses when both SRBC and DS were injected s.c. at the same site. Intraperitoneal injection of both SRBC and DS obeebction of both s.c. but at different sites did not result in increased delayed footpad swelling. DS was capable of augmenting footpad swelling responses when given s.c. as much as 6 days before SRBC. The optimal time for administration of DS was 2 days before SRBC. Injection of DS 2 days or more after SRBC resulted in no increase in delayed footpad swelling responses. The results of this study indicate that dextran sulphate is a potent adjuvant for cell-mediated delayed-type hypersensitivity immune responses in both mice and guinea-pigs.

We have investigated the ability of genetically athymic (nude) rats to develop an eosinophilia to stimuli which are known to cause an eosinophilia in normal rats. Nude rats developed eosinophilia following (a) the pulmonary embolization of bovine gammaglobulin (BCG)-coated latex particles, and (b) experimental infection with the nematode parasite Ascaris suum. Eosinophilia in nude rats was not dependent on the presence of specific homocytotropic antibodies, haemagglutinating antibodies or allergic bronchoconstriction. In the latex-BCG system, all normal litter-mates (rnu/+) demonstrated haemagglutinating antibodies and a percentage demonstrated IgE and IgG2a homocytotropic antibodies to BGG in conjunction with BGG-induced bronchoconstriction. Nude rats failed to display any of these phenomena yet developed lung eosinophilia with an identical time course to that seen in their normal litter-mates. Similar observations were made following infection with A. suum. IgE antibodies were only demonstrable in normal litter-mates, which also demonstrated significantly higher levels of haemagglutinating antibodies to the parasite compared to those seen in the nude rats. However, the time course and magnitude of eosinophilia following infection with Ascaris was similar in each group. In addition, nude rats developed a significantly higher peripheral blood eosinophilia compared with normal litter-mates following secondary infection with Ascaris. In view of the immunological studies demonstrating the thymus deficiency and T-cell deficiency of the nude rat, it can either be proposed that eosinophilia is not exclusively under T-cell control or that a sub-population of T cells capable of mediating an eosinophil response exists in these animals.

Immune cytolysis (lysis) of cells due to the action of antibody in the presence of complement is usually substantiated by the uptake of vital dye by the cells, or by the escape of radiolabel from the cells. Immune cytolysis has now been assayed by determination of cell volume distribution with a Coulter multi-channel particle size analyser used in conjunction with a Coulter counter. For Ehrlich ascites and sarcoma-180 cells, volume degradation corresponding to vital staining was obtained only if trypsin (final concentration 625 microgram/ml) was added immediately after the usual 1 h incubation period for cells, antibody and complement. For L1210 leukaemia cells, trypsin was added at 0 degrees just 1 min before Coulter evaluation, to avoid potentiation of antibody-mediated cell lysis by trypsin. Immune cytolysis of mouse thymic, splenic and lymph node lymphocytes required addition of pronase (final concentration 625 microgram/ml) at 0 degrees for further disruption of antibody-damaged cells, prior to determination of cell volume distribution in the Coulter equipment. Scanning electron micrographs of L1210 cells undergoing immune cytolysis illustrated the changes in cell volume recorded by the Coulter apparatus. This new method for determination of immune cytolysis provides detailed information about the volume distribution of target cells, which permits detection of subtle changes and gives insight into the process of cytolysis. It is not intended to displace other procedures in routine use, except that complete automation of the present method is possible in future.

The transfer of anaphylactic antibodies of classes IgE and IgGa from immunized female rats to babies was examined during primary and secondary immunization by injection, and also in rats infested with Trichinella spiralis. IgE antibodies were never detected in the blood of the babies, although they were sometimes detected in the milk in their stomachs. Similarly, no IgE antibodies were found in the serum of babies from mothers infested with Trichinella spiralis during pregnancy. IgGa antibodies were present in the serum and milk of the mothers following secondary immunization, and were readily absorbed through the intestine of the babies.

In mice primed with a mixture of bovine serum albumin (BSA) and adjuvant (capsular polysaccharide of Klebsiella pneumoniae (CPS-K)) cell-associated immunological memory was increased secondarily after a second injection of BSA alone, whereas a primary injection of BSA alone into normal unprimed mice did not result in detectable memory. The optimum antigen doses for expression of the primary and secondary memories of adoptively transferred cells from unboosted primed donors or boosted donors in in vivo culture systems were very similar, although those observed in intact mice were very different, as reported previously. The size of the secondary memory of adoptively transferred cells from boosted donors was more than ten times greater than that of the primary memory of adoptively transferred cells from unboosted primed donors. The lag period for increase of the secondary memory was shorter than that for the primary memory. Both primary and secondary memories increased during a long period (up to 3 months) after the antigenic stimulus. From the results of this study it was concluded that cell-associated immunological memory could be amplified in a secondary fashion upon contact with a second stimulus.

Random-bred TO mice have been selectively bred into two lines on the basis of the relative affinity (KR) of antibody produced to protein antigens, one line producing high and the other low KR antibody. After four generations of selective breeding the difference in KR between the two lines was highly significant (P less than 0-001). The selection on the basis of KR did not result in a corresponding selection for antibody levels (Abt), which were not significantly different in the two lines. These results indicate that antibody affinity is a genetically controlled parameter of the immune response. Furthermore, this control appears to be expressed by a mechanism which is independent of the amount of antibody produced.

Rats infected with the live vaccine strain (LVS) of Francisella tularensis develop in vivo and in vitro evidence of cellular hypersensitivity and a concomitant state of cellular resistance to infection. They key role of sensitized lymphocytes in cellular resistance was domonstrated in transfer experiments. Using this technique, it was shown that thoracic duct lymphocytes from Francisella immune donors conferred specific antimicrobial resistance on normal recipients, whereas antiserum afforded no protection whatsoever. Further evidence for the participation of sensitized lymphocytes in the host's defence emerged from experiments in which a comparative analysis was made of the immunogenic properties of living and heat-killed LVS organisms. Rats stimulated with the living parasite developed cellular hypersensitivity and specific antibodies. Throacic duct lymphocytes obtained from such animals could immunize adoptively. By comparison, rats stimulated with a substantially larger number of dead organisms failed to develop cellular hypersensitivity and their lymphocytes were devoid of protective activity. Dead organisms, however, provoked an antibody response similar to that observed in infected rats. The development of cellular hypersensitivity in Francisella-infected rats is associated with enhanced resistance to Listeria monocytogenes. This finding accords with the results of similar studies of infection immunity to other intracellular parasites, and implies that the expression of cellular resistance to F. tularensis is a cooperative venture involving specifically sensitized lymphocytes and non-specific inflammatory cells, presumably macrophages.

Immunochemical studies were made on the antibodies induced in rabbits against poly(ADP-ribose) and naturally-occurring antibodies in patients with systemic lupus erythematosus. Antibodies against poly(ADP-ribose) could also be induced in rabbits by oligo(ADP-ribose) associated with rat liver histones and by a complex of poly(ADP-ribose) with methylated bovine serum albumin (MBSA). The two types of antibody were inhibited to the same extent by poly(ADP-ribose). However, the antibody induced by oligo(ADP-ribose) associated with histones was inhibited by oligo(ADP-ribose) with an average chain length if 4 ADP-ribosyl units and by phosphoribosyl adenosine monophosphate (PR-AMP) but not by mono ADP-ribose, whereas that induced by poly(ADP-ribose) was practically not inhibited by these related compounds even in excess amounts. The sera of ten cases of systemic lupus erythematosus showing high antibody activity against poly(ADP-ribose) were also examined immunochemically. It was found that the antibodies of three patients showed a similar inhibitory pattern to that of antibody induced in rabbits by oligo(ADP-ribose) associated with histones, those of three patients showed a similar pattern to that of antibody produced in rabbits by poly(ADP-ribose), and the remainder did not show either pattern. These findings suggest that oligo(ADP-ribose) associated with histones may serve as antigen to elicit naturally-occuring antibodies to poly(ADP-ribose) in patients with systemic lupus erythematosus.

This study describes the use of the radioimmunoassay for the characterization of antibodies to basement membrane (type IV) collagen from bovine anterior lens capsule. The immunogen was extracted from calf anterior lens capsules by limited pepsin digestion and injected into rabbits. The antisera were characterized using gel diffusion, haemagglutination and the radioimmunoassay in which 125I-labelled types I, II, III, and IV bovine collagen were employed. In the direct radioimmunoassay there was no reaction with either native or denatured types I, II or III bovine collagen, whereas there were high titres towards both native and denatured type IV bovine collagen. Radioimmune inhibition studies using unlabelled types I, II, III and IV bovine collagen, collagenase digested and repepsinized type IV collagen showed that there was marked inhibition by either native, denatured or repepsinized type IV collagen, and slight inhibition by native type I collagen; native type II and type III, denatured types I, II and III, and collagenase digested type IV collagen had no inhibitory effects.

Serum thymic hormone activity (TA) was determined in hereditary hypopituitary dwarf mice (dw/dw) and their littermates (+/dw or +/+). It was found to be very low in the dwarf animals in comparison to their littermates. At 14 weeks of age, the dwarf littermates exhibited significant glomerular lesions characterized by deposits of IgG, IgG1, IgG2, IgA, IgM and C3, which were augmented by thymectomy of adult females. In contrast, hypopituitary dwarf mice had minimal glomerular deposits of immunoglobulins. Unlike these animals, their littermates showed antinuclear antibodies (ANA) and anti-deoxyribonucleic acid (DNA) antibodies in their serum. The present findings are discussed in relation to recent hypotheses on: (1) the role of the hypophysis in thymus-dependent immunological functions; and (2) the significance of T-cell deficiency in the development of autoimmunity.

The plaque-forming cell response and serum antibody titres to sheep red blood cells (SRBC) were enhanced in mice fed diets moderately deficient in the amino acids phenylalanine-tyrosine. These diets sustained normal growth during a 4 week study period. More severe limitation of phenylalanine-tyrosine or multiple essential amino acid restriction gave rise to a slight increas in the delayed hypersensitivity reaction to SRBC. These latter diets usually limit growth; nevertheless the humoral immune response to SRBC was not significantly depressed except in the mice fed a diet (diet 7) with severe restriction of seven essential amino acids. These results suggest that dietary restriction may suppress the production or function of an inhibitory cell, such as a suppressor T cell, whilst not affecting the effector cell to the same degree.

Three-week-old chickens were given sheep erythrocytes or bovine serum albumin intravenously. Seven days later their tears and saliva possessed low levels of antibody to those antigens. Concurrent infection with lentogenic Newcastle disease virus (NDV) caused a significant increase in transuded antibody in those fluids. In chickens with circulating antibody to NDV, induced by parenterally administered inactivated vaccine, respiratory infection with heterologous infectious bronchitis virus resulted in limited transudation of anti-NDV. In contrast, the tears, saliva and tracheal fluid of non-vaccinated chickens undergoing primary infection with NDV acquired considerable levels of specific anti-NDV. The difference between the two groups is attributed to locally synthesized antibody.

Macrophage uptake and presentation of thyroglobulin (TG) were investigated in rats with experimental autoimmune thyroiditis induced by immunization with TG and adjuvant. Peritoneal macrophages were shown to take up radiolabelled TG and this did not depend on the presence of thyroiditis in the animal or on the strain of rat used. Cell-associated antigen was largely unaffected by trypsin. Macrophages primed in vitro with TG produced a rise in the circulating TG autoantibody levels of convalescent animals when given intravenously (unlike free antigen), but did not induce disease in virgin animals. These findings show that macrophages can bind and present TG and suggest that this process may perpetuate otherwise transient autoimmune reactions.

The following fish orders, at different stages of phylogenetic development, were tested for serum antibody activity against actin, myosin, tubulin, thyroglobulin, haemocyanin, single-stranded DNA and trinitrophenyl (TNP): Elasmobranches (sharks), Chondrosteans (sturgeons), and Teleosts (bony fish). Significant antibody activity was found against all these self and non-self antigens but, in particular, high titres of natural anti-TNP antibodies were noted. Anti-TNP antibodies were then isolated by affinity chromatography from pooled sera of several representative fish species, and studied by a non-competitive and a competitive enzyme immunoassay. When the isolated antibodies were tested by the non-competitive assay, Teleost (tench) anti-TNP antibodies bound almost exclusively to TNP, whereas Chondrostean and Elasmobranche antibodies bound mainly to TNP but also to one or more other antigens. When the isolated antibodies were examined by the competitive assay, they could be separated into three groups: (i) anti-TNP antibodies whose binding could be inhibited by TNP only (Teleosts); (ii) anti-TNP antibodies mainly inhibited by TNP but also significantly inhibited by two or three other antigens (Chondrosteans); and (iii) anti-TNP antibodies binding to different antigens but only slightly inhibited by them (Elasmobranches). These antibodies were found by immunocytochemical staining to bind to normal cell constituents, and especially to cytoskeletal proteins. The results strongly suggest that natural antibodies against the seven antigens examined are present in normal fish serum, and that their specificities differ with the stage of phylogenetic development.

IgG1 immunoglobulin purified from sera 3 weeks following primary infection with 400 L3 larvae of Nematospiroides dubius was shown to be protective against challenge infection as assessed by worm recovery and worm length. No evidence was seen of blocking activity either using primary infection sera or IgG1 purified from primary infection sera in a variety of assay systems both in vivo and in vitro. In addition, immune complexes precipitated from primary infection sera by polyethylene glycol failed to inhibit the capacity of immune mesenteric lymph node cells to transfer immunity. It is concluded that an explanation for the prolonged survival of Nematospiroides dubius must be sought in mechanisms other than circulating blocking factors.

Partial suppression of cutaneous delayed hypersensitivity reactions to collagen in guinea-pigs was induced by pre-immunization with collagen and FIA. This suppression is specific since: (a) pretreatment with OA and FIA or FIA alone did not cause suppression of skin reactions to collagen; (b) suppression was observed only if the collagen used for pretreatment was from the same species as that employed for sensitization for delayed hypersensitivity reactions; and (c) animals with depressed skin reactivity to collagen reacted normally to PPD. The suppression is not mediated by inducible, circulating antibodies to collagen since: (a) antibody titres measured by passive haemagglutination did not correlate with the degree of suppression; (b) suppression was observed with collagen in random coil conformation which sensitizes guinea-pigs for delayed hypersensitivity skin reaction but does not induce antibodies to denatured collagen; (c) best suppression was obtained if the animals were pretreated with collagen and FIA 3 days before the sensitizing injection; and (d) passively transferred antibody from animals with suppressed skin reactivity did not suppress skin reactivity of animals made hypersensitive to collagen by injection of collagen and FCA.

Following appropriate immunization of mice with CGG or DNP-CGG spleen cells release antigen upon incubation in vitro. The release can be detected either by 'self-stimulation' of the primed cell population, so that upon adoptive transfer antibodies to CGG and DNP are produced without need for further administration of antigen, or by stimulation of indicator (primed) cells. Boosting mice with antigen in saline following initial immunization with antigen in adjuvant proves the most effective method of loading spleen cells with antigen. A small fraction (10- minus 4- minus 10- minus 5) of the antigen used for boost is retained. The antigen appears to be held on the cell surfaces in the form of antigen-antibody complexes, for it is retained on the cells for up to 13 weeks in the presence of circulating antibody, during which time its capacity to self-stimulate can be inhibited by host serum in vitro. Activated thymus cells take up detectable amounts of antigen-antibody complexes. Complexes obtained by immunization with antigen in adjuvant can be inactivated by trypsinizing spleen cells. Trypsinization of separated T and B cells reveals detectable amounts of complex on the T cells, Complexes detected on T cells in this way are believed to be equivalent to the blocking complexes operative in transplantation and tumour immunity.

Evidence is presented that one a way which adjuvants exert their effect is by initiating an enduring increase in the localization of labelled cells in draining lymphoid tissues. Agents not generally considered to be adjuvants (carbon, latex, sheep erythrocytes) but capable of altering lymphocyte recirculation are shown to prevent the induction of tolerance by soluble bovine gamma globulin (BGG). These properties fulfil the criteria for adjuvanticity as defined by Dresser (1968) and link the expression of adjuvanticity to alterations in lymphocyte circulation.