Glycomapping the fine specificity of monoclonal and polyclonal Lewis antibodies with type-specific Lewis kodecytes and function-spacer-lipid constructs printed on paper.

BACKGROUND: Lewis serologic reagents frequently give inaccurate phenotyping results. Furthermore these serologic reagents are often used in nonserologic assays such as inhibition and immunohistochemistry. In both scenarios knowledge of the fine specificity and cross-reactivity of these reagents will improve the quality of results obtained. STUDY DESIGN AND METHODS: A range of contemporary and historical workshop and developmental Lewis reagents including mouse monoclonal (MoAb) and human and goat polyclonal (PoAb) reagents were evaluated. All were evaluated both against Lewis kodecytes expressing only single Le(a) , Le(b) , ALe(b) , BLe(b) , Le(x) , Le(y) , ALe(y) , or BLe(y) antigens and against the same antigens inkjet printed on a paper-based microplate and analyzed by enzyme immunoassay. Nine clinical samples were also evaluated. A kodecyte antigen dilution sensitivity assay was used to establish the ratio of Le(b) antigen between group A1 /A2 and O RBCs. RESULTS: A continuum of cross-reactivity from Le(x) through to H was observed with MoAbs. All PoAb and few MoAb anti-Le(a) samples and reagents cross-reacted to some degree with Le(b) antigen. Some PoAb and MoAb anti-Le(b) did not cross-react with Le(a) . All polyclonal goat anti-Le(b) reagents showed substantial activity against ALe(b) and BLe(b) , while no MoAb reagent had this activity. A1 RBCs had less than half the Le(b) antigen of A2 /O RBCs. CONCLUSIONS: Substantial cross-reactivity of both MoAbs and PoAbs with related antigens highlights the risks of using serologic reagents in nonserologic assays or against synthetic antigens. The lack of ALe(b) activity in anti-Le(b) MoAbs explains their poor performance against blood group A1 Le(a-b+) phenotypes.

Acute hemolytic transfusion reaction due to anti-Le(b).

BACKGROUND: Anti-Le(b) is usually a clinically insignificant antibody of immunoglobulin M subclass most often found in the sera of pregnant women or individuals that are Le(a-b-). We report a case of an acute hemolytic transfusion reaction due to a hemolytic anti-Le(b) that was not seen in the pretransfusion antibody detection test, but was strongly reactive in posttransfusion testing. CASE REPORT: A 30-year-old African-American woman with metastatic renal cell carcinoma was receiving chemotherapy. She was anemic with hemoglobin (Hb) of 7.2 g/dL and had a negative antibody detection test by the solid-phase red blood cell adherence method. She was transfused with 2 RBC units without incident. Nine days later her Hb was 7.9 g/dL again with a negative antibody detection test. Transfusion of an additional RBC unit was begun. During the transfusion she developed chills, nausea, hypertension, and red-brown urine. The posttransfusion sample plasma was grossly hemolyzed with a strongly positive direct antiglobulin test (DAT) by gel. By comparison the pretransfusion plasma was normal appearing and the DAT was weaker. The eluate was negative on both occasions. Anti-Le(b) was detected in the posttransfusion sample by MTS gel (Ortho Diagnostics). Both RBC units she had received before the RBC unit that caused the reaction were Le(b+) as was the implicated RBC unit. CONCLUSION: This case illustrates that anti-Le(b) which is usually clinically insignificant can occasionally cause severe hemolytic transfusion reactions. Only three other reported cases of anti-Le(b) causing hemolytic transfusion reactions could be found in the literature, two of which were in abstract form only.

Distribution and impact of erythrocyte Lewis-system antigens on patients with ischemic heart diseases in the west of Georgia.

The aim of the research is to reveal the cases of Lewis System Antigens Phenotype in West Georgia and setting the connection between antigens expressivness and IHD. Therefore, we have phenotypically tested 393 people (236 healthy donors; average age 42±7,5 and 157 patients ill with ischemic heart diseases; average age 62,5±7,5). In accordance with the findings, the number of Lewis -antigens among healthy population is 46,6% (110 ±4,8; p<0.05) with Lea-b+ phenotype; 30,9% - with Lea-b- phenotype(73±2,9; p<0.03); 19% - with Lea+b- phenotype-(47±1,7; p<0.03) Only 2,6% cases of phenotype Lea+b+ (6±0,2; p<0.02),were revealed among healthy population. As for the patients with ischemic heart diseases we got the following results: 41% cases of Lea-b- phenotype (65±3,9; p<0.05); 32,8% - Lea-b+ (51±3,2; p<0.03); 21,1% - Lea+b- - phenotype 33±2,9; p<0.03) and 5,6% - Lea+b+ phenotype (8±1,2; p<0.02). On the whole, in the West Georgia the most frequent phenotype is Lea-b+ among healthy population and Lea-b- phenotype among people with ischemic heart diseases. Research was carried out in a control group according to Lewis antigen phenotype. People were separated in two groups; I group -healthy people with Lea-b- phenotype and II group - healthy people with Lea-b+ and Lea+b- phenotypes. On the basis of the research we concluded that people in the first group (with Lea-b- phenotype) had a high BMI, arterial hypertension and lower indexes of high density lipoprotein and triglyceride than the people in the second group(with Lea-b+ and Lea+b- phenotypes. These kinds of changes (characterised to the people with Lea-b- phenotype) are associated with a high risk of ischemic diseases and atherosclerosis. To sum up, people with Lea-b- phenotype have a high risk of ischemic heart disease. In accordance with the findings, Lewis phenotype research can be carried out to detect HID and other diseases as well (hypertension, ischemic insult and insulin-dependent diabetes mellitus).

Expression of Lewis (b) blood group antigen interferes with oral dienogest therapy among women with adenomyosis.

Adenomyosis is frequently observed in premenopausal women, and oral dienogest is the recommended treatment to target the underlying pathology and improve the symptoms. This retrospective study investigated the association of Lewis (b) antigen expression with outcomes of dienogest therapy among women with adenomyosis. Records from a total of 342 adenomyosis patients were analysed, who were prescribed with oral dienogest for a maximum of 16 weeks. Expression levels of Lewis (b) antigen were measured to categorize all patients into either Le (b)- and Le(b)+ groups. Treatment outcomes, in terms of uterine volume, menstrual flow, pain symptoms and quality of life, were compared between the two groups. While oral dienogest therapy showed considerable clinical efficacy in both groups of patients, the extent of improvements in treatment outcomes was significantly more pronounced in Le (b)- group than Le (b)+ group, with respect to treatment time, uterine symptoms, menstrual flow, pain symptoms and quality of life. No difference in adverse effects was observed between the two groups. Expression of Lewis (b) blood group antigen interferes with oral dialogist therapy among women with adenomyosis.

FUT3 and FUT2 genotyping and glycoconjugate profile Lewisb as a protective factor to Toxoplasma gondii infection.

The interaction between the ABO, FUT2 and FUT3 genes results in the synthesis of different glycoconjugates profiles expressed in gastrointestinal tract. Moreover, the protozoan Toxoplasma gondii, which causes toxoplasmosis, utilizes this organ as an infection route. We analyzed the frequencies of the different glycoconjugate profiles which were determined by phenotyping ABO and genotyping the status secretor (FUT2; substitution G428A) and Lewis (FUT3; substitution T202C and C314T) histo-blood systems, assessed by PCR-RFLP and PCR-SSP, respectively. A total of 244 pregnant women (G1: Seropositive; G2: Seronegative) for IgG T. gondii antibodies were enrolled. IgG anti-T. gondii antibodies were determined by ELISA. G1 was composed of 158 (64.8%) sample and G2 by 86 (36.2%). The glycoconjugate profile was accessed in 151 seropositive and 85 seronegative samples by the combination of ABO and Lewis phenotyping as well as FUT2 and FUT3 genotyping. In G1, 36 (22.8%) presented the glycoconjugate profile ALeb, 5 (3.3%) A, 13 (8.6) BLeb, 1 (0.6%) B, 41 (27.1%) Leb, 13(8.6%) H, 38(25.2%) Lea and 4 (2.6%) Lec. G2 was composed of 13 (15.3%) of ALeb, 15 (17.6%) BLeb, 1 (1.2%) B, 42 (49,4%) Leb and 14 (16.5) Lea. H and Lec glycoconjugate profiles were not found in G2. The frequencies of the glycoconjugates profiles Leb (p = 0.001) and H (p = 0.005) were significantly different compared between G1 and G2. The glycoconjugate profile H inferred from the ABO phenotyping and FUT3 and FUT2 genotyping is associated with infection by T. gondii in pregnant women and the Leb profile appears to protect the infection by this parasite.

Phenotyping of Lewis and secretor HBGA from saliva and detection of new FUT2 gene SNPs from young children from the Amazon presenting acute gastroenteritis and respiratory infection.

The Histo-blood group antigens (HBGA) are host genetic factors associated with susceptibility to rotavirus (RV) and human norovirus (HuNoV), the major etiological agents of viral acute gastroenteritis (AGE) worldwide. The FUT2 gene expressing the alpha-1, 2-L- fucosyltransferase enzyme is important for gut HBGA expression, and also provides a composition of the phenotypic profile achieved through mutations occurring in populations with different evolutionary histories; as such, it can be considered a genetic population marker. In this study, Lewis and secretor HBGA phenotyping was performed using 352 saliva samples collected from children between three months and five years old born in the Amazon (Brazil, Venezuela and English Guyana) presenting AGE or acute respiratory infection (ARI), the latter considered as control samples. The total of children phenotyped as secretors was 323, corresponding to 91.80%. From these, 207 (58.80%) had a Le (a + b+) profile. The HBGA profiles were equally found in children with AGE as well as with ARI. The rs1047781 of the FUT2 gene was not detected in DNA from saliva cells with a Le (a+b+) profile. However, mutations not yet described in the FUT2 gene were observed: missense 325A>T, 501C>T, 585C>T, 855A>T and missense substitutions 327C>T [S (Ser) > C (Cys)], 446 T>C [L(Leu) > P(Pro)], 723C>A [N(Asn) > K(Lys)], 724A>T [I(Ile) > F(Phe)], 736C>A [H(His) > N(Asn)]. The SNP distribution in the FUT2 gene of the analyzed samples was very similar to that described in Asian populations, including indigenous tribes.

Reduced frequency of blood group Lewis a-b- in female Type 1 diabetes patients.

AIMS: To examine a disputed association between the Lewis(a(-)b(-)) phenotype and Type 1 diabetes (T1D). METHODS: Lewis red blood cell phenotyping was performed for 97 T1D White patients and 100 control subjects using monoclonal antibodies. Two historical cohorts were also included as a control population. RESULTS: T1D patients had a lower frequency (4.1%) of Lewis(a(-)b(-)) blood group compared with simultaneously tested healthy control subjects (10.0%) and the historical control group (11.1%, P = 0.02). Male T1D patients showed a Lewis(a(-)b(-)) frequency of 8.0%, which was similar to both matched healthy male donors (9.8%) and historical (9.5%) male control subjects. Unexpectedly, none of the female T1D patients displayed Lewis(a(-)b(-)) phenotype, vs. 10.3% and 10.8% of female control subjects (P = 0.039 and 0.017). CONCLUSIONS: The Lewis(a(-)b(-)) phenotype occurs less frequently in T1D compared with healthy control subjects with a strong female gender bias.

ABH and Lewis antigen distributions in blood, saliva and gastric mucosa and H pylori infection in gastric ulcer patients.

AIM: To investigate the ABH and Lewis antigen expression in erythrocytes, saliva and gastric epithelium, as well as the association between H pylori and the presence of gastric epithelial lesions. METHODS: The distribution of ABH and Lewis blood group antigens in erythrocytes, saliva and gastric mucosa of H pylori-infected gastric ulcer patients was analyzed. Forty-two patients with gastric ulcer were studied, and fifty healthy individuals were used as control group. The blood group antigens were determined by direct hemagglutination, dot-ELISA and immunohistochemical methods in erythrocytes, saliva and gastric mucosa specimens, respectively. Diagnosis for H pylori infection was performed by conventional optical microscopy and ELISA. RESULTS: A higher seroprevalence of IgG H pylori specific antibodies was observed in gastric ulcer patients (90%) compared to the control group (60%). We observed a significant increase of phenotypes O, A2 and Lewis b in H pylori-infected patients. The expression of these antigens had progressive alterations in areas of ulcerous lesions and intestinal metaplasia. CONCLUSION: ABH and Lewis blood group antigens are a good indicator for cellular alterations in the gastric epithelium.

Evaluation of Secretory State in Patients with Oral Lichen Planus: A Case-Control Study.

BACKGROUND: Non-secretor individuals lack ABO blood group antigens in their secretions like saliva; these carbohydrate structures play an important role in protection of the oral cavity from exogenous pathogens; therefore these individuals are more susceptible to mucous membrane damages. The aim was to assess the secretory state of patients with oral lichen planus (OLP) in comparison with healthy controls. METHODS: Fifty patients and 100 age-gender matched control subjects were recruited to the study. Patients were visited in the outpatient clinic of dermatology at Shohada-e-Tajrish Hospital, Shahid Beheshti University of Medical Sciences from 2012 - 2014. Two-milliliter (mL) blood was collected from each subject to detect Lewis phenotypes. According to Lewis phenotype of each subject, secretory state was determined except in subjects with Le (a-b-) phenotype, in whom saliva was collected to determine the secretor status. RESULTS: Non-secretor status in patients with OLP was more frequent compared with healthy controls (37 out of 50 patients (74%) vs. 24 out of 100 healthy controls (24%), (P < 0.001)). There was no association between secretory state, and type of OLP and disease duration (P > 0.05). CONCLUSION: This study supported the possible role of cell surface histo-blood group antigens in protection of mucosal surface from exogenous pathogens. Therefore, it appears that non-secretor individuals are more prone to oral lichen planus.

Combined role of the Lewis antigenic system, Chlamydia pneumoniae, and C-reactive protein in unstable angina.

OBJECTIVES: The goal of this study was to assess the prognostic role of the Lewis antigenic system, Chlamydia pneumoniae (CP) seropositivity (CP+), and C-reactive protein (CRP) levels in unstable angina (UA). BACKGROUND: The role of CP infection in acute coronary syndromes is contradictory. The Lewis antigenic system, a genetically determined blood group system associated with infections and several disorders, including ischemic heart disease, might influence the susceptibility to CP infection, inflammatory response, and risk of cardiac ischemic events. METHODS: The CRP levels, Lewis antigens, and CP+ were measured in 54 patients with Braunwald's class IIIB UA. All patients were followed up for one year, and the occurrence of new coronary events (coronary death, myocardial infarction, and recurrence of instability) were recorded. RESULTS: Twenty-five coronary events occurred during follow-up. At univariate analysis CRP >3 mg/l (CRP+) (p = 0.0056), Lewis antigen b (Leb+) (p = 0.028), and the combination of Leb+ and CP+ (p = 0.006) and of CRP+ and Leb+ (p = 0.003) were associated with new coronary events, while CP+ alone was not. At multivariate analysis, CRP+ (p = 0.008) and combined Leb+CP+ (p = 0.03) were independent predictors of worse outcome. The event rate was 64% in CRP+ patients, 67% in Leb+CP+ patients, and 86% in CRP+Leb+CP+ patients. Combined Leb+CP+, but not Leb+ and CP+ alone, was related to CRP levels (p = 0.03). Among CP+ patients, CRP levels were higher in Leb+ than Leb- (p = 0.028). CONCLUSIONS: Our data demonstrate that in UA the Lewis antigenic system plays an important role, probably determining individual susceptibility to the detrimental effects of CP infection and by determining an enhanced inflammatory response.

Serum sialyl lewis X-i antigen levels in non-small cell lung cancer: correlation with distant metastasis and survival.

To evaluate the correlation between serum levels of sialyl Lewis X-i antigen and distant metastasis and survival in patients with non-small cell lung cancer (NSCLC), we measured the serum levels of the tumor marker in 371 patients with untreated NSCLC. The sialyl Lewis X-i antigen level was measured using a RIA kit. In patients with adenocarcinoma or other NSCLC subtypes, there was a correlation between serum sialyl Lewis X-i antigen and stage of the disease (P = 0.0001 and P = 0.0015, respectively). Levels of the marker varied significantly depending on the number of metastatic organs in adenocarcinoma (P = 0.0089) and in other NSCLC subtypes (P = 0.002). Univariate analysis showed that survival of NSCLC patients with high (more than 100 units/ml) sialyl Lewis X-i antigen levels was significantly poorer than that of patients with low antigen levels (P = 0.0001). Multivariate analysis using Cox's proportional hazard model showed that high sialyl Lewis X-i antigen levels correlated significantly with poor survival (P = 0.004). Our data suggest that a high serum level of sialyl Lewis X-i antigen seems to be an indicator of the presence of metastasis and might indicate the need for a careful investigation of all putative metastatic sites. The serum levels of sialyl Lewis X-i antigen may reflect the extension of metastasis and would be helpful in considering treatment options.

ABH secretor status, as judged by the Lewis phenotypes, in Norwegian survivors from meningococcal disease.

Survivors from meningococcal disease (serogroups B and C) and a control series (blood donors) were examined for their ability to secrete ABH blood group substance. The examination was done indirectly by determining their Lewis phenotypes. There was no significant difference in the secretor status between the two groups.

Hepatic neoexpression and increased plasma levels of Lewis Y, a carbohydrate antigen, in chronic inflammatory liver diseases.

The clinicopathologic relevance of the hepatic expression of Lewis Y (Le(y)), a carbohydrate antigen, and its plasma levels was studied in benign and malignant liver diseases. Tissue and plasma antigens, respectively, were determined with an avidin-biotin-peroxidase complex method and a radioimmunoassay using monoclonal antibody AH6. Normal liver cells and bile ductules did not express Le(y). In the inflammatory tissues, the liver cells and proliferated bile ductules expressed Le(y). The strongest expression by the liver cells was observed in chronic active hepatitis with severe activity and that by the ductules in liver cirrhosis. Only 1 of 16 hepatocellular carcinomas expressed Le(y). The plasma levels of Le(y) increased significantly but nonspecifically in chronic persistent hepatitis, chronic active hepatitis, liver cirrhosis, and hepatocellular carcinomas. It was concluded that (1) Le(y) is an inflammation-associated but not a cancer-associated antigen; (2) the more the tissue damage advances, the more the antigen is expressed; and (3) hepatic and plasma Le(y) are, however, nonspecific markers of necroinflammatory liver diseases.

Clinical relevance of tumour markers CA 19-9 and CA-50 in sera from patients with pancreatic duct carcinoma.

Serum levels of the tumour antigens CA 19-9 and CA-50 and their relation to the extent of the disease were studied in 97 patients with carcinoma of the pancreas. Of 13 patients with normal serum concentrations of CA 19-9, 11 (84.6%) had irresectable disease, whereas 87.5% of the patients with resectable disease expressed antigen levels above cut-off. Following attempted radical surgery, preoperatively elevated serum levels decreased in eight patients (50%). Unchanged and high levels were associated with residual disease and early death. Clinical signs of recurrence were preceded by elevated serum levels of both tumour antigens.

Predictive value of preoperative serum sialyl Lewis X-i antigen levels in non-small cell lung cancer.

BACKGROUND AND STUDY OBJECTIVE: Serum sialyl Lewis X-i antigen has been shown to be one of the most specific markers for monitoring non-small cell lung cancer(NSCLC). We evaluated the correlation between serum levels of sialyl Lewis X-i antigen and mediastinal lymph node involvement in NSCLC patients. METHODS: One hundred patients with untreated NSCLC were included in this study. All the patients received surgical treatment and the lymph nodes were evaluated pathologically. Pretreatment sera from these patients examined for the levels of sialyl Lewis X-i antigen, using a radioimmunoassay. RESULTS: Serum levels of sialyl Lewis X-i antigen levels in patients with N2 were higher than those of N0-1(p = 0.0049). According to the receiver characteristic curve analysis, the recommended cut-off level of the antigen between N0-1 patients and N2 patients was 38.0 U/ml. CONCLUSION: Measurement of serum levels of sialyl Lewis X-i antigen might provide preoperative information in patients with NSCLC.

[The Lewis antigen system as a marker of IHD risk]. / Sistema antigenov Lewis kak marker riska IBS.

Prevalence of Lewis antigens and phenotypes has been investigated in 60 healthy donors and 74 patients with ischemic heart disease. It was found that phenotype Le(a-b-) marks high risk and phenotype Le(a-b+) is a resistance marker for development of ischemic heart disease. It would be valid to elucidate relationships between Lewis system and pathogenesis of internal diseases.

Investigation of unexpected serum CA19-9 elevation in Lewis-negative cancer patients.

BACKGROUND: Cancer patients with a Lewis (a-b-) phenotype have no carbohydrate antigen 19-9 (CA19-9) in their serum. However, we found a small but distinct elevation in the serum CA19-9 level in three cancer patients with the Lewis-negative phenotype. Here, we investigated the reason of such phenomena. METHODS: Six cancer patients with a Lewis-negative phenotype were selected by very low CA19-9 concentrations: three showed a small elevation (Group A) and the other three showed no elevation (Group B) in the serum CA19-9. We investigated the difference by analyzing the Lewis/Secretor genotypes. RESULTS: All of the six patients with a Le (a-b-) phenotype were genuine Le-negative genotypes: four individuals were homozygous for le1 (le(59,508)), one patient was compound heterozygous for le1 (le(59,508)) and le2 (le(59,1067)) and one patient was compound heterozygous for le1 and le(202,314). As for the Secretor gene, the three patients in Group B were homozygous for Se2 (one patient) or compound heterozygous for Se2 and sej (two patients), while the patients in Group A were all homozygous for sej genotypes. CONCLUSIONS: Even genuinely Le-negative patients, who genetically lack the Le enzyme and theoretically never produce CA19-9, occasionally show a slight increase in serum CA19-9 level when they are homozygous for Se-negative genotypes and suffer from advanced cancer with overproduction of glycans as precursors of CA19-9. Although such cases are not frequent, we should be acquainted with the correlation between serum CA19-9 values and genotypes of Lewis and Secretor genes.

Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1).

BACKGROUND: In cancer patients, MUC1 glycoprotein may carry Lewis y which could be involved in immune response. PURPOSES: 1- to evaluate the presence of Lewis y and MUC1 in circulating immune complexes (Lewis y/CIC and MUC1/CIC, respectively) and their correlation; 2- to analyze the possible presence of Lewis y in carbohydrate chains of tumoral MUC1 glycoprotein and 3- to correlate serum and tissue parameters considered. METHODS: Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05). RESULTS: By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean +/- SD values expressed in OD units were: 0.525 +/- 0.304; 0.968 +/- 0.482 and 0.928 +/- 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples. CONCLUSION: Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.