High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR.

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.

Profile of autoantibodies against phosphorylcholine and cross-reactivity to oxidation-specific neoantigens in selective IgA deficiency with or without autoimmune diseases.

Immunoglobulin A deficiency (IgAD) is considered the most common form of primary immunodeficiency. The majority of IgA-deficient individuals are considered asymptomatic, even though IgAD has been associated with an increased frequency of recurrent infections, allergy, and autoimmune diseases. In this study we evaluate the Natural autoantibodies (NatAbs) reactivity to phosphorylcholine (PC) and to some pro-inflammatory molecules in IgAD with or without autoimmune disorders. We observed that in the absence of IgA there is an enhancement of IgG subclasses functioning as NatAbs against PC. Immunoglobulin G (IgG) against lipopolysaccharide, C-reactive protein, and IgA was found in IgAD, regardless of the autoimmune manifestations. Nonetheless, IgAD patients with autoimmune disease showed significantly higher IgG reactivity against pro-inflammatory molecules, such as cardiolipin, oxidized low-density lipoproteins, and phosphatidylserine, with positive correlation between them. In conclusion, the IgG NatAbs against PC may represent a compensatory defense mechanism against infections and control excess of inflammation, explaining the asymptomatic status in the IgA deficiency.

Role of Yersinia pseudotuberculosis outer proteins (Yops) in murine humoral immune response.

The infection of mice with the wild-type (WT) strain of Y. pseudotuberculosis did not induce polyclonal activation of B lymphocytes. Suppression in the production of certain isotypes of Ig was observed, provoked mainly by YopH, YopJ and YpkA. The WT strain induced a progressive increase in the serum-specific IgG, which peaked after 4 weeks after infection, IgM being produced only after 1 week. Autoantibodies against phosphorylcholine, myelin, thyroglobulin and cardiolipin could be detected in the serum of mice infected with the WT strain. The infection of mice provoked suppression in the production of immunoglobulins by splenic B cells and that YopH, YopJ and YpkA must be involved here.

Cross-reactivity of anti-phosphorylcholine antibodies to neuromuscular blockers in a murine model of immunization.

Hypersensitivity reactions to curare-like neuromuscular blocking agents (NMBA) used in anesthesiology are more frequent in females and often occur at the first exposure to these drugs. To evaluate the antibody response to a hapten sharing the same allergenic epitope with NMBA in a murine model of immunization with Nitrophenylphosphorylcholine (NPPC) coupled with Keyhole Limpet Hemocyanin (KLH). BALB/c mice from both sexes were intraperitoneally immunized with NPPC-KLH with alum and boosted twice, on 7 and 14 days. The antibodies were tested for specificity to PC and for cross-reactivity to the haptens, NPPC and PC, as well as to the NMBAs. Mice immunized with NPPC-KLH produced anti-PC antibodies mainly of IgM, IgG1 and IgG3 isotypes, at similar levels in both sexes. Different affinities for the haptens were detectable between isotypes, with anti-PC IgG3 antibody reactivity mostly related to the choline portion of the NPPC hapten. When comparing among sexes, females developed greater IgG2 affinity to the hapten than males. Cross-reactivity to NMBAs was predominant among the anti-PC IgG3 antibodies, mainly in females achieving 75% of inhibition with 16 mM of suxamethonium, while other isotypes achieved up to 30% and was absent for IgE. The investigation of antibody isotypes regarding sensitization to choline-derived structures could contribute to understanding the differential ability of females to produce antibodies that are cross-reactive with NMBAs.

Sphingosylphosphorylcholine activates dendritic cells, stimulating the production of interleukin-12.

Compared with other lysophospholipid mediators such as sphingosine-1-phosphate and lysophosphatidic acid, little is known about the physiological significance of the related bioactive lysosphingolipid sphingosylphosphorylcholine (SPC), which is present in high-density lipoprotein particles. The present study was undertaken to evaluate the effect of SPC on human immature dendritic cells (DCs). Reverse transcription-polymerase chain reaction and flow cytometry assays revealed that DCs express two putative receptors for SPC, ovarian cancer G-protein-coupled receptor 1 and G-protein-coupled receptor 4. Exposure to SPC induced a rapid and transient increase in intracellular free calcium concentrations but did not stimulate endocytosis or chemotaxis of DCs. SPC increased the expression of HLA-DR, CD86 and CD83 and improved the T-cell priming ability of DCs, as well as the ability of DCs to stimulate the production of interferon-gamma by allogeneic peripheral blood mononuclear cells during the mixed lymphocyte reaction. Consistent with these results, we also observed that SPC stimulated the production of interleukin (IL)-12 and IL-18 by DCs. Taken together, our results support the notion that the accumulation of SPC in peripheral tissues during the course of inflammatory processes may favour the development of T helper type 1 immunity.

Characterization of antigens of the nematode Nippostrongylus brasiliensis by monoclonal antibodies.

Hybridomas secreting monoclonal antibodies to Nippostrongylus brasiliensis antigens were generated by hybridization of IR983F myeloma cells with spleen cells from Lou/M/Wol rats infected with living third-stage larvae. Antibodies specific either for larval or worm antigens were identified by enzyme-linked immunosorbent assays with Nippostrongylus brasiliensis fragments, homogenates and secretions as antigens. The results demonstrate that all antibodies which recognized larval antigens (38 antibodies) also reacted with worm surfaces. Ten antibodies were specific only for worm antigens. Ten antibodies reacted with worm homogenate, three antibodies recognized components of worm secretion and 17 antibodies combined with acetylcholinesterase. The epitope specificity was investigated by the capacity of various glycosides, aminoacids, N-acetylneuraminic acid and phosphorylcholine to inhibit the binding to worm fragments. The analysis revealed that alpha-methylglucoside, alpha-methylmannoside, N-acetylglucosamine, N-acetylgalactosamine, fucose and the amino acids leucine, phenylalanine, tyrosine, serine, tryptophan did not combine with the antigen-binding sites of the antibodies. Proline, arginine and histidine, however, displayed inhibitory effects. With N-acetylneuraminic acid as inhibitor three groups of antibodies could be discriminated. At a concentration of 10-20 mM, phosphorylcholine was a potent inhibitor for all antibodies.

Phosphorylcholine antigens from Nippostrongylus brasiliensis. II.--Isolation and partial characterization of phosphorylcholine antigens from adult worm.

The phosphorylcholine antigens (C substance) were specifically isolated from Nippostrongglus brasiliensis adult worms. They formed a gorup of fairly closely related molecules, but it was not possible to evidence that the carrier molecule was unique. An indirect immunoenzymatic test using immobilized lectins (concanavalin A, phytohaemagglutinin Els, wheat germ agglutinin, recin types I and II, peanut agglutinin) gave some light on the carbohydrate composition of the carrier molecules, whereas the amino acid part of these molecules seemed to indicate an unique oligopeptide, the composition of which would be: Asx (4), Thr (2), Ser (4), Glux (6), Gly (6) Ala (3), Val (2), Ile (1), Leu (2), Phe (8), Lys (2), Arg (1). An epitope of the carrier molecules was demonstrated with anti-N. brasiliensis egg antisera. It was shared with various pathogens including Haemonchus contortus, Schistosoma mansoni and Dipetalonema vitae. It was also found on the purified pneumococcal C. polysaccharide. The C. substance from a variety of parasites can now be isolated by a combination of specific reactions using both anti-phosphorylcholine and anti-carrier molecule antibodies.

Phosphorylcholine antigens from Nippostrongylus brasiliensis. I.--Anti-phosphorylcholine antibodies in infected rats and location of phosphorylcholine antigens.

The anti-phosphorylcholine (PC) antibody synthesis was investigated in the rat after infection with the nematode Nippostrongylus brasiliensis. Serum IgM and IgG antibodies were demonstrated from day 2 or 3 post-infection. Intestinal IgA-antibody synthesis began shortly after the worms reached the intestine. Antigens containing PC were located with the fluorescent antibody technique in L3 infective larvae, adult worms and eggs of the parasites. They were always found to internal structures such as intestinal tract or gonads. It seems, therefore, that the anti-PC antibody synthesis resulted from the release of PC antigens by the parasite (moulting fluids, excretion, secretion or breakage products).