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.

Regulation of interleukin-6 fetoplacental levels could be involved in the protective effect of low-molecular weight heparin treatment on murine spontaneous abortion.

PROBLEM: CBA/J x DBA/2 abortion rate could be the consequence of a deficient local production of T helper (Th2) cytokines, which cause fetal wastage via fgl2 prothrombinase. Heparin reduces significantly the abortion rate in mice and recurrent spontaneous abortion (RSA) patients. We proposed to determine the effect of enoxaparin on the levels of local interleukin (IL)-6 during murine pregnancy. METHOD OF STUDY: Recombinant human IL-6 (rhIL-6) or enoxaparin were inoculated in CBA/J x DBA/2 pregnant mice on days 6.5-12.5. IL-6 levels in sera as well as in culture supernatants of day 9.5 fetoplacental units of CBA/J x BALB/c control mice or CBA/J x DBA/2 abortion combination were determined by enzyme-linked immunosorbent assay (ELISA) test. RESULTS: CBA/J x DBA/2 fetoplacental units secreted significantly lower levels of IL-6 with regard to CBA/J x BALB/c normal units. rhIL-6h and enoxaparin treatments decreased the resorption rate and regulated IL-6 fetoplacental levels. CONCLUSION: This study suggests that regulation of IL-6 fetoplacental levels could be involved in heparin-mediated anticoagulation protection against abortion.

Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved.

The aims of this study were to characterize human American tegumentary leishmaniasis, which includes cutaneous, mucocutaneous and mucosal leishmaniasis, in Northwest Argentina, to determine the prevalence of double infection with Trypanosoma cruzi and to identify the species of Leishmania in this area. Most of the 330 leishmaniasis patients presented cutaneous ulcers (96.1%), 2.4% mucocutaneous and 1.5% the mucosal form ('espundia'). The aetiological agents, determined by isoenzyme electrophoresis, were identified as Leishmania (Viannia) braziliensis in 16 out of 20 isolates and in the remaining 4 as Leishmania (Leishimania) amazonensis, the first ever-documented in Argentina. Sera analysed by ELISA and IFA using complex antigen from both T. cruzi and L. braziliensis showed a very high percentage of positives (66.3-78.2%). When antigens for specific diagnosis of Chagas' disease were used, 40.9% of the leishmaniasis patients were also found to be infected by T. cruzi. These results indicate that the strong immune response against T. cruzi gave no protection to Leishmania, in spite of the serological cross-reaction between these parasites. In addition, we showed that more than 40% of the patients would be misdiagnosed as chagasic if complex antigens, as epimastigotes or soluble fraction from epimastigotes, were used in IFA or ELISA. This is of paramount importance not only because patients' treatment would be associated to misdiagnosis but the fact that in many countries in Central and South America, a positive test for Chagas' disease means a rejection for those seeking employment.

Anticuerpos asimétricos: el arma de protección antigénica en la respuesta inmune regulada por Th2 / Asymmetric antibodies: the antigen protective arm in a Th2-regulated immune response

Está bien aceptado que, durante el embarazo, las infecciones por micro organismos extracelulares y los procesos alérgicos -aparte de otras condiciones-, los adultos inmunocompetentes montan respuestas inmunitarias Th2 que implican un aumento de la producción de anticuerpos. Considerando que los anticuerpos son capaces de activar mecanismos inmunitarios efectores tales como la fijación de complemento, el aclaramiento de antígenos y la fagocitosis, un simple desvío de las citocinas implicadas en ese cambio podrían no explicar completamente fenómenos tales como la supervivencia fetal y la cronicidad de ciertas infecciones. Hace unos veinte años que comenzamos nuestras investigaciones sobre anticuerpos asimétricamente glicosilados. Así probamos que la presencia de un carbohidrato extra en una de las regiones Fab de la molécula de IgG, afecta a la interacción con el antígeno y determina su univalencia característica y sus propiedades bloqueantes. En el presente estudio, revisaremos las principales propiedades de este tipo de anticuerpos y reportaremos nuestros hallazgos en distintos procesos fisiopatológicos. (AU)

Interleukin regulation of asymmetric antibody synthesized by isolated placental B cells.

PROBLEM: Protecting antibodies against trophoblast surface molecules were previously described. Here we analysed the synthesis of asymmetric IgG by placental B-lymphocytes. METHOD OF STUDY: B cells were isolated from human term placenta and cord blood, stimulated with anti-CD40 IgG and cocultured with transfected Fcgamma R-expressing mice Ltk-fibroblast. Interleukin-4, IL-6, IL-10, IL-11 and IL-13 were added to cultures for 14 days. Asymmetric IgG were assessed in culture supernatants by concanavalin A (Con A) fixation and enzyme-linked immunosorbent assay. RESULTS: When IL-6 was added to the cultures, the percentages of asymmetric IgG synthesized by placental B cells were: IL-6: 29 +/- 10; IL-6 + IL-10: 24 +/- 7; IL-4 + IL-10 + IL-6: 38 +/- 9. The last combination induced the highest increase in the asymmetric IgG synthesis as compared with control (19 +/- 10%, P < 0.05). Additionally, placental B cells synthesized more asymmetric IgG than umbilical cord blood B-lymphocytes (P = 0.0015). CONCLUSIONS: Isolated placental B-lymphocytes synthesized asymmetric IgG in response to Th2 interleukins, more notably IL-6 in combination with IL-4 and IL-10. The in vitro increase of protective asymmetric IgG synthesis in response to Th2-cytokines support the hypothesis that a local Th2-switch is beneficial for pregnancy outcome.

During pregnancy, in the context of a Th2-type cytokine profile, serum IL-6 levels might condition the quality of the synthesized antibodies.

PROBLEM: In recent years, the central role of cytokines in the immune response has been widely studied. It is considered that a T helper (Th)1-type cytokine profile is associated with the rejection phenomenon, whereas a Th2-type cytokine profile is associated with immunological tolerance. In pregnancy, the enhanced Th2/Th1 ratio seems to be necessary to fetal protection. Taking into account that a Th2-type response means antibody production by B cells, and that these antibodies could induce degradation of the paternal antigens, we investigated the quality of the antibodies produced during pregnancy and their regulation. METHOD OF STUDY: Review of previous data. RESULTS: The regulation of protective antibodies by IL-6 in a dose-dependent fashion is proposed as a hypothesis. CONCLUSION: Cytokines play a central role in the success (or failure) of pregnancy. However, the quality of the synthesized antibodies is also a regulatory key. The preferential synthesis of asymmetric immunoglobulin G antibodies during pregnancy could be one of the several pathways that lead to a successful pregnancy

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.

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.

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.

[Participation of transcriptional factors in the regulation of lymphocyte differentiation]. / Participación de factores de transcripción en la regulación de la diferenciación linfocitaria.

The cytomorphological changes as well as the expression of certain markers in the different stages of lymphocyte differentiation are well known. Studies carried out on expression patterns of specific genes in lymphoid cells and their regulatory mechanism have led to the identification of the fundamental mechanism of cell development and activation. A great deal of knowledge has accumulated concerning enhancers and promoters, the regulatory elements of those genes and the transcriptional factors to which they bind. The present paper analyzes these components and the participation of some of them such as PU.1, Ikaros, Aiolos, GATA-3, Egf-1, E2A, EBF-1, PAX-5 (BSAP), TFE-3, Oct-1, Oct-2, and Nf-kappa B in the regulation of the differentiation stages of cells belonging to the B and T lymphoid lineages.

Participación de factores de transcripción en la regulación de la diferenciación linfocitaria. / [Participation of transcriptional factors in the regulation of lymphocyte differentiation]

The cytomorphological changes as well as the expression of certain markers in the different stages of lymphocyte differentiation are well known. Studies carried out on expression patterns of specific genes in lymphoid cells and their regulatory mechanism have led to the identification of the fundamental mechanism of cell development and activation. A great deal of knowledge has accumulated concerning enhancers and promoters, the regulatory elements of those genes and the transcriptional factors to which they bind. The present paper analyzes these components and the participation of some of them such as PU.1, Ikaros, Aiolos, GATA-3, Egf-1, E2A, EBF-1, PAX-5 (BSAP), TFE-3, Oct-1, Oct-2, and Nf-kappa B in the regulation of the differentiation stages of cells belonging to the B and T lymphoid lineages.

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.

Paradoxical behavior of asymmetric IgG antibodies.

Changes in the quantity and quality of antibodies occur in the course of an immune response. This review describes the physicochemical and biological properties of asymmetric antibodies as well as their functions, beneficial or harmful to the host, according to the nature of the antigen and the particular situation in which they act. Asymmetric antibodies have two paratopes, one of high affinity, with K0 similar to that of symmetric antibodies, and the other one with an affinity for the antigen 100 times lower. Functional univalence is due to steric hindrance present in one of the paratopes by the carbohydrate moiety attached to the Fd fragment of the Fab region, so these antibodies are unable to form large antibody-antigen complexes and cannot trigger reactions, such as complement fixation, phagocytic activity and antigen clearance. When asymmetric IgG antibodies are specific for self-antigens, they prove beneficial for the host by exerting regulatory functions. In allergic manifestations, in autoimmune diseases and especially during pregnancy, despite the fact that the antigens responsible for the process are foreign to the host, they also perform beneficial activity. During pregnancy, the placenta secretes molecules or factors that regulate the synthesis of these antibodies, thus favoring fetal protection.

Incidence of rat-soluble placental factors on IgE and IgG2a synthesis.

PROBLEM: The in vivo effect of soluble factors present in placental culture supernatants (PCSs) on the synthesis of rat immunoglobulin E (IgE) and IgG2a isotypes was investigated. METHOD OF STUDY: Batches of Wistar SPF rats immunized with a 10-microgram dose of ovalbumin and Al(OH)3 were used: group I, consisted of virgin rats; group II, virgin females injected simultaneously with PCSs; and group III, pregnant females. As controls, nonimmunized batches were included. Serum samples were collected at days 0 (basal) and 10 after antigen challenge, determining levels of total and specific antiovalbumin of both IgE and IgG2a by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: In vivo and at least at the doses administered, PCSs exert an inhibitory effect on the synthesis of specific and total anti-ovalbumin IgE during the course of immune response to such challenge. However, PCSs did not modify serum values of total and specific IgG2a. CONCLUSIONS: These results suggest that PCSs exert selective influence on the synthesis of diverse immunoglobulin isotypes during immune response, through the balance of cytokines synthesized by placental cells.

Effect of rat placental culture supernatants on cellular and humoral immune responses.

PROBLEM: To evaluate the effect of rat placental culture supernatants (PS) on spontaneous, mitogen- and alloantigen-induced lymphoproliferation, antibody synthesis regulation, and symmetric/asymmetric antibody ratio. METHOD OF STUDY: The effect of PS was determined: (a) on cell proliferation of murine hybridoma cells and on spontaneous or ConA-induced proliferation of murine and rat splenocytes by thymidine incorporation; (b) on rat or mouse cell-mediated cytotoxicity (CMC) by 51Cr release; and (c) on antibody synthesis by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: With 20% PS, hybridoma cell inhibition was 37% and that of splenocytes up to 60%, whereas it was 75 and 43%, respectively, in the presence of ConA. Despite marked cell death, hybridoma proliferation index increased significantly. There was a drop in total antidinitrophenylated (DNP) immunoglobulin G1 (IgG1) antibody production and an increase in asymmetric antibody percentage, correlating with placental supernatant concentration. CONCLUSIONS: Rat placental culture supernatants inhibit cell proliferation in all cases, diminish total antibody production, and increase the percentage of asymmetric antibodies by the hybridoma, and they increase antibody production by rat splenocytes.

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.

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.

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.

Cross-reactivity studies and differential serodiagnosis of human infections caused by Trypanosoma cruzi and Leishmania spp; use of immunoblotting and ELISA with a purified antigen (Ag163B6).

Results of our studies on the reactivity of chagasic and leishmaniasic sera with the purified T. cruzi-specific antigen 163B6, as assessed by ELISA, and with complex antigenic mixtures from T. cruzi and Leishmania mexicana, by immunoblotting, are presented here. Our objective was to identify the antigens responsible for the exhibited cross-reactivity between trypanosomiasis and leishmaniasis, and to find a specific reactivity pattern corresponding to each parasitosis. In spite of the high cross-reactivity observed with the immunoblotting, the use of 7.5% A-B gels made it possible to identify a characteristic pattern for each parasitosis, that could be distinguished by the naked eye. The characteristic pattern corresponding to chagasic patients was ascribed to reactivity with T. cruzi bands of mol. wts 131, 125, 116, 111, 51-45 and 43 kD, that were not recognized by leishmaniasic sera. Trypanosoma cruzi antigens of mol. wts 85, 81, 70, 65-60, 37 and 32 kD were considered as crossing antigens, since they were recognized by leishmaniasis sera. With L. mexicana, most of the chagasic patients presented reaction with antigen of mol. wts 124, 107, 92, 59 and 32 kD, while bands of mol. wts 155, 140, 73, 56 and 48 kD were recognized only by leishmaniasic sera. In this study we found 12 out of 45 sera of patients with leishmaniasis, from a region endemic for both parasitoses, which exhibited a pattern of bands very similar to those corresponding to chagasic individuals, strongly suggesting a mixed infection. This hypothesis was verified by using a purified specific antigen of T. cruzi, Ag163B6, which would be the major cysteine proteinase of this specie (cruzipain). By ELISA, these 12 sera showed a positive reaction with this purified antigen, as those of chagasic patients, thus leading to the confirmation of the presence of a mixed infection.

Immune response against Trypanosoma cruzi antigens in Cebus apella monkeys.

The american primate Cebus apella has been used as an experimental model for the study of acute and chronic Chagas' disease. The antibody response elicited by 4 x 10(6) blood trypnomastigotes injected into four monkeys was analysed. Peak titres of IgM and IgG of anti-Trypanosoma cruzi antibodies were found at day 22, and between days 20 and 40 post-infection (p.i.), respectively. The ability of a Mr 37kDa (T37K) glycoprotein purified from T. cruzi epimastigotes to generate IgG anti-T. cruzi antibodies in monkeys, and protect them against a challenge with trypomastigotes, was also studied. Monkeys non-immunized with T37K reached peak values of parasitaemia between days 18 and 21 post-infection, whereas immunized monkeys had lower parasitaemias without important variation. Anti-T37K antibodies in immunized monkeys decreased from day 2 with the lowest titres between days 14 to 22 p.i., coincident with the peak of parasitaemia in control non-immunized monkeys. These results suggest that anti-T37K antibodies could be responsible for the low parasitaemia detected in immunized monkeys.