Specificity of cell-mediated transplantation reactions in rats detected by the macrophage adherence inhibition test

The macrophage adherence inhibition (MAI) test, previously described as a correlative to specific cellular immunity, has been used to study the specificity of primary cell reactions after allografting in rats. The adherence of sensitized peritoneal cells (PCs) from AVN rats (major histocompatibility haplotypeH-1 ^a ) bearing Lewis skin grafts (H-1 ^l ) was inhibited specifically by antigens from the graft donor and by those of the allogeneic strains BN (H-1 ⁿ ), BD V (H-1 ^d ), and DA (H-1 ^a ). Antigens of strain AVN, congenic strain Lewis. 1A (H-1 ^a ), and xenoantigens did not inhibit adherence. In general, positive reactions occurred during interaction of the sensitized PC with antigenic material sharing some components of theH-1 system with the graft donor. On the other hand, no crossreactions between individual rat strains were found when cytotoxic antibodies were tested after skin allografting. The negative MAI test in PC from AVN rats sensitized with skin allografts from the Lewis strain done with Lewis. 1A (H-1 ^a ) antigenic material, and the positive MAI test with antigenic material of the DA (H-1 ^a ) strain point to the possible existence of serologically undefined transplantation antigens participating in the cell-mediated reactions.     

Transfer Agent of Immunity

Detection of the cells which contain antibody was accomplished by a method of immune adherence of human erythrocytes to a single cell, termed SCIA (single cell immune adherence) reaction. Peritoneal exudate cells were collected from mice immunized with flagella of either Salmonella enteritidis or S. tennessee. Serologically specific antibody was detectable in some of the peritoneal exudate cells of such mice. An immune ribonucleic acid (RNA) was extracted from the peritoneal exudate cells of mice immunized with salmonella flagella. When mice were injected intraperitoneally with this preparation, serologically specific antibody was found in some of their peritoneal exudate cells by the SCIA method. This preparation was inactivated by treatment with ribonuclease, but was resistant to proteinases, deoxyribonuclease and anti-flagella antibody, suggesting that this agent is of RNA nature and does not contain antigen or fragment thereof.     

Production and immunological characterization of a monoclonal antibody to Trichophyton quinckeanum: interaction with phosphorylcholine-bearing components

A monoclonal antibody (Tq-1) that interacts with the phosphorylcholine (PC)-bearing antigens of Trichophyton quinckeanum was produced by fusion of myeloma cells (JKAg-8) with spleen cells of BALB/c mice immunized with an alum-precipitated fraction of T. quinckeanum cytoplasmic antigen. It was characterized as an IgM class antibody by immunodiffusion using anti-Ig heavy chain specific reagents, ELISA using immunoglobulin-specific peroxidase-conjugated antibodies, and by gel filtration chromatography; it showed high affinity for Staphylococcus aureus protein-A. Interaction of Tq-1 with PC-like antigens of T. quinckeanum was demonstrated by inhibition studies using ELISA, immunoblotting, immunoprecipitation and immuno electron microscopy techniques. The binding activity of Tq-1 antibody with a range of dermatophyte proteins was completely inhibited by prior incubation with PC hapten. Moreover, dermatophyte antigens reacting with the monoclonal antibody reacted strongly with sera from chronically infected mice. Dermatophyte antigens derived from both young (24 h) and old (20 d) cultures reacted with Tq-1 and this binding was inhibited by PC, suggesting that Tq-1 target antigen PC appears at an early stage during fungal growth and remains throughout its life.     

Circulating parasite antigen(s) in lymphatic filariasis: use of monoclonal antibodies to phosphocholine for immunodiagnosis

Hybridoma cell lines producing monoclonal antibodies (MAb) against a 200 kD antigen found circulating in the sera of microfilaremic patients infected with Wuchereria bancrofti were obtained by immunizing mice with a partially purified antigen preparation. A sensitive MAb (CA101)-based ELISA for measuring circulating parasite antigen was capable of detecting antigen in the sera of 93% of patients with microfilaremia, 46% of those with lymphatic obstruction, and 56% of patients with tropical pulmonary eosinophilia syndrome. Circulating antigen was absent from sera of normal controls, and "false positives" were recorded in only two of 17 patients with nonfilarial helminth infections. By ELISA and immunoblot analysis, it was shown that three of the monoclonals raised to this 200 kD antigen were directed to epitopes of phosphocholine (PC). Two MAb (CA86, CA101) were identified as having the T15 idiotype previously associated with antibodies to the PC of pneumococcal teichoic acid; one was untypeable. All three of these anti-PC MAb reacted with adult, microfilaria, and larval antigen preparations, and by immunoblotting showed multiple banding patterns that indicated the presence of PC determinants on many different antigenic molecules. On the other hand, target antigens of CA101 which were found in the circulation of infected patients were limited to three species with apparent m.w. of 200, 160, and 78 kD. The 200 kD antigen was seen more frequently than the other two antigens. Other T15 anti-PC MAb derived from mice not immunized with filarial antigen showed similar patterns of reactivity with circulating filarial antigen.     

Route-related variation in the immunogenicity of killed Salmonella enteritidis vaccine: role of antigen presenting cells

In order to assess the role of the route of immunization on the immunogenicity of killed Salmonella vaccine, mice were immunized with killed S. enteritidis by intraperitoneal (i.p.) and intradermal (i.d.) routes. Whereas the former was non-immunogenic, the i.d. immunization generated an excellent delayed-type hypersensitivity response; further, i.p. immunization could even suppress the subsequent i.d. immunization. Since the peritoneal macrophages (MO) are known to be particularly low in Ia or MHC-class II antigens, so essential for antigen presentation, the non-immunogenicity by i.p. route was thought to be due to their poor presentation efficiency. Poly I: poly C, an interferon inducer, is known to enhance the MHC-class II expression; hence effect of poly I: poly C treatment on the immunogenicity of the killed vaccine by i.p. route was tested and indeed the non-immunogenicity was corrected. Poor efficiency of presentation of S. enteritidis antigen by peritoneal cells and its improvement by prior poly I: poly C treatment was further confirmed by in vitro lymphocyte transformation test using primed T cells and peritoneal cells from normal and poly I: poly C treated mice. Poly I: poly C treatment also enhanced expression of Ia antigens on peritoneal cells.     

Assessment of cell-mediated hypersensitivity against coxsackievirus B3 viral-induced myocarditis utilizing hypertonic salt extracts of cardiac tissue.

Hypertonic KCl extracts prepared from heart tissues of adolescent CD-1 mice inoculated with coxsackievirus B3 (CVB3) were tested for antigenicity in evaluating cell-mediated sensitivity to CVB3 virus utilizing the agarose droplet cell-migration-inhibition assay. Immune mouse peritoneal exudate cells (IMPEC) from mice immunized against CVB3 virus and Freund's complete adjuvant were specifically inhibited in the cell-migration-inhibition assay with graded doses of KCl-extracted antigen and purified protein derivative (PPD). Unimmunized for "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited in the presence of the CVB3 KCl extracts. KCl heart extracts from mice inoculated with a cardiotropic strain of antigenically distinct mengovirus failed to inhibit CVB3 IMPEC, and noncardiac KCl extracts of liver and spleen from CVB3-inoculated mice also failed to inhibit cellular migration of CVB3 IMPEC. Reciprocal specificity experiments utilizing KCl-extracted antigens from mice infected with antigenically distinct cardiotropic mengovirus failed to inhibit cellular migration of IMPEC from mice immunized against the mengovirus. Serum-blocking power experiments indicate the antigenic KCl extracts failed to bind virus-neutralizing antibodies, indicating absence of detectable quantities of virion antigens. The results indicate that inoculation of mice with CVB3 virus results in the appearance of a new antigen(s) in cardiac tissue reacting with CVB3-IMPEC, but not with mengovirus IMPEC.     

Unsuccessful induction of low-zone tolerance to bovine serum albumin injected into the subarachnoid space

Introduction of T-dependent antigens into the subarachnoid space (isas) resulted in higher systemic antibody responses in mice than injections into the peritoneal cavity (ip) or other sites commonly used for immunization. Antibody production in isas immunized mice was not increased by treatment with cyclophosphamide (Cy) at doses known to abolish T-suppressor-cell activity, but such treatment increased antibody production in ip immunized mice toward the higher level which was observed in the isas immunized animals. Suppressor cell-dependent low zone tolerance (LZT) to TNP-BSA could not be induced by isas injections of deaggregated BSA (d-BSA). Conversely, mice which were unresponsive to ip injected d-BSA showed consistent systemic antibody responses when the antigen was injected isas. These observations indicate that immune responses initiated within the CNS are associated with relatively ineffective induction of systemic suppressor cell activity.     

Immune response to phosphorylcholine. VII. Functional evidence for three separate B cell subpopulations responding to TI and TD PC-antigens

A T-independent PC antigen, the C-polysaccharide, was used to induce a primary response to PC in splenic foci containing precursors taken from normal, unprimed BALB/c mice. The precursor frequency for the TI response was compared with the frequency for a T-dependent PC antigen, PPC-TGG-Hy. Idiotype analysis of the precursors for TD and TI responses indicate that the TD responses are more diverse with respect to clonal dominance of idiotype, TEPC-15, than that of TI responses. If splenic fragments were doubly immunized with both TI and TD antigens, the combined PC-specific precursor frequency was superadditive. TD-antigen-induced responses were found resistant to tolerogen and anti-idiotype suppression, whereas TI responses were extremely sensitive to both manipulations. Since under limiting dilution conditions precursors sort out independently, the superadditive response seen in dual immunized fragments can only be interpreted as evidence for an additional subpopulation of B cells that responds to the combined signals of TD and TI antigens. This postulated third B cell population is also extremely sensitive to tolerogen and anti-idiotype suppression.     

T lymphocyte-enriched murine peritoneal exudate cells. I. A reliable assay for antigen-induced T lymphocyte proliferation.

The in vitro activation of murine thymus-derived (T) lymphocytes by soluble protein and synthetic antigens has been difficult to assess because of the lack of a specific and reliable proliferation assay. The present report describes the development of an assay system which overcomes these problems by making use of a population of nylon wool column-purified T lymphocytes obtained from thioglycollate-induced peritoneal exudates of immunized mice. PETLES (peritoneal exudate, T lymphocyte-enriched cells) were composed mainly of T lymphocytes, eosinophils and small numbers of macrophages. Contamination with bone marrow-derived (B) lymphocytes averaged only 2%. When PETLES from immunized mice were stimulated in microtiter cultures with the immunizing antigen, large degrees of proliferation ensued as measured by incorporation of 3H-methyl-thymidine 5 days after initiation. As few as 1.25 x 10(4) cells and as little as 50 ng/ml of antigen gave significant stimulation. Maximum responses were obtained witn a series of 10 experiments under these optimal conditions, gave a mean incorporation of 70,900 cpm while the controls cultured without antigen showed only 3,600 cpm. PETLES from nonimmunized mice or from mice immunized to other antigens did not respond to DNP5OVA although they did respond to mitogens. The antigen-induced proliferation was shown to require the presence of immune T lymphocytes by two criteria: elimination of the response by treatment with anti-Thy 1.2 serum plus complement and failure to reconstitute the response when the few remaining immune B lymphocytes left after anti-Thy 1.2 treatment were added to nonimmune T lymphocytes. In addition, the system exhibited carrier specificity. Because of the paucity of B lymphocytes in the population, their contribution to the overall magnitude of the proliferative response was negligible as demonstrated by the small response to B cell mitogens. Thus, the assay appears to be a quantitative as well as a qualitative assay for one aspect of T lymphocyte function. This technique should prove useful for the study of murine T lymphocytes in vitro.     

Ribonucleic acid in the immune response

Summary In the studies of experimental salmonellosis, immunization of mice with a live vaccine SER of S. enteritidis was found to be effective against further infection with virulent S. enteritidis 116-54. Macrophages obtained from the peritoneal cavity, subcutaneous tissue or liver of immunized mice inhibited intracellular growth of bacteria and resisted cell degeneration caused by engulfment of virulent 116-54 bacteria. This immunity was called cellular immunity.We discovered by chance in 1961 a transfer agent of immunity (TA) from the culture fluid of immunized macrophages. This agent is RNA in nature and can be extracted from the spleen, peritoneal exudate cells or the lymph node of immunized animals and is called immune (i) RNA. We could demonstrate antibody activity in macrophages treated in vitro or in vivo with iRNA by the immune adherence hemagglutination technique.Cellular immunity against tumor cells could be transferred in vitro or in vivo to lymphocytes through iRNA prepared from the spleen cells of syngeneic, allogeneic and xenogeneic animals immunized with the tumor cells.We prepared iRNA against antigens capable of inducing humoral antibody production in animals, i.e., RBCs, bacterial toxin, bacterial flagella and hapten-protein conjugates. Serum antibody was not demonstrated in recipient animals of iRNAs by single or repeated injections of these agents. However, in these animals an increase in the number of specific antibody-carrying cells was found as rosette-formers. It was found further that prior injection of iRNA could induce immunologic memory and produced a high titer of humoral antibody after a boosting stimulation with a small dose of the corresponding antigen. The required interval between the first iRNA and the second antigenic stimulation, and the minimal effective doses of iRNA and antigen are described.We studied the interaction of iRNA with either T- or B-cells and with both cells using adoptive transfer system, athymic nude mice and neonatally thymectomized (NT) mice. Immune RNAs against T-dependent and T-independent antigens could not induce the proliferation of antibody-carrying cells in cyclophosphamide-treated (B-cell depleted) mice. But these agents could induce the proliferation of rosette-formers, implying that iRNAs can replace some role of T-cells even against T-dependent antigens. B-cells can be directly activated by treatment with iRNA against both T-dependent and T-independent antigens, and they differentiated into rosette-formers.Passive transfers of iRNA were successful in establishing immunity against infection with S. enteritidis, or immunity to Salmonella flagella, RBCs and hapten-protein conjugates. The ability of iRNA to confer a secondary response of antibody formation is serially and passively transmissible in recipient animals. These facts suggest the presence of some mechanism that is responsible for the amplification of antigenic stimulation in the immune response. The RNA-dependent RNA polymerase and RNA-dependent DNA polymerase are presented and their role in the immune response is discussed.     

Immunological aspects of murine infection with the rat nematode Strongyloides ratti Sandground, 1925

In a study of the immune response of the rat to infection with the nematode Strongyloidis ratti, the antigens of the infective larval stage (L3) and of the parasitic, parthenogenetic female (Fp) were investigated. From both the larvae and the adult females, one metabolic (exoantigen) and two somatic antigens were extracted. Of the two somatic antigens, one was soluble and obtainable by physical means while the other was separated by chemical means from the tegument of the parasite. Humoral responses to the various antigens were evaluated by immunodiffusion and ELISA techniques, while the overall immune response was assayed by the worm burden in the immunized and subsequently infected rats. Agar-gel double diffusion yielded precipitin bands only with larval somatic antigens. ELISA proved positive at a titer of 20,000 with larval metabolic antigen and sera of rats immunized against either larval metabolic or somatic antigens. By 20 days post challenge infection, however, this titer diminished to 4000. In vivo studies of worm burden in rats immunized with the various antigens and then exposed to the live L3 of the nematode showed that there were significantly fewer adult worms in the rats immunized with larval somatic antigen and adult metabolic antigen than in those immunized with adult somatic antigen or larval metabolic antigen.     

A new surface antigen (PC.2) expressed exclusively on plasma cells

Somatic cell hybridization of NS.1 nonsecretor myeloma cells with spleen cells of (DBA/2 X C57BL/6)F1 mice immunized against the myeloma MOPC 70A of BALB/c mice led to the establishment of five hybridoma clones which continuously secrete anti-MOPC 70A cytotoxic antibodies. The respective antigen detected by each of the five monoclonal antibodies is expressed both on plasmacytomas and on antibody-secreting cells as the only normal cell type. The tissue distribution of this new antigen is different from that reported for the alloantigen PC.1, and we have therefore designated it as PC.2. On the basis of immune elimination of direct and indirect plaque-forming cells, all mouse strains tested express PC.2 determinants, identifying PC.2 essentially as an autoantigen. Conventional anti-PC.1 alloantiserum contains antibodies to the PC.2 determinant, and these antibodies are distinguishable from the anti-PC.1 antibodies proper by the fact that only the latter are absorbed by liver cells. Monoclonal anti-PC.2 antibodies are not directed against MuLV-(murine leukemia virus)--associated antigens as over 20 ecotropic, several MCF (mink colony forming recombinant, and xenotropic viruses failed to react in immunofluorescence assays.     

A plant polyphenol-rich extract restores the suppressed functions of phagocytes in influenza virus-infected mice

Influenza infection was induced in white ICR mice by intranasal (i.n.) inoculation of the virus A/Aichi/2/68 (H3N2). The number, migration and phagocyte indices of alveolar and peritoneal macrophages (pM?) and of blood polymorphonuclear leukocytes (PMNs), as well as the inhibition of the PMN adherence in the presence of a specific antigen were followed for 9 days after infection. The effect of the i.n. application of a polyphenol-rich extract, designated as polyphenolic complex (PC), isolated from the medicinal plant Geranium sanguineum L., on the inspected immune parameters was studied in parallel with the virological parameters of the infection, e.g. rate of mortality, mean survival time (MST), infectious lung virus titre and consolidation of the lungs. It was found that the application of PC induced a continuous 2- to 2.5-fold rise in the number of both peritoneal and alveolar macrophages (aM?) in the infected and healthy controls. The migration of both peritoneal and aM? increased 1.5- to 2-fold in the group of infected PC-treated animals and four to fivefold in the control group, the maximum being on day 9. PC stimulated phagocyte activities of blood PMNs in both infected and healthy mice. The leukocyte adherence inhibition (LAI) index decreased in the infected and PC-treated animals. The restoration of the suppressed functions of phagocytes in influenza virus-infected mice (VIM) was consistent with a prolongation of MST and reduction in mortality rate, infectious virus titre and lung consolidation. The immunoenhancing properties of PC apparently contribute to the overall protective effect of the plant preparation in the lethal murine experimental influenza A/Aichi infection.     

Transfer of osteosarcoma-specific cell-mediated immunity in hamsters by rabbit dialyzable leukocyte extracts

We have investigated the transfer of specific cell-mediated immunity (CMI) to osteosarcoma-associated antigens (OSAA) to hamsters with dialyzable leukocyte extracts (DLE) from OSAA-immunized rabbits. The transfer of specific CMI was determined by leukocyte adherence inhibition (LAI) assay and skin testing. DLE was prepared from rabbits immunized with OSAA, purified protein derivative (PPD), or fibrosarcoma cell plasma membrane preparation (FSM). Control DLE was prepared from rabbits injected with 0.85% NaCl. Significant leukocyte adherence inhibition was observed with leukocytes from hamsters that had received OSAA-specific, PPD-specific, and FSM-specific rabbit DLE, when OSAA, PPD, and FSM were used as antigens, respectively. Similarly, significant ear swelling after injection of OSAA, PPD, or FSM was observed only in hamsters that had received DLE from rabbits immunized with OSAA, PPD, or FSM, respectively. These results suggest that CMI specific for OSAA, PPD, or FSM can be transferred to normal hamsters by DLE from immunized rabbits.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

Maturation of B cell subpopulations responding to phosphorylcholine

In this study, the order of appearance of B cell precursors responding to different phosphorylcholine (PC) antigens was investigated. Cells from neonatal BALB/c mice were transferred to male (CBA/N X BALB/c)F1 immunodeficient mice, and splenic fragment cultures were set up at different times after transfer. Because Xid F1 mice are unable to mount a primary anti-PC response, the time to prepare fragment cultures after cell transfer could be prolonged. This created an expanded maturation effect in which small differences in the response pattern of different precursor subsets are amplified. Splenic fragment cultures containing neonatal precursors were immunized with PG-TGG-HY (TD antigen), PnC (TI-2 antigen), or PC-TGG-LPS (TI-1 antigen), or with combinations of these antigens. The in vitro responses to these antigens were found to be additive, indicating the existence of different subpopulations. The detected precursors were also analyzed with respect to the T15 idiotype, which is the normally dominant idiotype of the response against PC in BALB/c mice. The analysis demonstrated a distinctly different maturation sequence of the three B cell subsets in which the order of appearance is: TI-1, TD, and TI-2 responding precursors. The T15 idiotype is expressed dominantly in all stages except the early stage of the TI-1 precursors.     

Experimental Salmonellosis X. Cellular Immunity and Its Antibody in Mouse Mononuclear Phagocytes

A cell-associated antibody was detected in the peritoneal mononuclear phagocytes (referred to as monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis, by use of immune transfer and immune adherence hemagglutination techniques. The cellular antibody inhibited the growth of a virulent strain of S. enteritidis with the aid of complement and lysozyme on nutrient agar plates. This type of bactericidal antibody could not be detected in the monocytes of mice immunized with killed vaccine of S. enteritidis. The antibody extracted from the peritoneal monocytes of mice hyperimmunized with live vaccine was identified as a macroglobulin by ultracentrifugal analysis.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. III. Correlation of resistance with induction of activated larvacidal macrophages

Mice protected against Schistosoma mansoni infection by intradermal (i.d.) immunization with nonliving larval or adult worm antigens plus bacterial adjuvant developed 24-hr skin test responsiveness to schistosome antigens with the histologic features of delayed hypersensitivity. Intraperitoneal antigen injection elicited a mononuclear cell-enriched exudative population containing macrophages activated for direct cytotoxicity against schistosomula and tumor cell targets. This was likely to be due to in vivo exposure to macrophage-activating lymphokines, since these cells were unresponsive to further lymphokine stimulation in vitro and splenocytes from immunized mice reacted to specific in vitro antigen challenge by production of lymphokines capable of conferring larvacidal activity upon control macrophages. In contrast, mice treated with schistosome antigens by i.v. injection, which were not protected against challenge infection, failed to develop delayed hypersensitivity or activated macrophages in response to specific antigen challenge in vivo, and the titers of macrophage-activating lymphokine produced by in vitro antigen-stimulated splenocytes from these mice were threefold to fourfold lower than those produced by cells from animals immunized by the i.d. route. Thus, sensitization for cell-mediated immune responses including lymphokine production and macrophage activation correlated with induction of resistance to S. mansoni in this model of vaccination.     

Specific binding of T lymphocytes to macrophages. V. The role of Ia antigens on Mphi in the binding.

The effect of anti-Ia alloantiserum on the capacity of selected peritoneal exudate lymphocytes (selected PEL) to bind to antigen-pulsed F1 (responder x nonresponder) macrophages was investigated. With the use of selected PEL for antigens under Ir gene control, it was shown that anti-Ia serum to the responder haplotype blocked adherence of selected PEL to antigen-pulsed macrophages whereas anti-Ia serum to the nonresponder haplotype did not. The target cell of the anti-Ia alloantiserum appeared to be the macrophage because anti-13 Ia in contrast to anti-2 Ia did not inhibit binding of F1 (2 x 13) DNP-GL selected PEL to DNP-GL pulsed strain-2 Mphi (responder strain). Taken together with previous experiments that indicate that an antibody to the native protein antigen employed is unable to block specific binding, the present results suggest that T cells may recognize fragments of exogenous antigen in association with Ia molecules.     

Taenia pisiformis: protective immunization of rabbits with solubilized oncospheral antigens

Antigens were derived from hatched and activated oncospheres of Taenia pisiformis which had been separated from embryophoric debris by centrifugation on Percoll. Crude oncospheral antigen was prepared by freeze-thawing and sonication of oncospheres at 4 C, and a supernatant of crude antigen was collected following centrifugation at 100,000g. Other antigens tested were the supernatants collected after 100,000g centrifugation of crude antigen solubilized in Triton X-100, butanol, lithium diiodosalicylic acid, KCl, sodium dodecyl sulfate, or sodium deoxycholate. When groups of rabbits were immunized with the various antigens and challenged with T. pisiformis eggs, both sodium deoxycholate- and Triton X-100-solubilized antigens stimulated a level of protection similar to the crude antigen. All other antigens failed to stimulate significant protective immunity. When sodium deoxycholate-solubilized antigen was fractionated using high-performance liquid chromatography, the major host-protective components were in the fractions with molecular weight greater than 140,000. Levels of the enzyme, glutamate dehydrogenase (EC 1.4.1.2), in the serum of rabbits challenged with T. pisiformis eggs closely reflected the degree of liver damage caused by migrating larvae, and were not markedly elevated in those rabbits effectively immunized using the crude or sodium deoxycholate-solubilized antigens.