José Dokmetjian

Interleukin Regulation of Asymmetric Antibody Synthesized by Isolated Placental B Cells

Canellada A, Färber A, Zenclussen AC, Gentile T, Dokmetjian J, Keil A, Blois S, Miranda S, Berod L, Gutiérrez G, Markert UR, Margni RA. Interleukin regulation of asymmetric antibody synthesized by isolated placental B cells. AJRI 2002; 48: 275–282

A possible explanation for the discrepancy between ELISA and neutralising antibodies to tetanus toxin

The structure and protective activity of tetanus antibodies elicited in rabbits after whole-cell pertussis diphtheria-tetanus vaccine (DTPw) vaccination was studied. ELISA antibody levels and toxin neutralisation activity (TNT) were measured in individual serum samples. The ratio of symmetric and asymmetric (functionally monovalent) IgG molecules was determined by concanavalin A (Con A) chromatography. This test is based on the fact that the carbohydrate group responsible for the molecular asymmetry has high affinity for the lectin Con A. Asymmetric molecule ratio was observed to increase with immunisation time, as well as differences between TNT and ELISA levels. All serum samples were overestimated by ELISA as compared to TNT assay, in line with the markedly higher proportion of asymmetric molecules which have lower toxin neutralising activity. Protective levels could not be predicted reasonably from ELISA results below 0. 222 IU/ml, because this methodology fails to discriminate between both types of antibodies and only an in vivo serum neutralisation procedure (TNT) reflects the true neutralising serum activity.

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.

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.

OCCURRENCE, PROPERTIES, AND FUNCTION OF ASYMMETRIC IgG MOLECULES ISOLATED FROM NON-IMMUNE SERA

We have previously demonstrated that 10–20% of the IgG isolated from non-immune sera is asymmetrically glycosylated, in such a way that it fails to trigger immune effector mechanisms. As a result, a major portion of the non-immune asymmetric IgG molecules of the host could be self-specific, acting as auto-protective antibodies. In order to test this hypothesis, we investigated whether asymmetric IgG molecules are capable of recognizing self-antigens. About 40% of F(ab′)2 fragment from normal rat IgG was able to react specifically with autologous rat cells. Moreover, upon being purified from normal rat sera, 78% of the asymmetric IgG sub-population showed self-reactivity. We demonstrated that about 14% of rat asymmetric IgG-F(ab′)2 fragments was able to react with bacteria isolated from the intestine of uninfected rats. Lastly, in order to test whether there is a correlation between the decline of immune responses during ageing and asymmetric antibody production, we assayed IgG isolated from sera of young and old rats. There was an increase in the asymmetric:symmetric IgG ratio with ageing. We therefore suggest that asymmetric antibodies may exert a beneficial action by protecting self-antigens as well as normal intestinal flora from a deleterious immune response.

Placental secreted factors: Their role in the regulation of anti-CII antibodies and amelioration of collagen induced arthritis in rats

Pregnancy induces collagen-induced arthritis (CIA) remission in rats. Placental hormones, cytokines and growth factors can regulate immune cell activity at the feto-maternal interface as well as at the systemic level. We assessed the effect of placental culture supernatants (PS) in CIA developed in rats after the inoculation of collagen type II (CII) in complete Freunds adjuvant. After the onset of CIA, animals were injected by ip route with seven doses of PS. On the 18th day of treatment with PS, serum anti-CII antibody (total IgG, IgG(1), IgG(2a), IgG(2b), IgG asymmetric molecules) and cytokine levels were determined by ELISA. An arthritic index was used by daily measure of joint swelling and visual signs of arthritis. Our results demonstrated that the PS treatment diminished CIA symptoms, reduced TNF-alpha, INF-gamma and anti-CII antibody serum levels, increased the proportion of asymmetric IgG anti-CII antibodies and affected IgG(1)/IgG(2a) and IgG(1)/IgG(2b) ratio. Two weeks after the last PS inoculation there was a recurrence of arthritis, a rise in IgG anti-CII and, simultaneously, the percentage of asymmetric IgG anti-CII fell. We concluded that PS have an effective CIA suppressor activity partly due to the modulation of humoral immune response and may be closely related to an inhibitory effect on TNF-alpha and INF-gamma production.

Structure and Protective Capacity of Tetanus and Diphtheria Antibodies Produced During Human Pregnancy and Transferred to New-Born

PROBLEM: The structure and protective activity of antibodies against tetanus (anti‐T) and diphtheria (anti‐D), produced during human pregnancy and transferred to new‐born, was studied.

Asymmetric IgG antibodies induced by different immunotherapies in a murine model of allergy.

Specific immunotherapy (SIT) is the only potentially curative treatment for those allergic processes mediated by IgE. We compared the effects of different SITs in mice sensitised with ovalbumin (OVA) Al (OH)3 : 1) OVA entrapped in particles of poly (D,L-lactic-co-glycolic acid) (PLGA-OVA), 2) Soluble OVA (OVA-sol) and 3) Polymerised OVA (OVA-pol). Serum levels of specific IgE, IgG1, IgG2a and asymmetric IgG, the cutaneous anaphylaxis test (PCA), and the IL-10, IFNγ and IL-4 levels in culture supernatants of splenocytes challenged with OVA were assessed. Mice treated with PLGA-OVA had higher levels of asymmetric antibodies than non-desensitised mice; a low IgG1 and high IgG2a level was observed together with inhibitory effect in the PCA reaction that reversed in the absence of asymmetric IgG. IL-10 and IFNγ levels were higher in supernatants from mice treated with PLGA-OVA and OVA-sol than those obtained from non-desensitised controls. Our results suggest that among the different SITs evaluated, PLGA-OVA is the one that best showed an increase in the asymmetric IgG molecules and an effective deviation of the immune response. Furthermore, the increase in the proportion of asymmetric antibodies would be of importance when designing new vaccination strategies for allergy.

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.