IgG-recognizing shed tumor-associated antigens can promote tumor invasion and metastasis

Tumors secreting glycoproteins that act as tumor-associated antigens have been described as highly invasive and metastatic. In this study, the consequences of the humoral immune response (HIR) against these antigens were investigated. Using an in vitro model of tumor cell invasion, results indicated that the invasiveness of tumor cells secreting antigenic secreted/shed tumor glycoproteins (STGP) increases in the presence of specific anti-STGP IgG, polymorphonuclear cells and monocytes. This in vitro model showed that the coincidental presence in the matrix of both STGP and specific anti-STGP IgG increases the local release of IL-1beta, IL-6 and vascular endothelial growth factor (VEGF) by stromal cells, but not by tumor cells. Using an in vivo model, the experiments show that immune-competent mice develop an anti-tumor HIR with anti-STGP IgG production. In this model, tumor growth was increased in parallel with the serum concentration of specific anti-STGP IgG. In athymic nude (nu/nu)-beige mice the same trend was observed, suggesting a T-cell-independent tumor-promoting effect induced by anti-STGP IgG. Tumor histology showed intense infiltration of IgG-positive plasma cells and lymphocytes. A severe combined immunodeficient-beige mouse-based in vivo model of tumors, experimentally infiltrated with monoclonal IgG plasmocytoma cells, showed that only specific anti-STGP-IgG-secreting cells could exacerbate tumor invasion, angiogenesis and metastasis. These results suggest that tumors shedding/secreting antigenic STGP can induce a host IgG immune response that can promote invasion and metastasis by inducing tumor infiltrating stromal cells to release proinflammatory cytokines and VEGF.     

Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4′-diisothiocyano-1,2′-dihenylethane-2,2′-disulfonic acid (³H₂DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of ³H₂DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the ³H₂DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (τ_½ = 360 min) component representing 33% of the radioactivity, and a fast (τ_½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ_½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither ³H₂DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.     

Contribution of humoral immune responses to the antitumor effects mediated by anthracyclines

Immunogenic cell death induced by cytotoxic compounds contributes to the success of selected chemotherapies by eliciting a protective anticancer immune response, which is mediated by CD4⁺ and CD8⁺ T cells producing interferon-γ. In many instances, cancer progression is associated with high titers of tumor-specific antibodies, which become detectable in the serum, but whose functional relevance is elusive. Here, we explored the role of humoral immune responses in the anticancer efficacy of anthracyclines. Chemotherapy reduced the number of tumor-infiltrating B cells, and failed to promote humoral responses against immunodominant tumor antigens. Although anthracycline-based anticancer chemotherapies failed in T cell-deficient mice, they successfully reduced the growth of cancers developing in mice lacking B lymphocytes (due to the injection of a B-cell-depleting anti-CD20 antibody), immunoglobulins (Igs) or Ig receptors (Fc receptor) due to genetic manipulations. These results suggest that the humoral arm of antitumor immunity is dispensable for the immune-dependent therapeutic effect of anthracyclines against mouse sarcoma. In addition, we show here that the titers of IgA and IgG antibodies directed against an autoantigen appearing at the cell surface of tumor cells post chemotherapy (calreticulin, CRT) did not significantly increase in patients treated with anthracyclines, and that anti-CRT antibodies had no prognostic or predictive significance. Collectively, our data indicate that humoral anticancer immune responses differ from cellular responses in, thus far, that they do not contribute to the success of anthracycline-mediated anticancer therapies in human breast cancers and mouse sarcomas.     

Inhibition of specific antibody binding to adult male Schistosoma mansoni by adsorbed host serum components

The ability of anti-schistosome antibody to bind to adult male Schistosoma mansoni was studied, using fluoresceinated Staphylococcus aureus to detect specific antigen-antibody interaction at the parasite's surface. Both freshly perfused parasites and parasites which had had their adsorbed host antigens removed by elution were employed in a series of experimental manipulations to ascertain under what conditions specific antibody binding occurs and what conditions or factors are necessary for the parasite to reconstitute its surface so that specific binding is precluded. Neither normal mouse serum nor normal mouse IgG bound in a specific manner to either fresh or eluted worms. Slight binding was noted with immune mouse serum on both fresh and eluted worms, while immune IgG produced weak binding on fresh worms, but strong binding to eluted worms. This strong binding was reduced to the level seen on fresh worms by pre-incubation of the eluted worm in normal IgG prior to incubation in immune IgG and binding was completely negated by pre-incubation in either normal mouse serum or normal mouse serum minus IgG. The binding of immune IgG to eluted worms was not diminished by pre-incubation in mouse albumin, bovine albumin, or fetal bovine serum. These studies demonstrate that adsorbed host serum components can inhibit specific antigen-antibody interaction at the parasite's surface and suggest that a degree of specificity exists in what the parasite adsorbs from the host. These data further suggest that the protective serum factor or factors may include, but are not limited to, host IgG.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

Immunization of Rhesus monkeys with a SialylTn–mAb17-1A conjugate vaccine co-formulated with QS-21 induces a temporary systemic cytokine release and NK cytotoxicity against tumor cells

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are frequently over-expressed on cancer cells and provide potential targets for cancer vaccination. Immunization of Rhesus monkeys with SialylTn coupled to a highly immunogenic carrier molecule and formulated on aluminum hydroxide induced a strong immune response against the carrier protein but only a moderate IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. The kinetics of the carbohydrate-specific IgG response correlated with a temporary release of cytokines such as IFNγ, IL-2, IL-1β, TNFα and GM-CSF which was measurable in the immune serum by xMAP Multiplex technology. Furthermore, tumor cell killing by activated natural killer cells was induced. These data demonstrate that immunization with a tumor-associated carbohydrate antigen in a highly immunogenic formulation results in a temporary release of type 1 cytokines which may be required for the induction of a specific IgG immune response against the carbohydrate antigen as well as for activation of effector cells against tumor cells.     

Role of cytokines in immune response to T-independent antigens, type 2

The influence of human and mouse cytokines on the induction of immune response to model T-independent (TI) antigens of type 2--DNP-dextran and DNP-ficoll--in the culture of mouse spleen cells was studied. For the first time this study revealed that the action of definite T-cell factors not only induced the polyclonal stimulation of B lymphocytes, but also the increased synthesis of specific antibodies, as well as switched over antibody isotypes from IgM to IgG. This confirmed the suggestion that T cells took part (directly or indirectly) in the regulation of immune response to so-called TI antigens. These results widened our knowledge on the mechanisms of the development of humoral immune response to TI antigens of type 2.     

Humoral immune responses induced by Gymnorhynchus gigas extracts in BALB/c mice.

The aim of this study was to determine if the plerocercoid larvae of Gymnorhynchus gigas, a common cestode of the ray's bream (Brama raii), possess antigenic compounds potentially capable of provoking anaphylactic episodes. A murine experimental model, using BALB/c mice, was developed to study the humoral immune response induced by G. gigas extracts. A highly specific humoral immune response was detected and cross-reactions were not observed between parasite and host antigens. The presence of IgM and IgG3 levels suggest the presence of thymus-independent antigens in the parasitic extract. The IgG antibody class showed the highest levels, with the IgG1 the predominant subclass. These IgG1 levels are in accordance with the supposed presence of a type I allergic reaction after the ingestion of G. gigas plerocercoids parasitizing fish, as well as inducing anaphylaxia in fish. These results indicate that somatic products released from ingested larvae of G. gigas could induce the development of a Th2 response capable of causing allergic disorders.     

Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes

Infections of animals with parasitic worms, such as Schistosoma mansoni, induce humoral immune responses to carbohydrate antigens, raising the possibility that such antigens might be useful targets for the development of vaccines and new diagnostic approaches. Here we describe the identification of fucosylated LacdiNAc (LDNF) [GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc-R] as a new carbohydrate antigen in S. mansoni that induces humoral immune responses in infected mice. The presence of antibodies was determined by ELISA using a neoglycoconjugate synthesized to express LDNF sequences. Sera from S. mansoni-infected, but not uninfected, mice contain IgM, IgG, IgA, and IgE antibodies to LDNF. The IgG antibodies are primarily of the IgG1 and IgG3 subclasses, with no detectable levels of the complement-fixing IgG2a and IgG2b isotypes. An IgM monoclonal antibody, designated SMLDNF1, was generated from the spleens of S. mansoni-infected mice, and the antibody exhibits specific recognition of LDNF sequences, but not other fucosylated glycans tested. Immunocytochemical analysis demonstrates that LDNF antigens are localized on the tegumental surface of adult S. mansoni. Western blot analysis indicates that LDNF sequences are expressed on numerous high-molecular-weight glycoproteins from the three major human schistosome species, as well as the bird schistosome Trichobilharzia ocellata. The identification of LDNF antigen on the tegumental glycoproteins of schistosomes and the ability to synthesize LDNF conjugates should aid in the development of glycan-based vaccines and immunodiagnostic tests for schistosomiasis and in determining the role(s) of the glycans in worm development and pathogenesis.     

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.     

Virus-enhanced modulation of cell surface antigens: effect on immune lytic susceptibility.

Monkey kidney cells, upon progressive subculture, became refractory to complement (C)-dependent immune cytolysis by anti-cell serum. Arbovirus infection restored these cells to a state of lytic susceptibility. Similar results were also abtained with antibody-dependent cellular cytotoxicity (ADCC), which is C independent. Antibodies raised against different subcultures varied considerably in lytic efficiency, indicating changing patterns of host cell expression during continous subculture. Taken together with the fact that arbovirus infection festored the lytic efficiency of all antibody preparations to the same degree suggested some form of host cell antigen re-expression as a mechanism. The results obtained in several exploratory experiments indicated that the antigenic re-expression responsible for the restoration of lysis was probably a local or selective rather than a generalized phenomenon. Thus, the amount of host cell surface antigen, measured by the use of mouse anti-cell serum and 125I anti-mouse globulin, was identical in both uninfected lytic susceptible and refractory cells, and decreased in both functional states following infection. Further, the binding of 125I concanavalin A, used to quantify surface glycoproteins, was similar in both lytic refractory and susceptible cells, and in both cases declined folowing virus infection. This result was incompatible with gross "masking" of cell surface antigens by exuberant production of surface coat material in lytic resistant cells. Finally, brief trypsinization of lytic resistant cells yielded an 8-fold increase in immune lysis, a result further consistent with local rather than generalized surface changes. The data were discussed interms of modulation of cell surface antigens affected both by repeated subculture and arboviral infection, and as a possible in vitro correlate of altered self-reactivity.     

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN- for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN- for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Immunological aspects of infection of 6 inbred strains of mice by 3 different strains of Trypanosoma cruzi

We evaluated humoral and cellular immune responses in 6 inbred mouse strains (BALB/c, B-10, C3H, A/J, AKR and DBA) infected with 3 Trypanosoma cruzi strains (Peruvian, 21 SF and Colombian), which are the standards for the 3 strains Types of Andrade's classification. Negative delayed-type hipersensitivity reactions to parasite antigens were evidence of suppressed cell-mediated immunity. An early drop of IgG1 and rise of IgM levels were observed in almost all mouse strains infected by any T. cruzi strain. Elevation of IgG2a and/or IgG2b levels was higher in resistant mouse strains. Anti-T. cruzi antibody levels (Indirect immunofluorescence and ELISA) did not correlate with survival. Despite some differences among mouse strains there was a definition of an overall pattern of host response and the maintenance of biological standards which characterize the basic types of T. cruzi strains.     

Immunoglobulin classes and subclasses in alloantisera to mouse thymocyte surface antigens

A variety of cytotoxic alloantisera directed against murine cell surface components were analyzed for their content of specific IgG1, IgG2a, and IgG2b antibodies by radioimmunoassay, and specific IgM antibodies by 2-mercaptoethanol inhibition of cytotoxicity. Results indicate: (1) that the IgM and IgG subclass content of specific antibodies in anti-Lyt antisera produced at M.I.T. and Sloan-Kettering using the same donor and recipient strains are similar; (2) that specific antibodies produced concurrently in the same mice against at least two different alloantigens (Lyt-3.1 and H-2^k) can differ in IgG subclass content; and (3) that preparation of alloantisera against a given antigen (Lyt-3.1) using different combinations of donor and recipient strains can yield specific antibodies which differ in IgG subclass and cytotoxic IgM content. Thus, it appears that humoral immune responses to cell surface alloantigens are not random, but reflect both the antigens being recognized and the strains employed for the immunization.     

Immunity to virus-free syngeneic tumor cell transplantation in the BALB/c mouse after immunization with homologous tumor cells infected with type C virus.

Syngeneic tumor cell lines free of endogenous type C virus or viral antigen antigen expression were derived from spontaneously occurring tumors of the BALB/cCr mouse. Two cell lines free of endogenous type C virus were examined and found to be highly tumorigenic in tumor growth kinetic studies. In vitro inoculation of these cell lines with Rauscher-murine leukemia virus (R-MuLV) resulted in their chronic infection in which 95 to 100% of the cells were scored as virus positive. These infected lines showed a highly significant increase in their immunogenicity as compared to their uninfected controls. Animals in which these virus-positive tumors regressed were then shown to be highly resistant to challenge with the uninfected tumor cell lines as well as to live R-MuLV. This observed resistance to uninfected tumor cell lines could not be induced by immunization of the mouse with uninfected tumor cells and R-MuLV simultaneously at the same injection site, nor could it be induced with lethally irradiated virus-infected tumor cells, subtumorigenic doses of uninfected cells, or inactivated R-MuLV or Gross leukemia virus (G-MuLV). Cell-mediated cytotoxicity studies revealed that spleen cells obtained from animals whose virus-infected tumors regressed were cytotoxic to homologous infected and uninfected tumor cells as well as to other uninfected tumor cell lines syngeneic to the BALB/c mouse. Correlation of in vitro cytotoxicity with in vivo immunity was provided by the Winn assay, by inoculation into susceptible mice of immune and nonimmune spleen cells premixed with uninfected tumor cells. The immune cells were highly effective in preventing this tumor cell transplantation. It was concluded that type-C virus infection of these syngeneic tumor cells resulted in their acquiring strong transplantation antigens that were in part due to the virion, but were at least in part due to alterations of antigens or haptens that are present in a less immunogenic form on the uninfected tumor cell.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. II. Lectin and Immunologic Studies*

SYNOPSIS. Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing α-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and α-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with α-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with α-1,4 or repetitive α-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.     

益生菌及其细胞组分的免疫调节作用

益生菌对肠道黏膜系统的调节作用是应用益生菌提高宿主抵抗某些病源菌感染及预防肿瘤发生的基础。但是,益生菌的作用机理目前还不完全清楚。益生菌细胞壁组分可能是引发免疫反应的功能性物质,而构成各种益生菌细胞壁组分的差异可能是益生菌免疫调节作用存在菌种特异性的主要原因。肠道派伊尔斑外层的M细胞是介导益生菌或其细胞组分进入上皮下层的主要通道,进入上皮下层的益生菌或其细胞残片可以被黏膜免疫系统的树突状细胞(DC)或巨噬细胞吞噬,并呈递给淋巴细胞。益生菌及其细胞组分在黏膜局部刺激黏膜免疫系统,也可以引起系统免疫反应,但其通过局部免疫刺激引起系统免疫反应的机理目前还不清楚。研究益生菌细胞组分构成和益生菌免疫激活作用之间的关系,及由局部免疫刺激引发系统免疫反应的机理是益生菌免疫刺激作用机理研究需要解决的两大问题。     

Autogenous Immunity to Endogenous RNA Tumor Virus: Differential Reactivities of Immunoglobulins M and G to Virus Envelope Antigens

The autogenous humoral immune response of mice to their endogenous leukemia virus has been examined in terms of the reactivities of individual classes of antibody present in normal B6C3F(1) serum. Whole serum and the immunoglobulin (Ig) M and IgG fractions of serum from animals of different age groups were compared by radioimmune precipitation assays and viral infectivity neutralization assays. Both IgM and IgG fractions were able to precipitate virus, although not as effectively as whole serum. Virus-specific antibody levels, as well as total antibody concentrations in whole serum, appeared to increase with age. Sodium dodecyl sulfate gel electrophoresis analysis was performed with immune precipitates obtained when whole serum or 19 or 7S fractions from animals of different age groups were reacted with disrupted virus. The 19S antibody fraction reacted with three antigenic determinants on the viral envelope. These antigens have apparent molecular weights of 17,000, 43,000, and 68,000. The last two appear to be glycoproteins and may correspond to the M(2) and M(1) antigens. In contrast, the 7S component reacted only with the 17,000-molecular-weight protein. Neutralization assays against BALB:virus-2, a xenotropic endogenous mouse type C virus, revealed that 19S and whole serum but not the 7S fraction possessed neutralizing activity. These findings indicate that there are differential reactivities of IgM and IgG antibodies in normal serum of B6C3F(1) mice, with respect to both recognition of viral envelope antigens and neutralization of endogenous MuLV. These results are consistent with the hypothesis that the autogenous humoral immune response is a systemic host function that may be important in the regulation of endogenous type C virus expression in vivo.