Plasma proteins and immunoglobulins in penguins (Pygoscelis adeliae).

Total and fractionated proteins were evaluated in the serum of normal penguins. Four proteins with antibody activity, determined by passive haemagglutination, were isolated by Sephadex G-200 filtration and DEAE-cellulose chromatography, from the serum of penguins (Pygoscelis adeliae) inoculated with dinitrophenylated human gamma-globulin. Immunoelectrophoresis and immunodiffusion showed that all these proteins exhibited both their own and shared antigenic determinants. On account of their electrophoretic mobility, elution conditions and characteristics of the precipitation arcs, they have been tentatively denominated IgG2 (gamma2), IgG1 (gamma1), IgM (gammam) and IgX (probably IgA).

Isolation and partial characterization of biologically active Fc receptor of chicken red cells.

It has previously been demonstrated that chicken red cells have a receptor with the capacity to bind aggregated IgG, IgM 7 S or antigen-complex IgG. This receptor was isolated from Nonidet P-40 soluble extracts of chicken red cells by immunoadsorption with either immobilized aggregated IgG or monomeric IgM (IgM 7 S) and further gel filtration through a Sephacryl S-300 column. The Fc binding material was characterized as a glycoprotein with a molecular weight of 30,000 which retained its Fc receptor activity after the isolation procedure. This was demonstrated by its capacity to inhibit the binding of 125I-IgM 7 S or 125I-labelled aggregated IgG to chicken red cells. After Bacillus cereus phospholipase C treatment the Fc receptor activity remained unchanged, but the molecular weight (15,000) did not, suggesting that the phospholipids cleaved by this treatment were not essential for the interactions of the receptor with specific ligands. However, this Fc-binding component was shown to have a molecular weight of 13,000 and a diminished Fc receptor activity after reduction with dithiothreitol, suggesting the presence of at least one disulphide bridge, necessary to maintain the total ligand-binding activity.

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.

Asymmetric antibodies: a protective arm in pregnancy.

In normal conditions, a simple change in the pattern of cytokines towards a Th2 response is associated with the production of aggressive antibodies. This fact could not completely explain phenomena such as the fetal survival or the chronicity of certain infections. However, it has been demonstrated that Th2 cytokines increase the proportion of asymmetric antibodies, which are unable to activate effector immune mechanisms (complement fixation, clearance of antigens and phagocytosis). Investigations of asymmetrically glycosylated antibodies demonstrated that these IgG molecules have an extracarbohydrate in one of the Fab regions. This glycosylation affects their antigen interaction turning them into a functionally univalent and blocking antibodies. It has been established that their synthesis is increased under different physiopathological situations involving Th2 responses: chronic infections by extracellular microorganisms, pregnancy and allergic processes. In this review we summarize the experiments performed by our research group over the last years as well as the advances made concerning the role and mechanism of asymmetric antibodies.

The asymmetric IgG non-precipitating antibody. Localization of the oligosaccharide involved, by concanavalin A interaction.

Binding to Con A-Sepharose 4B of the low-affinity Fab fragment from sheep IgG1 anti-Dnp non-precipitating antibody was previously determined. This communication reports the results obtained when the concanavalin A interaction with Fd' fragments and L chains from non-precipitating and precipitating antibodies was examined. When Fd' fragments and L chains from non-precipitating and precipitating antibodies were tested as inhibitors of the binding of 125I-labelled concanavalin A to guinea pig erythrocytes, inhibition of such binding was only achieved by Fd' fragments from non-precipitating antibodies. Forty eight per cent of this Fd' fragment was bound by Con A-Sepharose 4B. From these results and our previous studies we conclude that mannose and/or glucose residues must be present and exposed at the Fd' fragment from the low-affinity Fab arm of sheep IgG1 anti-Dnp non-precipitating antibody. Glycosylation differences may explain the difference in precipitation behaviour of the 2 types of antibody.

N-glycanase treatment of F(ab')2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity.

The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab' glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab')2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab')2 fragments.

Analysis of oligosaccharides involved in the asymmetrical glycosylation of IgG monoclonal antibodies.

The aim of this study is to identify the oligosaccharide residues involved in the asymmetric glycosylation of immunoglobulins. We have studied two anti-DNP monoclonal antibodies of the IgG1 isotype. Results show both qualitative and quantitative differences in the carbohydrates of both monoclonal antibodies and their fragments F(ab')2, Fab' and Fd. One of the antibodies -112D5-, which appears to be homogeneous in the Scatchard plot, has oligosaccharide residues in the L chain and in the Fd of one of the Fab'. On the other hand, 112B2 mAb, which also appears to be asymmetrically glycosylated, shows the bimodal curve characteristic of antibodies with different combining site affinities.

Effect of pregnancy and placental factors on the quality of humoral immune response.

Asymmetrical IgG molecules are characterised by the presence of a mannose-rich oligosaccharide group in only one of the two Fab fragments, which impairs the corresponding paratope, causing such molecules to behave as univalent antibodies and therefore as antigen blockers [1-3]. During human and murine pregnancy, an increase has been detected in asymmetrical IgG molecules in serum and those bound to the placenta, which normally releases factors capable of modulating the immune response. It thus seemed of interest to investigate the effect of placental culture supernatants (PCS) on in vivo and in vitro synthesis of rat immunoglobulin IgG1, IgG2a, IgG2b and IgG2C, particularly the ratio of symmetrical and asymmetrical molecules in each isotype. The effect of PCS was determined in vivo by means of passive transfer to virgin females and in vitro by analysing the supernatants of spleen cells cultured in the presence of PCS. The results showed that neither pregnancy status nor PCS were capable of modifying serum levels of IgG2a, IgG2b or IgG2c, whereas the level of IgG1 was reduced. When PCS were added to the spleen cells cultures, an in vitro increase was observed in IgG2a, IgG2b and IgG2c production. The separation of symmetrical from asymmetrical IgG molecules was performed by affinity chromatography in Concanavalin A-Sepharose, as such lectin binds high mannose sugars present only in asymmetrical IgG molecules. It is shown that pregnancy and PCS induce an increase in IgG1 and IgG2 molecules asymmetrically glycosylated, capable of binding to ConA-Sepharose. Therefore, the placenta is capable of releasing factors which can regulate the relative proportion of asymmetrical IgG molecules and induce quantitative and qualitative modifications of the in vitro and in vivo produced antibodies.

Purification and properties of anti-Trypanosoma cruzi antibodies isolated from patients with chronic Chagas' disease.

Three purified human IgG antibodies against Trypanosoma cruzi (anti-F, anti-Mc and anti-Cs) showed different reactivities in vitro. While all of them agglutinated specifically sensitized sheep red cells the complement-fixing capacity of anti-Fc and anti-Cs was higher than that of anti-Mc antibody. This one also showed a minimal precipitating activity. When the ability to neutralize mice bloodstream trypomastigotes was analyzed, the results obtained indicated that anti-F antibody was the most effective.

Identity of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi and an antigen (Ag163B6) isolated with a monoclonal antibody.

Cruzipain, purified by conventional methods, and Ag163B6, isolated by affinity chromatography with a monoclonal antibody raised against a T. cruzi extract, are glycoproteins with a similar electrophoretic mobility, which reacted with sera from most chronic chagasic patients. Their behaviour in SDS-PAGE, Western blotting, isoelectric focusing, two-dimensional electrophoresis (IEF and SDS-PAGE), Ouchterlony's double diffusion, and enzyme activity in SDS-PAGE gels containing 0.1% gelatin suggests that they are identical.

IgG asymmetric anti-ovalbumin antibodies synthesized by virgin and pregnant rats.

A study on the synthesis of asymmetrical IgG molecules 'with no specific activity' and with anti-ovalbumin activity was carried out both in virgin rats and in rats inoculated with ovalbumin and made pregnant by syngeneic and allogeneic males. Before pregnancy, female rats synthesize about 23% of asymmetrical IgG molecules, when the level of these molecules is assessed in total IgG, in anti-ovalbumin IgG and in the supernatant from the adsorption of anti-ovalbumin antibodies. On the other hand, anti-ovalbumin antibodies isolated are predominantly of the symmetrical IgG type; they are also precipitants and effectors of the biological mechanisms that the host operates to preserve pathogenic antigens (bacteria, parasites). In rats pregnant by syngeneic and allogeneic males, the ratio asymmetric/symmetric IgG molecules increases, and the anti-ovalbumin antibodies are mainly of the asymmetrical IgG type, which aid antigen-blocking. Similar results are found in virgin rats, immunized with ovalbumin and intraperitoneally transferred simultaneously with supernatants of placental cultures. These results suggest that, during pregnancy, there is an increase of the IgG asymmetric/symmetric molecule ratio, produced by placental factors, whatever the immunogen specificity may be. Speculations about this fact are presented.

The placental regulatory factor involved in the asymmetric IgG antibody synthesis responds to IL-6 features.

Pregnancy success is attributed to the joint action of several factors such as regulatory placental molecules and components of the mother's immune system, among others. Asymmetrical glycosilated and functionally univalent IgG antibodies are suggested to influence the immune balance between the mother and foetus, playing a meaningful role on the foetal survival in the maternal uterus. Placental secretory factors might be responsible for the increase of these molecules during gestation. Since placental factors appeared to be the inducers of these high Concanavaline A-affinity (Con-A) IgG molecules our work was focused on the identification of such factors. The chromatographic separation of placental culture supernatants (PCS) allowed the detection of fractions capable of increasing the high Con-A affinity monoclonal antibody (Mab) ratio synthesised by a hybridoma. The presence of multimeric placental forms of interleukin 6 (IL-6) could be identified in these fractions. Considering that IL-6 modulates protein glycosylation we decided to investigate its effect on the monoclonal IgG glycosylation. When placental IL-6 containing fractions or rIL-6h were added to hybridoma cultures, the proportion of asymmetric IgG antibodies increased substantially.

Preferential synthesis of asymmetric antibodies in rats immunized with paternal particulate antigens. Effect on pregnancy.

The effect of immunization of female Fischer rats with particulate (spleen cells) (group I) or soluble (supernatant of disintegrated spleen cells) (group II) paternal antigens previous to mating with Buffalo rats was investigated. The percentage of asymmetric IgG molecules in the serum of rats inoculated with particulate antigens was 38% while in those injected with soluble antigens it was 29% and 28% in non-immunized animals. These percentages further increased during pregnancy to 45%, 38% and 37%, respectively. The antipaternal antibody titres, as determined by indirect immunofluorescence (IIF), was much higher in the animals immunized with particulate antigens but the effector activity, judged by complement fixation, was similar in both groups. The same values were observed at the time of mating (after 3 months of immunization) and at day 17 of pregnancy. Fetus and placenta weights and offspring survival were equally greater in group I than in group II or non-immunized rats (group III). The results obtained indicate the preferential synthesis of antipaternal IgG asymmetric antibodies in rats injected with particulate antigens previous to mating and suggests a beneficial effect of these antibodies in pregnancy.

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.

Polymerase chain reaction reveals Trypanosoma cruzi infection suspected by serology in cutaneous and mucocutaneous leishmaniasis patients.

The existence of patients suffering a double infection caused by Trypanosoma cruzi and Leishmania spp. has been suggested by several authors. Since the conventional serological tests now available for the diagnosis of Chagas' disease lack specificity due to the cross-reactivity between these two parasites, a serological confirmation of a T. cruzi infection cannot be made unless specific antigens are used. An enzyme linked immunosorbent assay (ELISA) to detect antibodies against a specific T. cruzi antigen, named Ag163B6, and immunoblotting using T. cruzi epimastigotes, are non-conventional serological techniques that could be employed for specific diagnosis of Chagas' disease. Using these two methods 34 cutaneous or mucocutaneous leishmaniasis patients were classified into two groups: (A) patients with serological evidence of T. cruzi infection, i.e. those who tested positive in at least one assay (18/34); and (B) patients with no serological evidence of T. cruzi infection, i.e. those who were negative for both assays (16/34). Taking into account the difficulties of xenodiagnosis and its low sensitivity (less than 50%) for a direct diagnosis in the chronic period of the disease, we used polymerase chain reaction (PCR) to confirm a T. cruzi infection in those leishmaniasis patients who presented positive results with the non-conventional serological techniques. Of the 18 patients with serological evidence of T. cruzi infection, 17 gave positive results when genomic DNA primers were used. Using minicircle primers, 15/18 of that group were positive. Nevertheless, all the patients suspected of being double infected were positive in at least one PCR test. Just one patient with no serological evidence of T. cruzi infection gave a positive PCR result when amplifying the minicircle sequence. The proof of the existence of a T. cruzi infection by PCR in leishmaniasis patients suspected to be chagasic when non-conventional serology was used, strongly supports the use of the specific Ag163B6 and immunoblotting with epimastigotes as specific serological diagnostic tools to determine a T. cruzi infection.

Analysis and in vivo assay of B. abortus agglutinating and non-agglutinating antibodies.

Studies were made of physicochemical and immunochemical characteristics of Brucella abortus agglutinating and non-agglutinating antibodies in the sera of cattle repeatedly injected with living B. abortus (Strain 1119). Both agglutinating and non-agglutinating antibody were shown to be IgG1, and by immunodiffusion against rabbit anti-cattle gamma-globulin, agglutinating antibody gave a precipitation line of identity with that given by non-agglutinating antibody. Whilst agglutinating antibody increased clearance of antigen from the blood of passively protected mice, non-agglutinating antibody did not enhance clearance. Determination of the spleen infection index in mice pre-treated with agglutinating and non-agglutinating antibody showed that in animals passively immunized with non-agglutinating antibody the number of living (infecting) bacteria was approximately 4 times higher than in the case of agglutinating antibody. The possible potentiation of chronic B. abortus infection by non-agglutinating antibody is discussed.

Structural studies of sheep IgG1 precipitating and co-precipitating antibodies.

Descriptions have previously been given of the physiochemical and immunobiological behaviour of IgG co-precipitating antibodies, isolated from different animal species, compared with that of the precipitating ones belonging to the same immunoglobulin class. From those investigations it seems reasonable to assume that only one of the combining sites present in the two binding arms of the co-precipitating antibody molecule firmly binds the antigen. Consequently, the antibody does not form insoluble Ag-Ab complexes. The peptide maps, diagonal peptide maps and high voltage electrophoresis developed with peptides obtained after previous reduction and radioactive alkylation did not show differences between sheep IgG1 precipitating and co-precipitating antibodies. When diffused against rat anti-sheep IgG1 precipitating antibody serum, sheep IgG1 precipitating antibody gave a band of precipitation which was identical to the band given by sheep IgG1 co-precipitating antibody. Similar results were obtained when rat anti-sheep IgG1 co-precipitating antibody serum was used for precipitation. By immunodiffusion with cross absorbed sera no antigenic differences between the two antibodies could be demonstrated. The results obtained indicate that the different behaviour of precipitating and co-precipitating antibodies when interacting with antigen would probably not be a consequence of differences in the primary structures of their constant fragments. Both antibodies would belong to the same class, sub-class or type of immunoglobulin.

IgG precipitating and non-precipitating antibodies in rabbits repeatedly injected with soluble and particulate antigens.

The immune response of precipitating antibodies and non-precipitating antibodies of high affinity (co-precipitating) of the IgG class was analyzed in rabbits repeatedly injected with egg albumin (as a soluble antigen) B. abortus-egg albumin and polymerized egg albumin (as particulate antigens). The results showed that the levels of anti-egg albumin non-precipitating antibodies induced by the soluble antigen were never higher than 10-15% of total antibodies throughout the experimental time. When particulate antigens were injected, the levels of non-precipitating antibodies increased up to 30-70% of the total antibody levels. This phenomenon is related to the way in which the antigen is available to the immune system (particle or aggregated), and is independent of the response induced by the particulate carrier. Components from the cell wall or bacterial membrane that could act as coadjuvants do not participate in this phenomenon. The results obtained seem to indicate that possibly there was a suppression of the synthesis of precipitating antibodies, and this would produce a relative increase in the non-precipitating antibodies.

Humoral and cellular parameters of the immune system of Cebus apella monkeys. Cross reactivity between monkey and human immunoglobulins.

The humoral and cellular immunological parameters of the New World non-human primate Cebus apella were analysed. The study included: serum protein immunoelectrophoretic analysis; cross reactivity between monkey and human immunoglobulins by immunoprecipitation, ELISA and indirect immunofluorescence tests; immunoglobulin quantitation by radial immunodiffusion; and assays with peripheral blood lymphocytes involving tests for E and EAC rosettes and detection of surface markers (surface immunoglobulins and CD4-CD8 antigens). The results obtained showed that (a) at least three immunoglobulins with electrophoretic mobility corresponding to IgG, IgA and IgM which showed cross reactivity with the human ones were present in serum; (b) it was possible to evaluate the relative monkey immunoglobulin concentration using specific antibodies against human immunoglobulins and to obtain absolute values using adequate conversion factors; (c) lymphocytes forming E and EAC rosettes were found in peripheral blood in a similar proportion to that reported in man; (d) lymphocyte surface immunoglobulins were detected using anti-human immunoglobulin serum; (e) it was not possible to demonstrate the presence of T helper and T suppressor/cytotoxic lymphocytes using OK T4 and OK T8 monoclonal antibodies.

Antibodies against a tumour-associated antigen in an AKR lymphoma conditioned to grow in BALB/c mice.

An experimental model was used in which AKR lymphoma cells (L15) were conditioned to grow in BALB/c mice leading to tumour-bearing (progressor) and tumour-rejecting (regressor) animals. The behaviour of antibodies present in the sera of these animals was studied using as antigen L15 cells or a soluble tumour-associated antigen TEs. Both sera showed similar IIF and haemagglutinating activity. However differences were observed for the complement cytotoxicity assay. Regressor serum as well as a rabbit anti-tumour-associated antigen serum were strongly cytotoxic for the AKR lymphoma cells while progressor serum showed markedly lower activity. Specific antibodies against the tumour-associated antigen were purified. In both sera they were located mainly in IgG1 but also in IgG2. The purified antibodies agglutinated specifically sensitized sheep erythrocytes and reacted by indirect immunofluorescence with L15 cells but not with AKR thymocytes. It is suggested that two qualitatively different humoral immune responses are involved in the mechanisms leading to tumour enhancement or rejection.