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O. Kurtenkov [1] V. Brjalin [1] K. Klaamas [1] L. Miljukhina [1] L. Shljapnikova [1] A. Lipping [1] L. Engstrand [2] [4/15] A Level of Natural anti-Alpha-galactosyl Antibodies in H. [...]

Many investigators have demonstrated alteration of gastric mucins in H. pylori infected individuals. The inflammatory environment induced by H. pylori leading to aberrant glycosylation of MUC1 and demasking of core peptide MUC1 epitope could enhance immune responses to MUC1. IgG and IgM immune response to MUC1 in patients with gastric cancer (n = 214) chronic gastroduodenal diseases (n = 160) and healthy blood donors (n = 91) was studied with ELISA using bovine serum albumin-MUC1 60-mer peptide as antigen. H. pylori serologic status was evaluated with ELISA and CagA status by immunoblotting. Gastric mucosa histology was scored according to the Sydney system. Compared to H. pylori seronegative individuals, higher levels of IgG antibody to MUC1 were found in H. pylori seropositive patients with benign gastric diseases (p < 0.01) and blood donors (p < 0.03). Higher MUC1 IgG antibody levels were associated with a higher degree of gastric corpus mucosa inflammation in patients with chronic gastroduodenal diseases (p < 0.0025). There was a positive correlation between the levels of anti-H. pylori IgG and MUC1 IgG antibody levels in blood donors (p = 0.03), and in patients with benign diseases (p < 0.0001). In patients with gastric cancer (n = 214) a significantly higher level of anti-MUC1 IgG than in blood donors was observed (p < 0.001) irrespective of H. pylori status or stage of cancer. MUC1 IgM antibody levels were not related to the H. pylori serology. IgG immune response to tumor-associated MUC1 is up regulated in H. pylori infected individuals. This increase is associated with a higher IgG immune response to H. pylori and with a higher degree of gastric mucosa inflammation. High levels of MUC1 IgG antibody irrespective of H. pylori serologic status characterized patients with gastric cancer. The findings suggest that, in some individuals, the H. pylori infection may stimulate immune response to tumor-associated MUC1 peptide antigen thus modulating tumor immunity. [ABSTRACT FROM AUTHOR]

α-macroglobulin (α2M) preparations purified from the plasma of healthy persons and gastric carcinoma patients were further analysed by immunochemical techniques. It was found that only α2M from the plasma of gastric carcinoma patients gave a positive reaction with E-receptor sheep antiserum in rocket immunoelectrophoresis. The α2M fraction giving the positive reaction with E-receptor sheep antiserum was isolated and analysed by the immunoblotting technique with monoclonal antibodies to TII antigen. The presence of the α2M-CD2 complex in the plasma of gastric carcinoma patients was demonstrated. This finding may explain the immunosuppressive effect of the plasma α2M in cancer patients. [ABSTRACT FROM AUTHOR]

The E2 envelope glycoprotein of the hepatitis C virus (HCV) is a major target of broadly neutralizing antibodies that are closely related to a spontaneous cure of HCV infection. There is still no data about the diversity of E2-specific antibodies (Abs) glycosylation. The aim of this study was to analyze the level and sialylation of E2 IgG Abs, the relation of the respective changes to hepatic fibrosis (F) progression and their possible association with the efficacy of interferon- α -2a plus ribavirin (IFN-RBV) antiviral therapy. One hundred three HCV infected treatment-naive patients were examined using ELISA with E2 recombinant protein as antigen and sialic acid-specific Sambucus nigra agglutinin. The efficacy of the IFN-RBV treatment of patients with HCV dominant 1b and 3a genotypes (GT) was evaluated. A significant decrease of E2 Abs sialylation in the late stages of fibrosis was found irrespective of HCV genotype. On this basis, the F4 stage of fibrosis can be discriminated from its F0 or F1-3 stage by an about 75-79% accuracy. HCV infection of 1b genotype is associated with the production of lower sialylated E2 Abs, a higher frequency of F4 stage fibrosis, and a worse response to antiviral therapy. The increased SNA reactivity of E2 Abs was observed in patients with a sustained virological response (SVR). The proportion of SVR responders was significantly higher among patients with 3a genotype. However, for both dominant HCV genotypes (3a and 1b), an increased sialylation of E2 IgG was associated with a higher rate of patients with sustained virological response to antiviral therapy. Thus, the association of alterations of anti-E2 IgG Abs sialylation with hepatic fibrosis stage, HCV genotype, and the efficacy of antiviral therapy enables using these changes as novel noninvasive predictive biomarkers. The clinical potential of these findings is discussed., Competing Interests: The authors have approved this version of the manuscript and declare that there is neither conflict of interest nor financial gain regarding the publication of this paper., (Copyright © 2020 Oleg Kurtenkov et al.)

The Thomsen-Friedenreich (TF) antigen is expressed in a majority of human tumors due to aberrant glycosylation in cancer cells. There is strong evidence that humoral immune response to TF represents an effective mechanism for the elimination of cancer cells that express TF-positive glycoconjugates. The presence of naturally occurring antibodies to tumor-associated TF and cancer-specific changes in their levels, isotype distribution and interrelation, avidity, and glycosylation profile make these Abs a convenient and ubiquitous marker for cancer diagnostics and prognostics. In this review, we attempt to summarize the latest data on the potential of TF-specific Abs for cancer diagnostics and prognostics., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Oleg Kurtenkov.)

Total serum IgG level is a surrogate marker of hepatitis C (HC) severity. Antibodies (Abs) to microbial glycans could be markers of HC severity caused by the translocation of microbial products. The level of anti-glycan (AG) Abs was analysed in serum samples of patients ( n = 128) with chronic HC in ELISA using fourteen synthetic glycans present in microbes and adhesins to evaluate the association of Abs with clinical parameters and the efficacy of antiviral treatment. The anti-GlcNAc β IgG level was significantly higher in patients with fibrosis ( P = 0.021) and severe portal inflammation ( P < 0.001) regardless of other clinical parameters. The ROC curve analysis showed sensitivity of 0.59, specificity of 0.84, and AUC of 0.71 in discriminating F0 from F1-4 (HCV genotype-1b-infected patients). The level of anti-GA2 Abs before Peg-IFN/RBV treatment was significantly higher in nonsustained viral response (non-SVR) to treatment than in SVR ( P = 0.033). ROC analysis showed sensitivity of 0.62, specificity of 0.70, and AUC of 64. Correlations of AG Abs to clinical parameters were found. The quantification of anti-GlcNAc β Abs deserves attention in assessment of the hepatic damage while anti-GA2 Abs may be a sign of immune response related to the antiviral treatment.

Alterations in the glycosylation of serum total immunoglobulins show these antibodies to have a diagnostic potential for cancer but the disease-related Abs to the tumor-associated antigens, including glycans, have still poorly been investigated in this respect. We analysed serum samples from patients with breast carcinoma (n = 196) and controls (n = 64) for the level of Thomsen-Friedenreich (TF) antigen-specific antibody isotypes, their sialylation, interrelationships, and the avidity by using ELISA with the synthetic TF-polyacrylamide conjugate as an antigen and the sialic acid-specific Sambucus nigra agglutinin (SNA) and ammonium thiocyanate as a chaotrope. An increased sialylation of IgG and IgM, but a lower SNA reactivity of IgA TF antibodies, and a higher level and avidity of the TF-specific IgA were found in cancer patients. Other cancer-related signatures were the highly significant increase of the IgG/IgA ratio and the very low SNA/IgA index in cancer, including patients with an early stage of the disease. These changes showed a good diagnostic potential with about 80% accuracy. Thus, the level of naturally occurring anti-TF antigen antibodies, their sialylation profile, isotype distribution, and avidity displayed cancer-specific changes that could serve as novel noninvasive Ab-based biomarkers for early breast cancer.

Aim: To determine whether the structural and functional diversities of naturally occurring antibodies to the Thomsen - Friedenreich (TF) antigen may be of diagnostic and prognostic value in colon cancer., Materials and Methods: Serum samples were taken from patients with colon cancer (n = 94) and healthy controls (n = 64). The level of TF-specific antibody isotypes and their sialylation were determined using ELISA and lectin-ELISA with synthetic TF-polyacrylamide conjugate as an antigen and a sialic acid-specific Sambucus nigra agglutinin (SNA). The avidity was determined using ammonium thiocyanate as a chaotrope. The accuracy of diagnostics was evaluated using the receiver operator characteristic curve analysis and the survival analysis employing the Kaplan - Meier method., Results: Compared to healthy controls, patients with colon cancer exhibited a lower level of anti-TF IgG antibodies, significantly lower ratios of TF-specific IgG/IgM and IgG/IgA, an increased SNA reactivity of anti-TF antibodies, mostly on account of IgG, and a lower avidity of TF-specific antibodies, especially their SNA-reactive subset. An increased SNA reactivity of anti-TF IgG was observed already at the early stages of cancer (p = 0.0004). The decrease of the ratio of IgG/IgM and IgG/IgA showed a good accuracy of diagnostics with about 60% sensitivity at 90% specificity. A similar potential was found for the SNA binding/IgG level index. The high level of TF-specific IgA antibodies was associated with a lower survival rate (hazard ratio = 0.34)., Conclusion: This is the first report ever on the colon cancer-related signatures of anti-TF antibody diversity which show diagnostic potential, including in early cancer, and prognostic value. The hypersialylation of TF-specific antibodies appeared to be a common phenomenon in cancer. The signatures may be used as non-invasive antibody-based markers for colon cancer.

Natural antibodies to the tumor-associated Thomsen-Friedenreich antigen (TF) are related to tumor immunosurveillance and cancer patients' survival. Hidden IgG antibodies (HAbs) to TF, their lectin reactivity, avidity, and clinical relevance were studied. HAbs were present in cancer patients and controls. A decreased level of IgG HAbs was detected in cancer. The HAbs level positively correlated with the sialospecific SNA lectin binding in purified total IgG (tIgG) in donors and cancer patients, indicating that HAbs are higher sialylated. The avidity of anti-TF IgG in tIgG samples was lower in cancer patients ( P = 0.025) while no difference in the avidity of free anti-TF IgG was established. A negative correlation between the avidity of anti-TF IgG in tIgG and SNA binding in both groups was observed ( P < 0.0001). The HAbs level negatively correlated with the anti-TF IgG avidity in tIgG only in donors ( P = 0.003). Changes in the level of HAbs and Abs avidity showed a rather good stage- and gender-dependent diagnostic accuracy. Cancer patients with a lower anti-TF IgG avidity in tIgG showed a benefit in survival. Thus the TF-specific HAbs represent a particular subset of anti-TF IgG that differ from free serum anti-TF IgG in SNA reactivity, avidity, diagnostic potential, and relation to survival., Competing Interests: The authors declare that they have no competing interests.

Aim: To determine whether the naturally occurring Thomsen-Friedenreich (TF) antigen-specific antibodies differ in avidity between cancer patients and controls to find a novel biomarker for stomach cancer., Methods: Serum samples were taken from patients with cancer and controls. The level of TF-specific antibodies and their sialylation were determined using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA). The avidity was determined using ammonium thiocyanate as a chaotrope., Results: A significantly higher SNA lectin binding to anti-TF antibodies was found in cancer patients irrespective of disease stage. The avidity of only IgM TF-specific antibodies was significantly higher in cancer patients compared to controls. The SNA-positive anti-TF antibodies of cancer patients showed a significantly higher avidity, P < 0.001. The sensitivity and specificity of this increase for gastric cancer were 73.53% and 73.08%, respectively, with a 73.2% diagnostic accuracy. The higher avidity of SNA-reactive anti-TF antibodies was associated with a benefit in survival of stage 3 cancer patients., Conclusion: The SNA-reactive TF-specific antibodies display a significantly higher avidity in gastric cancer patients compared to controls, which can be used as a potential serologic biomarker for gastric cancer. It appears that IgM is the main target responsible for the above changes.

Unlabelled: The elevated anti-GalNAcβ IgG level of serum was shown to be associated with the significantly better survival of patients with gastrointestinal cancer., Aim: To characterize the specificity of IgG antibodies to GalNAcβ-terminated glycans of long-term gastric cancer survivors., Methods: Serum antibodies and affinity-isolated antibodies were analysed by the indirect and competitive ELISA using glycan-polyacrylamide (PAA) conjugates as well as by isoelectric focusing and Western blotting., Results: In the serum probes, a partial cross-reactivity of antibodies to GalNAcβ, GalNAcβ1-3Galβ (X2di), GalNAcβ1-3GalNAcβ (PFdi) and GlcNAcβ was observed. The isolated anti-GalNAcβ IgGs demonstrated the cross-reactivity to the X2di glycan mainly. The affinity of the X2di-PAA to anti-GalNAcβ IgGs was 11-21 times lower than that of the GalNAcβ-PAA. Anti-X2di and anti-PFdi IgGs demonstrated monoreactivity to their key glycans-PAA used in isolation. The IC50 values of key glycoconjugates ranged from 1 to 5 · 10(-7) M. No polyreactivity of antibodies to the unrelated antigens (ferritin, casein and DNA) was found. The polyclonal or oligoclonal distribution of IgG bands was established and the monoreactivity of antibodies was not associated with the clonal distribution of bands., Conclusion: The cross-reactivity of anti-GalNAcβ antibodies to X2di and related glycans deserves attention in the clarification of the role of antibodies in cancer progression and enhancement of the prognostic potential in the combined determination of antibody markers.

Aim: To study whether alterations in the sialylation of antibodies (Ab) specific to the Thomsen-Friedenreich (TF) glycotope have a diagnostic and prognostic potential in gastric cancer., Methods: Serum samples were taken from patients with gastric carcinoma (n = 142) and controls (n = 61). The level of TF-specific antibodies and their sialylation was detected using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA)., Results: The level of TF-specific IgM was significantly decreased in cancer compared with controls (P ≤ 0.001). Cancer patients showed a higher level of SNA binding to anti-TF IgM and IgA (P ≤ 0.001) irrespective of disease stage, tumor morphology, and gender. Changes in the SNA/Ab index demonstrated moderate sensitivity (66-71%) and specificity (60-73%) for stomach cancer. The best diagnostic accuracy (100%) was achieved in 29% patients with high SNA binding and low anti-TF IgM level. This subset of patients demonstrated the poorest survival., Conclusion: Our findings are the first evidence that the increased sialylation of TF-specific Abs combined with a low level of anti-TF IgM is strongly linked to gastric cancer and patients survival, which can be used as a novel biomarker for cancer detection and prognosis.

Objective: To evaluate the relation of the level of serum anti-TF, -Tn and -αGal carbohydrate antibodies to survival in gastrointestinal cancer patients., Methods: The level of anti-TF (Thomsen-Friedenreich antigen), -Tn and -αGal IgG was analysed in the serum of patients with gastric (n = 83) and colorectal (n = 51) cancers in the long-term follow-up, using ELISA with polyacrylamide glycoconjugates. To evaluate overall survival and the risk of death, the Kaplan-Meier method and the Cox proportional hazards model were used in the univariate analysis of patients groups., Results: A significantly better survival was observed: (1) in patients with an increased level of anti-TF antibodies (all, stage III, T2-4, N1-2 and G3; P = 0.004-0.038, HR = 0.16-0.46); and (2) in patients with an increased level of anti-Tn antibodies (G1-2 tumors; P = 0.034-0.042, HR = 0.34-0.47). A significantly worse survival was observed in gastrointestinal, gastric and colorectal groups with an increased level of serum anti-αGal antibodies. This association depended on the patho-morphology of tumors (all, stages I-II, III, T2-4, N0, N1-2 and G1-2; P = 0.006-0.048, HR = 1.99-2.33). In the combined assessment of the anti-TF and -αGal antibodies level of the whole gastrointestinal group (n = 53), P = 0.002, HR = 0.25, 95% CI 0.094-0.655. In the follow-up, the survival time was shorter in patients whose level of anti-αGal antibodies rose (P = 0.009-0.040, HR = 2.18-4.27). The level of anti-TF antibodies inversely correlated with neutrophil/lymphocyte ratio (NLR, r = - 0.401, P = 0.004, n = 49). Patients with a higher level of anti-αGal antibodies and NLR values demonstrated a significantly worse survival (P = 0.009, HR = 2.98, n = 48)., Conclusions: The preoperative levels of anti-TF, -Tn and -αGal antibodies and their dynamics are of prognostic significance. The method for the determination of circulating anti-carbohydrate antibodies may be a useful supplement in clinical outcome assessment.

Aim: To study whether alterations in the glycosylation of immunoglobulin G (IgG) specific to the Thomsen-Friedenreich glycotope (TF) have diagnostic and prognostic potential in gastric cancer., Methods: Serum samples were obtained from patients with histologically verified gastric carcinoma (n = 89), healthy blood donors (n = 40), and patients with benign stomach diseases (n = 22). The lectin-enzyme-linked immunosorbent assay-based glycoprofiling of TF-specific IgG (anti-TF IgG) was performed using synthetic TF-polyacrylamide conjugate as antigen, total IgG purified by affinity chromatography on protein G sepharose, and lectins of various sugar specificities: mannose-specific concanavalin A (ConA), fucose-specific Aleuria aurantia lectin (AAL) and sialic acid-specific Sambucus nigra agglutinin (SNA). The sensitivity and specificity of the differences between cancer patients and controls were evaluated by receiver operator characteristic (ROC) curve analysis. Overall survival was analyzed by the Kaplan-Meier method. Time-dependent ROC curve statistics were applied to determine cut-off values for survival analysis. All calculations and comparisons were performed using the GraphPad Prism 5 and SPSS 15.0 software., Results: The level of TF-specific IgG was significantly increased in cancer patients compared with non-cancer controls (P < 0.001). This increase was pronounced mostly in stage 1 of the disease. Cancer patients showed a higher level of ConA binding to anti-TF-IgG (P < 0.05) and a very low level of SNA lectin binding (P = 0.0001). No appreciable stage-dependency of the binding of any lectin to anti-TF IgG was found. A strong positive correlation between the binding of AAL and SNA was found in all groups studied (r = 0.71-0.72; P < 0.0001). The changes in ConA reactivity were not related to those of the fucose- or sialic acid-specific lectin. Changes in the SNA binding index and the ConA/SNA binding ratio demonstrated good sensitivity and specificity for stomach cancer: sensitivity 78.79% (95%CI: 61.09-91.02) and 72.73% (95%CI: 57.21-85.04); specificity 79.17 (95%CI: 65.01-89.53) and 88.64% (95%CI: 71.8-96.6), for the SNA binding index and the ConA/SNA binding ratio, respectively. The other combinations of lectins did not improve the accuracy of the assay. The low level of ConA-positive anti-TF IgG was associated with a survival benefit in cancer patients (HR = 1.56; 95%CI: 0.78-3.09; P = 0.19), especially in stages 3-4 of the disease (HR = 2.17; 95%CI: 0.98-4.79; P = 0.048). A significantly better survival rate was found in all cancer patients with a low reactivity of anti-TF IgG to the fucose-specific AAL lectin (HR = 2.39; 95%CI: 1.0-5.7; P = 0.038)., Conclusion: The changes in the TF-specific IgG glycosylation pattern can be used as a biomarker for stomach cancer detection, and to predict patient survival.

Unlabelled: Serum anti-(GalNAcβ) and anti-para-Forssman disaccharide (PF(di), GalNAcβ1--3GalNAcβ) IgG levels were earlier found to be related to histological grading and progression of gastrointestinal cancer., Aim: To study the relation of serum antibodies level to survival in patients with gastrointestinal cancer., Methods: The level of anti-GalNAcβ, and PF(di) IgG was analysed in the serum of patients with gastric (n = 78) and colorectal (n = 48) cancers in the long-term follow-up using ELISA with polyacrylamide glycoconjugates. Survival rate and hazard ratio (HR) were assessed by the Kaplan -- Meier method and Cox univariate analysis in different patho-morphological groups., Results: Better survival was observed in patients with an increased preoperative level of GalNAcβ antibodies. These were the gastrointestinal group in stages II, III or tumors T2--4 (n = 90--104, P = 0.007, HR = 0.48--0.49, 95% CI 0.27--0.83, and the group with gastric cancer in stages I, II (n = 49, P = 0.051, HR = 0.39, 95% CI 0.14-1.04). The survival time was significantly longer in the gastrointestinal group in patients whose GalNAcβ antibodies level rose in dynamics (stage III or N1--2: p = 0.031--0.039, HR = 0.29--0.31, 95% CI 0.09--1.00). No significant difference in survival of patients was observed in the evaluation of PF(di) antibodies. We suggest that the level of antibodies and its change reflect the enteric microbiota colonization, which may influence cancer progression via different interrelations between microbiota, the tumor and immune system., Conclusion: The preoperative level of GalNAcβ antibodies and its dynamics may be of prognostic significance for clinical outcome assessment.

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.

Host genetic factors responsible for the interindividual differences in naturally occurring antibody responses to the human epidermal growth factor receptor 2 (HER-2) in human beings have not been identified. The aim of the present investigation was to determine whether immunoglobulin gamma heavy-chain marker (GM) and kappa light-chain marker (KM) allotypes, which are genetic markers of IgG heavy chains and κ-type light chains, respectively, contribute to these differences. A total of 152 Estonian women with breast cancer were characterized for IgG antibodies to HER-2 and allotyped for several GM and KM markers. IgG3 determinant GM 13 was significantly associated with higher HER-2 IgG levels (median IgG titer 800 vs 400, p = 0.007). Other GM allotypes, known to be in linkage disequilibrium with GM 13, were also associated with higher anti-HER-2 antibody levels, albeit not as strongly. These results show that GM allotypes are associated with humoral immunity to HER-2, a finding with potentially significant implications for immunotherapy of breast cancer., (Copyright © 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes-genetic markers of IgG heavy chains and kappa-type light chains, respectively-contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin gamma2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.

Glycan structures of IgG strongly influence the affinity for Fcgamma receptors and antibody effector functions. However, no particular attention has been paid yet to the glycosylation of tumor antigen-specific IgG. The objectives of this study were (i) to investigate the concanavalin A lectin (ConA) reactivity of human anti-Thomsen-Friedenreich (TF) and anti-alphaGal specific IgG in gastric cancer patients and healthy controls and (ii) to evaluate whether the ConA-reactivity of anti-TF and anti-alphaGal specific IgG is associated with the survival rate of patients with cancer. Total IgG was purified from the sera of patients with gastric cancer and healthy blood donors. The anti-TF and anti-alphaGal glycotope specific IgG were detected with ELISA using synthetic saccharide-polyacrylamide conjugates as antigen. In parallel plate, the ConA reactivity of the anti-TF or anti-alphaGal IgG was determined and the ConA index was calculated. Results show that serum anti-TF specific IgG antibodies of patients with cancer contain significantly higher content of ConA positive IgG glycoform compared to IgG of controls. No correlation between the ConA reactivity of anti-TF IgG and anti-alphaGal IgG was observed. High level of anti-TF IgG ConA reactivity was associated with a significantly lower survival rate of patients with gastric cancer.

There are significant interindividual differences in naturally occurring antibody responses to the tumor-associated antigen mucin 1 (MUC1), but the host genetic factors that might contribute to these differences have not been identified. The aim of the present investigation was to determine whether the variation in naturally occurring antibody levels to MUC1 in patients with gastric cancer is associated with GM and KM allotypes, genetic markers of IgG heavy chains and kappa-type light chains, respectively. A total of 169 Caucasian subjects with gastric cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. GM 3 23 5,13 phenotype was highly significantly associated with MUC1 IgG levels; subjects with this phenotype had lower antibody levels compared with those lacking this phenotype (median IgG level 65.5 relative units versus 91.0 relative units, P = 0.0058). In addition, this phenotype had an interactive effect with KM phenotypes on the levels of IgG antibodies to this antigen (P = 0.0081). Levels of MUC1 IgM antibodies were not associated with these genetic markers. These results show, for the first time, that GM and KM allotypes contribute to the interindividual differences in humoral immunity to MUC1.

All human immunoglobulins are glycosylated. The changes in IgG glycosylation are associated with autoimmune disorders and pregnancy. Little is known about IgG glycosylation in patients with cancer. A lectin enzyme-linked immunosorbent assay (LELISA) based method was developed for measuring the Concanavalin A - positive IgG in the serum. Its rationale is as follows: PtA was used as a capture agent for binding IgG via the Fc fragment. Then IgG and the ConA-positive glycans on the IgG were detected using an anti-human IgG-F(ab)2 alkaline phosphatase conjugate or biotinylated ConA, respectively. The index ConA binding/total IgG was calculated. Serum samples from patients with gastric carcinoma (n=53) and healthy blood transfusion donors (n=24) were analysed. The protein A-agarose and ConA-sepharose affinity chromatography was applied to the purification of IgG, ConA-positive IgG, and Fab fragments. The LELISA, SDS-PAGE and Western blot methods were used to analyse the purified IgG and Fab fragments. A significantly higher ConA binding to IgG was found in patients with cancer compared to that of blood donors (ConA index = 1.07+/-0.08 (95% CI) and 0.81+/-0.08, respectively; P=0.0002). In donors, a significant correlation between the level of IgG bound to PtA and the ConA binding (r=0.85; p<0.001) was observed. Patients with gastric cancer showed a less pronounced, though significant correlation (r=0.33; P=0.02). Only the Fd fragment of the Fabs derived from both total serum IgG and ConA-positive fraction of IgG contained the ConA-positive glycans. The comparison of the purified IgG and Fab fragments derived from healthy blood donors and patient with gastric cancer showed no difference in either SDS-PAGE, immunoblotting or LELISA pattern. The LELISA is simple, reproducible and suitable for the evaluation of IgG glycosylation changes. The level of ConA positive serum IgG was found to be increased in patients with cancer. No convincing evidence of the presence of asymmetrically glycosylated F(ab)2 fragments was found. A trend towards a better survival of patients with a lower level of the ConA-positive IgG was observed suggesting a possible blocking effect of the latter on tumor immunity.

Unlabelled: Earlier we found two unusual IgG-antibody specificities to GalNAc beta and GalNAc beta1-3GalNAc beta (para-Forssman disaccharide, PFdi) carbohydrate ligands in human serum. The aim of the study was to evaluate whether elevated antibody levels are related to the progression of gastrointestinal cancer and the histopathological grading., Methods: Specific IgG levels were tested in 159 patients with gastric cancer, 88 patients with colorectal cancer and 96 blood donors by the ELISA using synthetic polyacrylaamide (PAA) conjugates, GalNAc beta-PAA and PFdi-PAA. Biochemical and haematological analyses were performed using automatic equipment., Results: The anti-PFdi IgG levels were significantly higher in patients with gastric and colorectal cancer than in donors: in stages II-IV, P = 0.0002 - 0.04 (U-test). The elevated anti-PFdi IgG level was associated with the advanced gastric cancer: in stages II, III, IV vs stage I (P = 0.004 - 0.06) and in case of the tumor size T2 + T3 vs T1 (stages I, II; P = 0.03). Differences in anti-GalNAc beta IgG level were insignificant. No relation between antibody levels and the regional and distant metastases of gastric or colorectal cancer was found. The lower anti-GalNAc beta IgG level was associated with lower-differentiated carcinomas (P = 0.01 - 0.04). Prolonged postoperative changes in the levels of both antibodies during the follow-up were established. An elevation of both antibody levels in patients with gastrointestinal cancer was revealed after a surgical removal of G3-tumors (P = 0.003 - 0.01). The anti-PFdi IgG levels correlated with the levels of the C-reactive protein: r = 0.50, P = 0.003. The anti-GalNAc beta IgG levels correlated with the percentage of peripheral blood monocytes: r = 0.42, P = 0.002., Conclusion: The association of the anti-PFdi IgG level with cancer progression suggests the implication of antibodies in the pathogenesis of gastrointestinal cancer. Further studies are required to identify natural targets of antibodies, their relation to other diseases, prognostic significance in cancer.

Aim: To study the humoral immune response to tumor-associated carbohydrate epitopes (TF, Tn and alphaGal) in patients with breast cancer and healthy donors, the putative impact of the chemotherapy and to evaluate if the level of antibody to these epitopes might be beneficial or detrimental for the patients with breast cancer., Materials and Methods: The humoral immune response to TF, Tn and alphaGal was studied in 133 patients with breast cancer, including the patients at stage II-III (n = 44) before and after neoadjuvant chemotherapy (10 patients received cyclophosphamide/methotrexate/fluorouracyl (CMF) chemotherapy regimens, 34 patients received cyclophosphamide/doxorubicin/fluorouracil (CAF)), and in controls (healthy donors and patients with fibroadenoma). Fully synthetic carbohydrate hapten-polyacrylamide conjugates were used as antigens in ELISA for anti-carbohydrate antibody determination. The correlation analysis between the level of anti-carbohydrate antibodies and the stage of cancer, histological grade, expression of TF and alphaGal epitopes in tumor tissue, patient's survival was performed., Results: The level of anti-carbohydrate antibodies varied between individuals with no significant correlation between IgG immune response to the three epitopes. Lower levels of antibodies were observed at advanced stages of cancer. Neoadjuvant chemotherapy stimulated antibody production to Tn and alphaGal epitopes (increase > 50%) in about one third of patients. Immunosuppression, decrease in antibody levels, was observed only in 4.5-13.6% of cases. High levels of TF-antigen specific IgG antibody before surgery were associated with a better survival time of stage II breast cancer patients., Conclusion: The widely used regimens of neoadjuvant chemotherapy (such as CMF, CAF) can stimulate the immune response to tumor-associated carbohydrate epitopes in some patients. The high levels of anti-TF antibody before surgery are associated with a better survival of stage II breast cancer patients. This may indicate that the selection of immunopotentiating regimens of neoadjuvant chemotherapy might be beneficial for the host.

The Galalpha1-3Galbeta (alphaGal) hapten is xenogeneic for humans; natural anti-alphaGal antibodies are present in human serum. To study the possible abnormal expression of the alphaGal in humans and the pathophysiological role of antibodies, the method of affinity purification of human anti-alphaGal IgG was developed. The specificity of antibodies was evaluated using polyacrylamide (PAA)-based glycoconjugates in direct and competitive enzyme-linked immunosorbent assays (ELISA). The purified antibodies exhibited alphaGal-restricted specificity. The IC50 value for alphaGal-PAA was equal to 4 x 10(-8) M. In a competitive assay, the Galalpha1-3(Fucalpha1-2)Galbeta-PAA (trisaccharide of blood group B) was found to be one hundred times less active inhibitor than alphaGal-PAA. The multivalent alphaGal-PAA was 1100 times more potent an inhibitor than the monovalent spacered alphaGal-saccharide. The antibodies did not show any reactivity to the negatively charged antigens (DNA, human tumor-derived mucins). At a concentration of 2 microg/mL, the antibodies agglutinated rabbit erythrocytes but not hare erythrocytes. The high reactivity of antibodies to the alphaGal-glycosphingolipids of rabbit erythrocytes and the pig kidney was shown by a modified sensitive method of thin-layer chromatography with immunodetection.

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.

The survival of patients with histologically verified gastric carcinoma at stage I (n = 44) and stage II (n = 43) was analysed by the Kaplan-Meier method depending on H. pylori serological status and a level of IgG and IgM antibody to tumor-associated Thomson-Friedenreich antigen (T Ag). In cancer patients at stage I, significantly better survival for H. pylori seropositive patients was observed compared to H. pylori seronegative patients (median SE survival time: 60.0 +/- 3.8 mths and 37.0 +/- 7.8 mths, respectively; P < 0.0004, log-rank test). Patients with higher level of T Ag-specific IgG antibody (strong responders) showed significantly and dramatically better (P < 0.00001) survival rate than weak responders. However, an association of better survival with a higher level of anti-T antibody level was limited to the H. pylori seropositive patients exclusively (P < 0.00001) with no difference for H. pylori seronegative group of patients. The level of IgM anti-T Ag antibody was not significantly related to the survival of patients at both stages of the disease, though better survival was noted in H. pylori seropositive IgM strong responders at approximately 40-60 months of observation. Statistically insignificant associations between survival and H. pylori status or anti-T antibody levels were also observed in a group of gastric cancer patients at stage II. In summary, the survival of patients with early gastric cancer (stage I) is significantly better in H. pylori seropositive patients, and this phenomenon may be in part explained by up-regulation of T Ag-specific IgG immune response in H. pylori infected individuals.

We tested the hypothesis that the gastric cancer associated bacteria, Helicobacter pylori (H. pylori) express the cancer-related Thomsen-Friedenreich (T) antigen. We also analysed whether infection with H. pylori alters the amount of natural anti-T antibodies in the patients' sera. Cell surface membrane extracts of H. pylori NCTC 11637 strain and clinical isolates of H. pylori (n = 13) were analysed by immunoblotting and cell-ELISA with five different T antigen-specific monoclonal antibodies (MAbs). Two major protein bands of approximately 68 kDa and 58 kDa were immunostained on blots of H. pylori extracts with T specific MAbs but not immunostained with unrelated MAb. The specificity was shown in that immunostaining was blocked with peanut agglutinin (PNA) and rabbit antiserum to T antigen. The binding of T specific MAb to the 58 kDa protein band was also blocked by rabbit antiserum against heat shock proteins of H. pylori. The relative expression of T antigen-related proteins differed among H. pylori strains, with 68 kD associated T antigen expression higher in patients with more severe pathology. The level of IgG antibody to T epitope in patients with gastric cancer (n = 66) and normal blood donors (n = 62) were compared and the level of anti-T Ab in gastric cancer patients was significantly lower than that in normal blood donors. A significant positive correlation between T specific antibody in serum and H. pylori IgG antibody level was found in H. pylori-infected normal blood donors (P < 0.001), but this correlation was not found in H. pylori-infected cancer patients. In summary, the cancer related T epitope is expressed in H. pylori and modulation of T antigen-specific immune response in H. pylori-infected individuals suggests that H. pylori infection may alter natural immune mechanisms against cancer.

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.

A possible association of serum anti-T IgM and IgG antibody levels with Lewis blood-group phenotype was investigated in 168 blood donors and 132 gastric cancer patients using ELISA with synthetic T-disaccharide-polyacrylamide conjugate as antigen. The donors of Le(a-b+) phenotype showed the highest anti-T IgM level irrespective of ABO(H) blood group. A significant decrease in anti-T IgM in serum was observed among cancer patients of Le(a-b+) phenotype: 95% of weak responders versus 17.5% for related groups of donors (p < 10(-6)). In contrast, no significant difference between patients and donors was found for Le(b-) individuals. Thus, a level of natural anti-T antibodies in serum of blood donors and its decrease in patients with gastric cancer are related to Le(a,b) phenotype. This should be taken into account where anti-T antibody level in the serum is used as a tumour marker or for monitoring patients during cancer immunotherapy with mucin-type vaccines.

The level of IgM antibodies to the Thomsen-Friedenreich hapten (TF) relative to the total IgM level in the blood sera of gastric and breast carcinoma patients and healthy persons was determined using enzyme-linked immunosorbent assay. The following TF glycoconjugates were tested: TF-polyacrylamide (PAA)(with 10 mol.% of TF hapten Gal beta 1-3GalNAc alpha 1-O(CH2)3NH per number of monomeric units in the polyacrylamide), TF-human serum albumin (HSA)(Gal beta 1-3GalNAc alpha 1-O-p-C6H4-HSA containing approximately 15 carbohydrate residues per HSA molecule), asialo-kappa-caseinoglycopeptide, and asialoglycophorin. The total IgM level was determined using antibodies to the mu-chain of human IgM. The statistically significant difference between cancer patients and healthy donors was revealed with two conjugates: TF-PAA and TF-HSA. In the case of TF-PPA, the sensitivity of the assay was 75-83%, and the specificity was 77%. Thus, TF-PAA is the most suitable conjugate for measuring the level of serum anti-TF-IgM antibodies.

Helicobacter pylori infection is considered to be a risk factor for gastric cancer. A high prevalence of H. pylori infection and high gastric-cancer incidence are characteristic of the Estonian population. To evaluate the relationship between these 2 events, we studied the seroprevalence of H. pylori in gastric cancer patients (n = 182) and in healthy blood donors (n = 306). A relative anti-H. pylori IgG antibody activity, as detected by ELISA and immunoblot patterns, was correlated with age, stage of the disease and tumor morphology. A significantly higher H. pylori seroprevalence was found in patients in the early stages of tumor development compared with both advanced cancer patients and controls. No significant difference in H. pylori seroprevalence between patients with the intestinal and diffuse types of tumor growth was observed. A decline in the recognition of putatively cross-reacting (33-66 kDa) antigens was noted in the cancer group. The response to vacuolating toxin-related 85-kDa and CagA 120-kDa protein antigens was not altered and was observed more often in the younger group of cancer patients.

The TFA is a tumor-associated, blood-group-related glycosidic precursor structure [Gal(beta 1-3)GalNAc]. Its expression in carcinomas is accompanied by a decrease of natural TFA antibodies in serum. The relationship between the ABO(H)-blood-group phenotype and natural anti-TFA immune response in patients with gastric cancer was studied. The level of TFA agglutinins in the sera of patients with gastric cancer and of healthy controls was examined by the hemagglutination of neuraminidase-treated blood-group-O donor erythrocytes. Individuals were classified as weak or strong TFA responders. They were also classified by ABO(H)-blood-group status, age, cancer stage, tumor morphology and level of isohemagglutinins. The proportion of weak TFA responders (WR) in cancer patients was 33, 50, 50 and 20% (for O, A, B and AB blood groups respectively), as compared with 11.7, 14.5, 13.9 and 26.1% for blood-group-related controls. The difference between cancer patients and controls was significant for all blood groups except group AB. Further analysis showed age-dependence in blood-group-O and -B controls, with a high level of WR in the older group. Blood-group-A cancer patients had the greatest and uniform suppression of the level of TFA agglutinins, irrespective of age, cancer stage or tumor morphology, and lower levels of anti-B isohemagglutinins.

Decreased natural killer (NK) activity and its inverse correlation with the level of circulating immune complexes were shown. Lowered NK activity was found to be associated with unfavorable prognosis.

Positive reaction to autologous tumor extracts in leucocyte migration inhibition test was established in one-third of stomach cancer patients under study. Responses with donor serum and autologous sera of blood were not identical. A significant correlation between response to autologous tumor extract in the presence of donor serum and survival time was found. Positive reaction with autologous serum proved to be bad prognosis. The results point to the prognostic value of antitumor immune reactions in autologous system in stomach cancer. Autoimmune reactions to organic antigens of the mucosa are indispensable in the interpretation of antitumor specificity of reactions.