The characterization of IgG antibodies to GalNAc beta-terminated glycans of gastric cancer survivors.

UNLABELLED: The elevated anti-GalNAcß 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 GalNAcß-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 GalNAcß, GalNAcß1-3Galß (X2di), GalNAcß1-3GalNAcß (PFdi) and GlcNAcß was observed. The isolated anti-GalNAcß IgGs demonstrated the cross-reactivity to the X2di glycan mainly. The affinity of the X2di-PAA to anti-GalNAcß IgGs was 11-21 times lower than that of the GalNAcß-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-GalNAcß 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.

The level of anti-(GalNAc beta) and anti-para-Forssman disaccharide IgG antibodies in patients with gastrointestinal cancer: relation to survival.

UNLABELLED: Serum anti-(GalNAcß) and anti-para-Forssman disaccharide (PF(di), GalNAcß1--3GalNAcß) 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-GalNAcß, 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 GalNAcß 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 GalNAcß 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 GalNAcß antibodies and its dynamics may be of prognostic significance for clinical outcome assessment.

Association of CD2 with fibrinogen in human plasma: depletion of the soluble E-receptor in blood clotting.

The soluble E-receptor (SER) of lymphocytes that is related to CD2 was detected in human plasma and serum using immunoelectrophoresis with sheep antiserum. All plasma samples (n=18) demonstrated reactivity with antiserum, whereas the reactivity of the corresponding sera remained low or undetectable. The depletion of SER in clotting is associated with fibrinogen, as shown by crossed-affinity immunoelectrophoresis with antisera to plasma proteins. The SER-associated fibrinogen was purified and analysed by the SDS-polyacrylamide gel electrophoresis and immunoblotting. A band close to 66 kDa was detected with monoclonal antibodies to CD2. The association of CD2 and other soluble receptors with fibrinogen via domains is suggested. It is recommended that the fresh plasma, not serum, should be used to study circulating receptors because coagulation may appreciably diminish their physiological level in blood samples.

The relation of serum anti-(GalNAc beta) and -para-Forssman disaccharide IgG levels to the progression and histological grading of gastrointestinal cancer.

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.

The isolation and characterization of human natural alphaGal-specific IgG antibodies applicable to the detection of alphaGal-glycosphingolipids.

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.

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates.

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.

[Evaluation of the immune status of patients with cancer of the stomach]. / Otsenka immunnogo statusa bol'nykh rakom zheludka.

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.

[Characteristics of the carbohydrate structure of alpha 2-macroglobulin in stomach cancer]. / Osobennosti uglevodnoi struktury al'fa-2-makroglobulina pri rake zheludka.

A comparative study was made for alpha 2M preparations obtained from plasma of patients with gastric carcinoma and healthy individuals. No significant differences were found in trypsin-binding activity of alpha 2M, in isoelectric focusing microheterogeneity and in binding with lectins of Limulus polyphemus hemolymph. The study of alpha 2M-phytohemagglutinin (PHA) binding in sera of patients with gastrointestinal carcinoma and healthy people revealed highly significant differences. The alpha 2M preparations from the gastric carcinoma patients had less affinity for PHA. The data suggest the existence of an abnormal carbohydrate structure of alpha 2M in gastric carcinoma patients.

[Sensitization of the leukocytes of stomach cancer patients to extracts of the tumor]. / Sensibilizatsiia leikotsitov bol'nykh rakom zheludka k ékstraktam opukholi.

The parallel LAI-tests using tumor cell (Tu) and normal mucosa (Muc) extracts were run in cases of gastric carcinoma (110), benign lesions of the stomach (46) and healthy controls (33). A high incidence (37-43%) of positive reactions in mucosa extract series was observed in both groups of patients. Four possible types of the response were analysed: Tu + Muc+, Tu--Muc--, Tu+Muc- and Tu--Muc+. The peak of Tu--Muc+ type reactivity was observed at stage III of the disease (p less than 0.05). Response to Tu extracts only (Tu+Muc--) was registered mainly in cancer patients but never occurred in the benign group. The best prognosis was observed in cases of Tu+Muc+ type response (p less than 0.02).

[Cellular antitumor immunity in an autologous system in stomach cancer]. / Kletochnyi protivoopukholevyi immunitet v autologichnoi sisteme pri rake zheludka.

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.

[Human antitumor immunity according to leukocyte adherence inhibition test data]. / Protivoopukholevyi immunitet u cheloveka po dannym testa tormozheniia prilipaniia leikotsitov.

A microvariant of the test of leucocytes adherence inhibition, which results were assessed by fluorescence, has revealed antitumor immune reactions to allogenic tumor antigens in gastric cancer (n = 38) and breast cancer (n = 35) in 51.7 and 68.5% of patients respectively. A correlation between the reaction and stage was found only in breast cancer patients. Postive reactions to heterogenous tumor antigen were noted in 17-25% of cases. The reactions to tumor antigens in nontumor lesions of the corresponding localizations were noted in 29.2 and 25%. The data obtained indicated that homogeneity of the antigenic tumor pattern is high enough within the limits of a particular localization irrespective of the degree of morphological tumor differentiation. It seems essential to take into account some possible autoimmune reactions to normal organotypical antigens to interprete the tumor specificity of the reactions under study.

[Cell immunity indices in carcinoma of the gastrointestinal tract]. / Pokazateli kletochnogo immuniteta pri rake zheludochno-kishechnogo trakta

A group of 137 patients with carcinoma and benign diseases of the gastrointestinal tract was tested for a specific immunity to tumor, mucosa antigens and PHA. A response of peripheral blood lymphocytes to PHA was suppressed in patients with cancer. An assay of the specific reactivity against tumor antigens was done either by a lymphoblastic transformation or by the leucocytes migration inhibition agarose technic. More than one third of cancer patients showed positive ractions to tumor extracts in autologous as well as allogeneic systems. No differences were noted between the effect of the mucosa or tumor extract in a group of benign diseases by migration inhibition technic, but there were 11 of 23 patients who showed a blasttransformation reaction to tumor antigen. The autologous mucosa extract had no effect on lymphocytes of cancer patients, but it was found to inhibit the leucocytes migration. The action of tumor extracts was comparable in autologous and allogeneic systems. The presence of common or cross-reactive tumor antigens in gastrointestinal cancer is assumed. Some possible reasons of the changes are discussed.

[The effect of local non-specific sensitization with 2,4-dinitrochlorbenzene on chemical carcinogenesis]. / Vliianie mestnoi nespetsificheskoi sensibilizatsii 2,4-dinitrokhlorbenzolom na khimicheskii kantserogenez

The induction of benzpyrene skin carcinogenesis was suppressed by repeated administrations of DNCB in C57Bl mice. DNCB was found to protect the development of the immunodepression induced by benzpyrene. The most appreciable effect was observed when DNCB applications were started two months before the benzpyrene administration. An insignificant inhibition or therapeutic effect was noted during later stages of the benzpyrene carcinogenesis.