Anaphylactic response to parasite antigens: IgE and IgG1 independently induce death in Trichinella-infected mice.

The response of animals infected with different Trichinella species (T. spiralis, T. britovi, T. pseudospiralis) to antigens of different Trichinella species was evaluated in outbred mice (CD1) and inbred mice (BALB/c, C3H, SJL, C57BL/6). In mice deficient for IgE or IgG1, T. spiralis only was used. In homologous conditions (i. e., same Trichinella species for antigens and infectious agent) mortality was very high (up to 100% in SJL and C57BL/6). The anaphylactic response in heterologous conditions was much lower and death was observed only in C57BL/6 and SJL mice. Experiments with deficient mice have shown that both specific IgE and IgG1 independently induce a fatal anaphylactic response.

Modulation of the immune response by progesterone-induced lymphocyte factors.

Rat spleen and peripheral blood lymphocytes express progesterone receptors whose concentration is increased greatly during the early phase of pregnancy. After stimulation of progesterone the expression of receptors was augmented 2-3 times. When cells were cultured in the presence of progesterone they released a soluble factor that inhibited cellular immunoreactions (MLR, CRC) and cellular proliferation as measured by thymidine incorporation by spleen-cell culture. This factor also inhibited the synthesis of anti-DNP antibodies by a mouse hybridoma and diminished the proportion of cells in phase S. However, the percentage of asymmetric molecules produced by the hybridoma remained unaltered. These results support the hypothesis that soluble factors released by rat lymphocytes modulate the immune response of the mother and participate in the mechanism that protects the fetus against antipaternal antibodies.

Modulation of the humoral immune response by placental secretory factors.

PROBLEM: To investigate how the factors secreted by human placenta modify the quality and the quantity of the antibody produced by the hybridoma as well as its cellular proliferation. METHOD: Supernatants of cultures of human placenta (PS) were added to a mouse IgG1 hybridoma culture producing anti-DNP antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. RESULT: It was found that PS augmented by 40-50% the quantity of total antibody produced, increased the proportion of asymmetric (blocking) antibodies from 15% to 30%, and diminished the cellular proliferation, as measured by 3H-thymidine incorporation. CONCLUSION: These results, together with other similar observations already described in human, rat and mouse pregnancies, suggest that secretory factors produced by the placenta do modify the immune response of the mother against paternal antigens and participate in the mechanisms that make possible the survival of the allogenic fetus.

Influence of mouse placental factors on in vitro antibody synthesis.

Placental culture supernatants (PS) obtained from various mouse crossbreedings were added to mouse IgG1 hybridoma cultures producing anti-DNP antibodies. The quantity of monoclonal antibody (mAb) produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. It was observed that the supernatants increased or diminished the production of mAb, depending on the genetic origin of the placentae. This effect was the same using placentae from primiparous or multiparous females and it was not due to modifications of the cellular proliferation of the hybridoma, as shown by 3H-thymidine uptake. It was also found that placental supernatants induced an increase in the proportion of asymmetric, blocking antibodies synthesized by the hybridoma. This effect was detected with supernatants from both allogeneic or syngeneic crossbreedings, but only when placentae were obtained from multiparous females. These observations indicate that placentae produce at least two soluble factors that participate in the regulation of antibody synthesis and suggest that these factors play an important role in the immune equilibrium between mother and fetus.

The immunological aspects of parasitic diseases.

Parasitic infections present a paradoxal immune situation: the host develops a normal immune response against the parasite but, in general, this response does not result in its elimination. An equilibrium is established which is essential to the survival of the parasite, involving a diminished state of the host, but not necessarily lethal. The mechanisms that allow the parasite to escape the immune defence are varied and include antigenic variation, mimetism, anatomical seclusion, immunomodulation, etc.

Trichinella spiralis: modifications of the cuticle of the newborn larva during passage through the lung.

A scintigraphic method was developed to study the distribution of radioactivity after iv injection of 131I-labeled Trichinella spiralis newborn larvae into normal rats. It was found that the radioactivity was immediately retained in the lungs and thereafter slowly released, with a mean transit time in excess of 9 hr, as calculated by image analysis. At various times after iv injection of newborn larvae into normal mice, the lungs were removed and parasites were recovered and counted. Fifty to seventy percent of the larvae injected were recovered after 30 sec, between 10 and 30% after 1 min, and less than 4% at 15 min. These results indicate that during the very rapid passage of newborn larvae through the lungs, labeled components of the cuticle are detached and retained. It is suggested that the modifications produced in the cuticle of the newborn larva during its passage through the lung may increase its resistance to the nonspecific defense mechanisms of the host.

Asymmetric non-precipitating antibodies in commercial hyperimmune gamma-globulin for therapeutic use.

The presence of asymmetric (non-precipitating or co-precipitating) antibodies has been studied in three commercial preparations of antitetanus gamma-globulin. It was found that 27% of the specific antibodies are of asymmetric type, a value two to three times higher than that found in normal IgG. In toxicity tests in mice with tetanus toxin, the asymmetric antibodies were 2.7 times less effective than the symmetric ones. It is concluded that the protection capacity of the tetanus gamma-globulin preparations is dependent on the ratio of symmetric to asymmetric antibodies. The high content of asymmetric antibodies is probably due to the fact that immunization is performed with the antigen in a particulate form.

IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human.

The proportion of symmetric and asymmetric IgG molecules was studied in 10 mothers at delivery. IgG was obtained from peripheral blood and placental blood sera and by elution at 4 M KCl from placenta cell membranes. The percentage of symmetric and asymmetric molecules was determined in the IgG and in their corresponding F(ab')2 fragments by absorption to Con A-Sepharose. The presence of antipaternal antibodies was investigated by IIF and MC tests using paternal lymphocytes. The average percentage of asymetric IgG molecules in the sera was 24.4, which is about double the value of that found in normal subjects. In the IgG eluted from the placenta, the proportion of asymmetric IgG was much higher, averaging 44.4%. Antipaternal antibodies were detected in 5 mothers by IIF and MC and in two mothers only by IIF. In three mothers no antibodies could be detected. It was found that the concentration of antipaternal antibodies was about three times higher in the asymmetric IgG fraction than in the summetric one. Considering the percentage of asymmetric IgG molecules with antipaternal antigen specificity eluted from placenta and the possibility that they function as blocking antibodies, their participation in fetal protection is suggested.

Asymmetric Fab glycosylation in guinea-pig IgG1 and IgG2.

The presence of asymmetric antibody molecules has been investigated in both IgG1 and IgG2 subclasses of guinea-pig immunoglobulins. It was found that about 20% of the IgG1 and 10% of the IgG2 were of asymmetric type. The proportion was essentially the same in the sera of normal animals, animals hyperimmunized with dinitrophenyl-bovine gamma globulin (DNP-BGG) and Freund's adjuvant, and animals infected with Trichinella spiralis. In the case of animals immunized with DNP-BGG, no differences were observed in the proportion of asymmetric molecules between the specific antibodies and the IgG not specific for the immunizing antigen. It is concluded that the asymmetric glycosylation occurs to a different extent in each subclass and that it is not affected by the antigen specificity of the antibodies studied.

Asymmetrically glycosylated IgG isolated from non-immune human sera.

When human IgG or its F(ab')2 fragment purified from a pool of non-immune sera was passed through a Con A-Sepharose column, 12% of the molecules bound to concanavalin A. While 44% of Fab' and 72% of Fd' fragments obtained from F(ab')2 retained by concanavalin A and eluted with methyl alpha-D-mannoside bound to concanavalin A, the Fab' and Fd' fragments obtained from non-retained F(ab')2 and the L chains and Fc fragments did not interact with the lectin. Only Fd' fragment obtained from the F(ab')2 retained by concanavalin A inhibited the fixation of guinea-pig erythrocytes to concanavalin A. These results are similar to those previously observed for IgG antibodies of different animal species and indicate that partial asymmetric glycosylation is a general phenomenon that is not restricted exclusively to IgG molecules with known specificity.

Antibody-dependent in-vitro cytotoxicity of newborn Trichinella spiralis larvae: nature of the cells involved.

The cytotoxic reaction of normal peritoneal mouse cells, containing less than 3% eosinophils, against newborn Trichinella spiralis larvae, in the presence of antibody, was studied using newborn worms less than 2 h of age, or newborn worms that had been maintained in culture at 37 degrees C for 20 h. Newborn worms 2 h old were killed, whereas 20 h newborn worms were not. The cells that initially adhered to the larvae were examined by electron microscopy. Only eosinophils adhered to 2 h newborn worms and only macrophages to 20 h ones. The attached eosinophils degranulated and died after a few hours. The macrophages that adhered to, but did not kill the 20 h newborn worms were morphologically in good state and no lysis of the larvae was observed. These results suggest that different antibody classes are involved in eosinophil and macrophage adherence, and strongly support the hypothesis that eosinophils mediate larval destruction. They also show that rapid changes are taking place after birth in the structure of the larval cuticle and that the age of Trichinella newborn worms is a major factor in the antibody-dependent cellular cytotoxicity reaction.

Production of an antibody-like factor in the sea star Asterias rubens: involvement of at least three cellular populations.

Cells from the axial organ of sea stars stimulated in vivo with TNP coupled to polyacrylamide beads and subsequently cultured in vitro were able to produce an antibody-like factor which induced the lysis of mammalian red cells sensitized with TNP. The axial organ cells were fractionated in two populations, adherent and non-adherent to nylon wool. The release of the antibody-like factor required the contact of both populations. When the adherent cells were disrupted by sonication the factor was not produced, but the non-adherent cells could be substituted by their membranes. Destruction by silica of the phagocytic cells present in the adherent population inhibited the production of the factor. The addition of mercaptoethanol to the cultures was essential and did not neutralize the effect of silica. It is concluded that at least three types of cells are involved in the production of the antibody-like factor adherent and non-adherent cells, and phagocytes.

Structure of asymmetric non-precipitating antibody: presence of a carbohydrate residue in only one Fab region of the molecule.

The reactions between purified precipitating and non-precipitating anti-DNP sheep and rabbit antibodies and the antigens DNP-BSA and DNP-GABA-BSA have been studied by immunodiffusion, complement fixation and an inhibition test. Both antigens reacted identically with precipitating antibodies. On the contrary, non-precipitating antibodies did not precipitate and did not fix complement with DNP-BSA but were able to do so with DNP-GABA-BSA. A different behaviour with both antigens was also demonstrated by an inhibition test. The properties of these antibodies were also studied after treatment with endo-beta-N-acetylglucosaminidase H. Non-precipitating antibody was able to give precipitin bands in gel diffusion and to fix complement with DNP-BSA after treatment with the enzyme. The treated antibody was able to agglutinate sensitized erythrocytes. Studies by fluorescence quenching showed that the affinity for the ligand DNP-GABA was significantly increased after hydrolysis of the carbohydrate residue. The properties of precipitating antibody were not modified by the endoglycosidase. Affinity chromatography of the F(ab')2 and Fab fragments obtained from precipitating and non-precipitating antibodies was made with Con A-Sepharose. The Con A retained all the F(ab')2 and 50% of the Fab from non-precipitating antibody, which were subsequently eluted with alpha-methyl-D-mannoside. The fragments from precipitating antibody were not retained at all. It is concluded that the asymmetry of the non-precipitating antibody molecule is due to a carbohydrate moiety which is present in only one of the Fab regions. This carbohydrate affects the reaction between the combining site and the antigen, and renders the molecule functionally univalent.

Immunocompetent cells in the starfish Asterias rubens. An ultrastructural study.

Cells from the axial organ of the starfish Asterias rubens were fractionated into two populations, adherent and non-adherent to nylon wool. In both populations the ultrastructural study revealed the presence of cells resembling the lymphocytes of the vertebrates, as well as phagocytic, peroxidase positive cells. The lymphocyte-like cells in the non-adherent population (average diameter 4 mu) have a high nucleo-cytoplasmatic ratio and are morphologically similar to Th lymphocytes while the adherent cells (average diameter 5.5 mu) are more similar to Bm lymphocytes. These observations are in line with the hypothesis that there exist, in the starfish, a primitive immune system with characteristics resembling those of the immune system of vertebrates.

Interaction of purified precipitating and non-precipitating (coprecipitating) antibodies with hapten and with haptenated protein. Evidence of an asymmetric antibody molecule.

The interaction of monovalent hapten dinitrophenyl epsilon-amino caproic acid (DNP-EACA) with purified IgG1 sheep anti-DNP precipitating and non-precipitating antibodies, and their F(ab')2, F(ab') and Fab fragments, was studied by fluorescence quenching and by a radioimmunoassay. The Scatchard plots of whole non-precipitating antibody and its F(ab')2 fragment showed a bi-modal curve that could be interpreted as due to the existence of two populations of sites with very different affinity for the ligand, each population representing 50% of the total number of sites. The F(ab) fragments of the non-precipitating antibody could be fractionated by immunoadsorption into two populations of high and low affinity whose association constants differed by more than 2 logs. The study of the interaction of whole antibodies with DNP-bovine serum albumin (BSA) demonstrated that each molecule of precipitating antibody can combine with two molecules of antigen but non-precipitating antibody cannot combine with more than one molecule of antigen. It is concluded that the molecule of non-precipitating antibody is asymmetric and has a site of high affinity and another of low affinity. As a consequence of this structure the non-precipitating antibody behaves functionally as univalent and is unable to form precipitates with the multivalent antigen and to activate effector mechanisms.

Specific immune response in the sea star Asterias rubens: production of "antibody-like" factors.

Axial organ cells from the sea star (Asterias rubens) inoculated 7 days before with TNP- or FITC-haptenated PAA beads and subsequently stimulated in vitro with the same antigen, produced and released a specific, soluble "antibody-like" substance that induced lysis of haptenated sheep erythrocytes. Fresh normal rabbit or guinea pig serum was essential for the lysis, suggesting the participation of complement components. The factor was produced by the total population of axial organ cells but not by nylon-wool adherent (B-like) or nonadherent (T-like) cells. These results provide further evidence of the existence, in the sea star, of a primitive immune system with characteristics reminiscent of the immune system of vertebrates.