Profiling of microorganism-binding serum antibody specificities in professional athletes.

The goal of this work was to elucidate similarities between microorganisms from the perspective of the humoral immune system reactivity in professional athletes. The reactivity of serum IgG of 14 young, individuals was analyzed to 23 selected microorganisms as antigens by use of the in house ELISA. Serum IgM and IgA reactivity was also analyzed and a control group of sex and age matched individuals was used for comparison. The obtained absorbance levels were used as a string of values to correlate the reactivity to different microorganisms. IgM was found to be the most cross reactive antibody class, Pearson's r = 0.7-0.92, for very distant bacterial species such as Lactobacillus and E. coli.High correlation in IgG levels was found for Gammaproteobacteria and LPS (from E. coli) (r = 0.77 for LPS vs. P. aeruginosa to r = 0.98 for LPS vs. E.coli), whereas this correlation was lower in the control group (r = 0.49 for LPS vs. P. aeruginosa to r = 0.66 for LPS vs. E.coli). The correlation was also analyzed between total IgG and IgG subclasses specific for the same microorganism, and IgG2 was identified as the main subclass recognising different microorganisms, as well as recognising LPS. Upon correlation of IgG with IgA for the same microorganism absence of or negative correlation was found between bacteria-specific IgA and IgG in case of Lactobacillus and Staphylococcusgeni, whereas correlation was absent or positive for Candida albicans, Enterococcusfaecalis,Streptococcus species tested in professional athletes. Opposite results were obtained for the control group. Outlined here is a simple experimental procedure and data analysis which yields functional significance and which can be used for determining the similarities between microorganisms from the aspect of the humoral immune system, for determining the main IgG subclass involved in an immune response as well as for the analysis of different target populations.

Effects of orally applied Fes p1-displaying L. plantarum WCFS1 on Fes p1 induced allergy in mice.

Group I grass pollen allergens are major contributors to grass pollen-related seasonal allergic rhinitis, and as such a primary target for allergen specific immunotherapy. In this study the potential therapeutic role of oral application of Lactobacillus plantarum WCFS1, directing cell wall attachment of the recombinant Fes p1 allergen, from Festuca pratensis was tested in a mouse model of Fes p1 allergy. For surface expression of Fes p1 allergen in L. plantarum WCFS1 pSIP system with inducible expression was used. Balb/c mice were sensitized with Fes p1 protein in alum and subsequently received live recombinant L. plantarum orally. Antibody levels (IgE, total IgG, IgG1, IgG2a, and IgA) were determined by ELISA. Differential eosinophil count in peripheral blood was performed. Reduced peripheral blood eosinophilia and increased serum IgG2A levels was detected in both groups which received live L. plantarum orally. Specific serum IgA levels were increased only in mice treated with the recombinant bacteria. Oral application of L. plantarum WCFS1 has a beneficial therapeutic effect in a mouse model of Fes p1 allergy. Cell surface expression of Fes p1 allergen potentiates this phenomenon in an allergen specific way.

Brain and liver fatty acid composition changes upon consumption of Lactobacillus rhamnosus LA68.

Recent reports suggest that the metabolic activity of the enteric microbiota may influence the fatty acid composition of the host tissue. There are many studies dealing with the influence of lactobacilli on various pathological conditions, and some of the effects are strain-specific. This study was designed to test the effects of a particular Lactobacillus strain, Lactobacillus rhamnosus LA68 on fatty acid composition of the liver and the brain of C57BL/6 mice in the absence of an underlying pathological condition. Female mice were supplemented with live L. rhamnosus LA68 bacteria for the duration of 1 month. Serum biochemistry was analyzed and liver and brain fatty acid composition was assessed by gas-liquid chromatography. Significant changes in liver and brain fatty acid composition were detected. In the liver tissue we detected an increase in palmitoleic acid (p = 0.038), while in the brain compartment we found an increase in palmitic (p = 0.042), stearic (p = 0.017), arachidonic acid (p = 0.009) and docosahexaenoic acid (p = 0.004) for control versus experimental group. These results show discrete changes caused by LA68 strain consumption. Even short duration of administration of LA68 influences the fatty acid composition of the host which adds to the existing knowledge about Lactobacillus host interaction, and adds to the growing knowledge of metabolic intervention possibilities.

Autoantibody response and pregnancy-related pathology induced by combined LPS and tetanus toxoid hyperimmunization in BALB/c and C57BL/6 mice.

Recent data concerning antiphospholipid syndrome (APS) induction have shown that ß2-glycoprotein I (ß2GPI) binds lipopolysaccharide (LPS), which results in conformational changes, exposition of a cryptic epitope and possible pathological anti-ß2GPI antibody production. In order to investigate the effects of LPS on the induction of APS-related pathology, we performed hyperimmunization of BALB/c and C57BL/6 mice with LPS, alone or in combination with tetanus toxoid (TTd), a protein structurally similar to ß2GPI. We report that, although high affinity pathological anti-ß2GPI antibodies were produced in all groups of animals, the reproductive pathology was recorded only in mice that received both LPS and TTd, implying on the important roles of both infections and molecular mimicry in APS pathogenesis. Moreover, APS-related reproductive pathology was more pronounced in BALB/c (lowered fertility and fecundity) than C57BL/6 mice (lowered fecundity), which correlated well with the disruption in natural antibody network observed in BALB/c mouse strain.

Lactobacillus rhamnosus LA68 and Lactobacillus plantarum WCFS1 differently influence metabolic and immunological parameters in high fat diet-induced hypercholesterolemia and hepatic steatosis.

In this study, two Lactobacillus strains (L. rhamnosus LA68 and L. plantarum WCFS1) were evaluated for their effects on high fat diet induced pathology in mice. The aim was to determine whether the administration of lactic acid bacteria had beneficial effects on ameliorating pathology. C57BL/6 mice fed a high fat diet were orally administered with the Lactobacillus strains. Both the metabolic and immunological parameters were analyzed. The administration of both of the strains had beneficial effects on mouse weight, serum cholesterol, TNF-α levels and liver histology. LA68 lowered the total cholesterol and HDL levels more prominently, whereas WCFS1 was more potent in lowering the TG and LDL levels. Leptin and adiponectin levels were increased in all experimental groups to different extents. The administration of L. plantarum WCFS1 led to a marked increase in leptin levels, as well as an increase in CD3+CD4+ and CD3+CD8+ cells, and a decrease of CD25+ cells, and had a lowering effect on IL-6 production and cell metabolic activity. In conclusion, active administration of both Lactobacillus strains had a positive effect on HFD-induced pathology. Although both of the tested strains had beneficial effects, oral administration of WCFS1 increased leptin levels and had a more prominent immunomodulatory effect, which should be taken into consideration in case of humane usage.

Effects of Lactobacillus rhamnosus LA68 on the immune system of C57BL/6 mice upon oral administration.

Probiotic bacteria have been used in human nutrition for centuries and are now attracting more attention. In order to examine the immunological aspects of probiotic consumption, Lactobacillus rhamnosus LA68 was orally administrated using gavage to healthy C57BL/6 mice. After one month splenocytes were isolated, and analysed by flow cytometry. The magnitude of splenocyte proliferation upon stimulation with lipopolysaccharide and peptidoglycan and cytokine levels (IFN-γ, IL-6, IL-10 and IL-17) was assessed. Cytokine levels in the serum were also analysed. Oral application of strain LA68 leads to a significant decrease of CD3+, CD25+ and CD19+ cells, and an increase of CD11b+ and CD16/CD32+ positive cell populations in the mouse spleen. Increased sensitivity to stimulation through proliferation and IL-6 secretion was detected. Increased serum IFN-γ and decreased IL-10 levels were found. Our results show increased responsiveness of splenocytes, activation of the Th1 type of immune response, and a shift of leucocyte populations towards monocyte/granulocyte populations.

Infection-induced autoantibodies and pregnancy related pathology: an animal model.

In addition to being the main cause of mortality worldwide, bacterial and viral infections can be the cause of autoimmune and pregnancy disorders as well. The production of autoantibodies during infection can be explained by various mechanisms, including molecular mimicry, bystander cell activation and epitope spreading. Conversely, bacterial and viral infections during pregnancy are especially dangerous for the fetus. It is documented that infection-induced inflammatory processes mediated by Toll-like receptors (TLR) represent the main cause of preterm labour. We used two crucial bacterial components and TLR ligands, namely peptidoglycan and lipopolysaccharide, to stimulate BALB/c mice before immunisation with tetanus toxoid. Tetanus toxoid is an inactive form of the toxin produced by bacterium Clostridium tetani and shares structural similarity with plasma protein ß2-glycoprotein I. Treatment with peptidoglycan and lipopolysaccharide in combination with tetanus toxoid induced the production of pathological autoantibodies, different fluctuations in natural autoantibodies and different types of reproductive pathology in treated animals, with peptidoglycan treatment being more deleterious. We propose that the production of pathological autoantibodies, TLR activation and changes in natural autoantibodies play crucial roles in infection-induced reproductive pathology in our animal model.

Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice.

One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein ß2GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-ß2GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-ß2GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.

Role of molecular mimicry and polyclonal cell activation in the induction of pathogenic ß2-glycoprotein I-directed immune response in Balb/c mice upon hyperimmunization with tetanus toxoid.

It is known that tetanus toxoid (TTd)-hyperimmunization induces increased titer of sera ß2-glycoprotein I (ß2GPI)-specific antibodies (Abs) in Balb/c mice. The concentrations of such induced anti-ß2GPI Abs as well as their pathogenic potential are strongly influenced by the context of TTd application. ß2GPI-specific immune response is established as a part of TTd-specific immune response by molecular mimicry mechanism due to structural homology between TTd and ß2GPI. This finding is supported by the following facts: (1) cross-reactive Abs that recognize both TTd and ß2GPI epitopes are present in Balb/c mice sera; (2) anti-TTd Abs secretion in splenic cultures is induced after ß2GPI stimulation and vice versa. However, analyses of (1) IL-10 production following in vitro stimulation of immunized Balb/c mice splenocytes by TTd, ß2GPI or glutaraldehyde-treated ß2GPI and (2) specific impact of ConA and agonists of TLR2, TLR4, and TLR9 on anti-TTd and autoreactive Abs secretion strongly imply that these two branches of the TTd-induced immune response do not use identical cell populations and are regulated in a different way. Results presented in this paper describe that structural homology between foreign and self-antigens could focus mounted autoreactive immune response toward specific self-structure, but the context of antigen application, including a history of previous immune stimulations and adjuvants applied together with the antigen, are the main factors which determine the outcome of the induced immune response.

Production, characterization and applications of a tetanus toxin specific monoclonal antibody T-62.

Tetanus neurotoxin (TeNT) represents a potent toxin that binds to its receptors on neurons and inhibits the release of neurotransmitters. Additionally, its fragments are used to transport pharmacological substances to neuronal cell bodies. The main objective of this study was the development of a suitable model system to study internalization of the TeNT. We have produced a monoclonal antibody (MoAb) specific for TeNT by hybridoma technology, after immunization of BALB/c mice with tetanus toxoid, and have named it T-62. The immunochemical characteristics of MoAb T-62 were tested using ELISA, PAGE and immunoblotting. Finally, we have used an immunohistochemical method to detect specific binding of MoAb T-62 to TeNT bound to PC 12 cells. Our results show that MoAb T-62 is highly specific for TeNT, even when it is bound to its receptor, and that it could be of considerable importance in studies regarding fundamental research on TeNT receptors, intracellular transport of TeNT, as well as retrograde transport of pharmaceutical substances and non-invasive delivery of polypeptides through the blood brain barrier. In addition, MoAb T-62 is an invaluable tool in TeNT vaccine production as it can be used for the detection of reverse toxicity, which could drastically reduce the need to use animals in these experiments.

Induction of decreased fecundity by tetanus toxoid hyper-immunization in C57BL/6 mice depends on the applied adjuvant.

It has already been shown that tetanus toxoid (TTd) hyper-immunization is a suitable experimental method for creating the animal model of antiphospholipid syndrome (APS) in BALB/c mice. The severity of APS pathology in BALB/c mice mainly correlates to the affinity of anti-ß(2) glycoprotein I (ß(2)GPI) antibodies. In this study we have investigated reproductive pathology induced in C57BL/6 mice by TTd hyper-immunization using a combination of different pretreatments (complete Freund's adjuvant or glycerol) and adjuvants (alhydrogel or glycerol). A decrease in fecundity was recorded in only C57BL/6 mice immunized with alhydrogel adjuvant, irrespective of the kind of applied pretreatment; it was associated with an increase in abundance of low affinity anti-ß(2)GPI IgG antibodies and Th1 prevalence.

Hexameric immunoglobulin M in humans: desired or unwanted?

Immunoglobulin M (IgM) is the first antibody produced upon infection, and is often suggested as the first line of defense of human immune system. In addition to being present on the surface of naïve B cells as a monomeric molecule, IgM is always secreted as a polymer. The most abundant IgM polymer in humans is pentamer, composed of five monomeric units, joined together by so-called joining or J chain. On the other hand, it is well known that hexameric IgM can be also found in human sera. Its presence is often related to different dissorders (Waldenström's macroglobulinemia, cold agglutinin, and recurrent urinary bacterial infections), although it is believed that small amounts of hexamer are present in normal human sera as well. Unlike pentamer, IgM hexamer contains six monomeric blocks and completely lacks J chain. Although it has been decades since its discovery, the precise function of IgM hexamer is still unknown. Since it was documented that hexamer is very potent in activating complement, it is suggested that its production in humans must be under strict control, and that it is produced in special conditions, when strong activation of complement is absolutely needed. However, the question is whether hexameric IgM is really a secret weapon or just an undesirable molecule in humans. According to structural and known functional characteristics of both pentamers and hexamers of IgM, it can be concluded that hexamers are, in addition to being maybe too reactive to be around, probably not that efficient in protecting us from bacterial and viral infections.

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.

Induction of APS after TTd hyper-immunization has a different outcome in BALB/c and C57BL/6 mice.

PROBLEM: The antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by vascular thrombosis and/or pregnancy complications (lower fecundity and lower litter size), as well as by an increase in anti-ß(2) glycoprotein I (ß(2) GPI)-specific autoantibody titer. We have investigated how the genetic background of the immune system [T helper (Th) prevalence] and the type of animal model of APS influence the induced pathology. METHOD OF STUDY: Antiphospholipid syndrome induced by tetanus toxoid (TTd) hyper-immunization and by intravenous application of monoclonal anti-ß(2) GPI-specific antibody 26 was compared in C57BL/6 (Th1 prone) and BALB/c (Th2 prone) mice. RESULTS: Tetanus toxoid hyper-immunization of BALB/c mice led to reduction in fertility, but in C57BL/6 mice a decrease in fecundity occurred. In both cases, pathology was caused by anti-ß(2) GPI antibodies, the production of which was adjuvant and strain dependent. CONCLUSION: We conclude that TTd immunization and i.v. application of monoclonal antibody 26 induced the same reproductive pathology and that the type of pathology is strain dependent.

[Correlation of sera concentrations of thyroperoxidase autoantibodies measured by two radioimmunoassays].

INTRODUCTION: Thyroid peroxidase-specific autoantibodies (TPO Abs) are mostly measured in patients with autoimmune thyroid diseases. The aim of this study was to compare TPO Ab concentrations measured by two radioimmunoassays. MATERIAL AND METHODS: Our investigation included 38 patients. Sera concentrations of TPO Abs were measured by using Cis biointernational (France) and Immunotech (Czech Republic) assays. RESULTS: Concentrations obtained by two assays were extensively different. The values measured by Cis biointernational assay were higher than ones obtained by Immunotech assay. The statistical arrangement of results showed the direct correlation between the two assays, with the coefficient of agreement R = 0.6239 (p < 0.001). The analysis of relative values (ratio of measured and upper limit values given by the manufacturer) demonstrated the statistically significant difference (p = 0.003) between values measured by Cis biointernational (18.94 +/- 37.22) and by Immunotech assay (4.22 +/- 8.22) concerning the distinction between normal and raised concentrations of TPO Abs. The agreement of results (enhanced or normal TPO Ab concentrations in both tests) was shown in 30 sera samples (78.95%), but in residual 8 sera (21.05%) normal TPO Ab concentrations were obtained by Immunotech, and enchanced by Cis biointernational assay. There is no difference in capability of distinction between normal and pathological results between the two tests (chi12 = 3.484, p > 0.05). The highest concentration of TPO Ab measured by Cis biointernational assay was not the highest one in Immunotech assay, which might be a reflection of different specificity of antibodies used in two diagnostic tests. CONCLUSION: TPO Ab concentrations obtained by Cis biointernational and Immunotech assays are very different. In several sera samples, normal concentrations of TPO autoantibodies were obtained by Immunotech assay and enhanced by Cis biointernational assay. The highest value obtained by one is not the highest value measured by another assay we used.

Changes in composition of IgM polymers in patients suffering from recurrent urinary bacterial infections after bacterial immunization treatment.

IgM is the first antibody produced during the immune response to infection or immunization, and it can be secreted as pentamer (containing a small polypeptide, termed as J chain) or hexamer (lacking J chain). In this paper we have analyzed structural characteristics (by electrophoresis and immunoblot) and anti-bacterial specificity (by enzyme-linked immunosorbent assay) of IgM antibodies purified from female patients suffering from recurrent bacterial infections of lower urinary tract and therapeutically immunized with the mixture of heat-inactivated uropathogenic bacteria. We report on changes in the composition of IgM polymers in tested patients. The immunization induced the raise of the levels of hexamers in patients that did not respond to immunization (non-responders), while the IgM polymers remained on the pentameric level in immunization dependent responders. We propose that the composition of IgM polymers could influence the immunization outcome and should be taken in count regarding the treatment of recurrent bacterial infections.

In vitro stimulation of Balb/c and C57 BL/6 splenocytes by a recombinantly produced banana lectin isoform results in both a proliferation of T cells and an increased secretion of interferon-gamma.

Lectins are widely used in many types of assay but some lectins such as banana lectin (BanLec) are recognised as potent immunostimulators. Although BanLec's structure and binding characteristics are now familiar, its immunostimulatory potential has not yet been fully explored. The synthesis by recombinant technology of a BanLec isoform (rBanLec) whose binding properties are similar to its natural counterpart has made it possible to overcome the twin problems of natural BanLec's microheterogeneity and low availability. This study's aim is to explore the immunostimulatory potential of rBanLec in the murine model. Analyses of the responses of Balb/c- and C57 BL/6-originated splenocytes to in vitro rBanLec stimulation were performed to examine the dependency of rBanLec's immunostimulatory potential upon the splenocytes' genetic background. It is shown that the responses of Balb/c- and C57 BL/6-originated splenocytes to rBanLec stimulation differ both qualitatively and in intensity. The hallmarks of the induced responses are T lymphocyte proliferation and intensive interferon-gamma secretion. Both phenomena are more marked in Balb/c-originated cultures; Balb/c-originated lymphocytes produce interleukin (IL)-4 and IL-10 following rBanLec stimulation. Our results demonstrate that any responses to rBanLec stimulation are highly dependent upon genetic background; they suggest that genetic background must be an important consideration in any further investigations using animal models or when exploring rBanLec's potential human applications.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization.

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

The context of tetanus toxoid application influences the outcome of antigen-specific and self-directed humoral immune response.

Results are presented concerning our attempts to create a suitable model system for studying the connection between microbial antigen (micAg), autoimmunity and autoimmune disease on the basis of hyper-immunization and application of micAg in different contexts. Our research was focused on tetanus toxoid (TTd) as a model micAg. Non-pretreated and complete Freund's adjuvant pretreated BALB/c mice were immunized with high doses of TTd mixed with glycerol or aluminum hydroxide as adjuvants. The main aims of the experiments were to evaluate the properties of induced humoral immune responses, evaluate the pathological potential of induced immune responses and determine possible correlations between the properties of a humoral immune response and its pathological potential. The production of TTd-specific and self-reactive beta(2)-glycoprotein I (beta(2)-GP I)-specific antibodies (Abs) was detected in all groups but with specific, context-related properties. Analysis of pregnancy-related pathology (anti-beta(2)-GP I Abs-associated) showed differences in the pathological potential of the induced immune response. It was demonstrated that severity of pathology is positively correlated to the abundance of IgG that recognizes beta(2)-GP I adsorbed onto phosphatidylserine, and to IgG affinity. Furthermore, it was demonstrated that molecular mimicry, which results in generation of anti-beta(2)-GP I Abs upon TTd immunization, is necessary but not sufficient for the development of pregnancy-related pathology.