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Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara 3 ), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara 3 . None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Lacto-N-fucopentaose I (LNFP I) is an abundant and important fucosylated human milk oligosaccharide (HMO). Here, an efficient LNFP I-producing strain without by-product 2'-fucosyllactose (2'-FL) was developed by advisable stepwise de novo pathway construction in Escherichia coli. Specifically, the genetically stable lacto-N-triose II (LNTri II)-producing strains were constructed by the multicopy integration of β1,3-N-acetylglucosaminyltransferase. LNTri II can be further converted to lacto-N-tetraose (LNT) by LNT-producing β1,3-galactosyltransferase. The de novo and salvage pathways of GDP-fucose were introduced into highly efficient LNT-producing chassis. Specific α1,2-fucosyltransferase was verified to eliminate by-product 2'-FL, and binding free energy of the complex was analyzed to explain the product distribution. Subsequently, further attempts aiming to improve α1,2-fucosyltransferase activity and the supply of GDP-fucose were carried out. Our engineering strategies enabled the stepwise de novo construction of strains that produced up to 30.47 g/L of extracellular LNFP I, without accumulation of 2'-FL, and with only minor intermediates residue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

2'-Fucosyllactose (2'-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2'-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2'-FL from glucose and lactose. Thus, the overproduction of 2'-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this study., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (< 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 and p = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Background: The secretor type α(1,2)fucosyltransferase gene (FUT2) is known to be rich in population-specific polymorphisms. However, genetic variations of FUT2 have not been well examined in Latin American populations in which nonsecretors are rare., Methods: Conventional polymerase chain reactions and direct sequencing were performed to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) of FUT2 in Mexicans including Americans of Mexican ancestry, Puerto Ricans, Caribbeans, and Colombians. FUT2 alleles were determined by cloning into plasmids or PHASE software. The impact of uncharacterized missense SNPs on the enzyme activity were examined by transient transfection assays and estimated by several software programs., Results: Three alleles, Se 357 , Se, and se 428 , were common, and the frequency of nonsecretors was relatively low in the studied populations. We also encountered several alleles specific to Africans, Europeans, and South and East Asians including a South Asian-specific se del . In contrast to the in silico prediction, a transient expression study suggested that both of two missense SNPs, 235G > A and 304G > A, did not impair the enzyme activity., Conclusions: The allelic polymorphism of FUT2 suggests that the modern Latin American populations were formed via genetic admixture among Native Americans and populations whose ancestors migrated from other continents. In this study, we have observed a discrepancy between in silico and functional analyses for FUT2 for the first time. Therefore, experimental functional analysis is required for evaluation of SNPs of FUT2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Carbohydrate antigen 19-9 (CA19-9) is the best-validated biomarker for pancreatic cancer. The National Comprehensive Cancer Network (NCCN) guideline asserts that "CA19-9 will be undetectable in Lewis antigen-negative individuals". However, reports of CA19-9 secretion and its significance in Lewis (-) patients with pancreatic cancer have been inconsistent. This study was to examine serum CA19-9 levels in patients with pancreatic cancer according to Lewis status., Methods: Patients with pancreatic cancer (1482 cases) were retrieved from a prospectively maintained database. Patients with benign pancreatic disease (210 cases) and normal subjects (315 cases) were used as controls. Lewis genotypes were examined by fucosyltransferase 3 (FUT3) sequencing., Results: In patients with pancreatic cancer, 8.4% of subjects were Lewis (-), but only 41.9% of Lewis (-) subjects had CA19-9 values ≤ 2 U/mL. CA19-9 was even elevated (>37 U/mL) in 27.4% of Lewis (-) patients. The area under the receiver operating characteristic (ROC) curve for CA19-9 as a diagnostic biomarker was 0.842 in Lewis (-) patients with pancreatic cancer, which is closing to that of CA19-9 applied in all of patients with pancreatic cancer (0.898). Lewis (-) status was an independent prognostic factor for shorter survival in a multivariable analysis (hazard ratio (HR), 1.30, 95% confidence interval (CI), 1.03-1.64; P = 0.028)., Conclusions: Not all Lewis (-) patients with pancreatic cancer are non-secretors of CA19-9. Contrary to general understanding, CA19-9 can retain its utility as a biomarker in these patients in spite of Lewis (-) genotype., (Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Autoreactive B-cell activation and antibody production are critical events for the development of bullous pemphigoid (BP). However, the mechanism that is involved in the modulation of B-cell activation and autoantibody generation has not been fully understood. Semaphorin 4D (Sema4D, or CD100) plays important roles in immune regulation related to B cells, but its implications in BP remain obscure. The aim of our study was to characterize Sema4D and the underlying mechanism contributing to the autoimmune features of BP. We found that soluble Sema4D (sSema4D) levels were elevated and correlated with disease severity and activity in serum and blister fluids from patients with BP. Additionally, Sema4D-expressing cells accumulated in subepidermal blisters of BP lesions. In patient-derived peripheral blood mononuclear cells, by promoting the differentiation of B cells into plasmablasts, sSema4D boosted anti-BP180/anti-BP230 antibody production in a time- and dose-dependent manner, which may be attributed to CD72-mediated activation of Akt/NF-κB phosphorylated (p-)65/ERK cascades in B cells. We determined that a disintegrin and metalloproteinase 10 is a proteolytic enzyme for the cleavage of sSema4D from CD15 + granulocytes instead of T cells, which is probably responsible for the high concentration of sSema4D in BP blister fluid and serum. These findings suggest that Sema4D is a crucial participant in BP pathogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Current scientific consensus about the physiopathology in the progression from severe sepsis to septic shock and death focuses on myocardial contractile dysfunction. Nevertheless, objective parameters to establish a pathological correlate of a fatal outcome are lacking; then a cause of death due to sepsis can remain an unsolved problem. We first reviewed all death cases recorded at our institutions during the period from 2007 until 2015. Then, we conducted a retrospective study of a selected autopsy series of people who had received "sepsis" as cause of death. Two pathologists re-examined the heart sections while the most suitable myocardial sample for each case was stained for immunohistochemistry with antibodies targeted for specific inflammatory-related molecules. We used specific antibodies for the following markers: alpha-smooth muscle actin (alpha-SMA); fibronectin; matrix metallopeptidase 9 (MMP-9); intercellular adhesion molecule 1 (ICAM-1); caspase-3; lactoferrin (LF); cluster differentiation 15 (CD15). The statistical significance of differences was assessed using student's t-test for unpaired data or non-parametric Mann-Whitney or Wilcoxon tests for skewed variables or one-way analysis of variance and post hoc Scheffe's test for continuous variables and Pearson's χ 2 -test for discrete variables. Linear regression analysis was used to determine the presence of a correlation between continuous variables. At our institutions, 2220 deaths have been recorded during the period study. Sepsis accounted as a cause of death for the 20% of total. We finally enrolled 56 cases; of these, only 20 were positive for microbiological analysis. At histological examination, clear inflammation was detectable in the 32% of cases; otherwise, immunohistochemical reaction showed a positive reaction for LF and CD15 in more than a half cases (56%). We still ignore all the underlying mechanisms of sepsis and all its pathophysiological connections with cardiac metabolism; in this sense, we aim to corroborate the diagnostic value of anti-LF and anti-CD15 staining for the post-mortem detection of myocardial inflammation.

Peri-implantitis is an infectious disease characterized by inflammation of the tissues surrounding the implant, bleeding on probing with or without suppuration, and bone loss. Peri-implant lesions contain a leukocyte infiltrate of plasma cells, lymphocytes, macrophages and neutrophils. A survey of the literature did not show any studies reporting an association between hypoxia and peri-implantitis. The aim of the present cross-sectional study was to evaluate histological changes and immunostaining for CD15, CD57 and HIF-1α in the peri-implant mucosa of patients with and without peri-implantitis. Mucosal biopsies were obtained from 18 patients with peri-implantitis and 10 control subjects without peri-implantitis at a private health care center between 2010 and 2012. The sections were fixed in 10% buffered formalin, processed and embedded in paraffin for histopathological and immunohistochemical study. Acanthosis, spongiosis and exocytosis were observed in both groups, with no significant difference between them. The peri-implantitis group showed increased immunostaining for CD15, a neutrophil marker, and HIF-1α, a tissue hypoxia marker, but no significant difference in immunostaining for CD57, a Natural Killer cell marker. The increase in neutrophil (CD15) and hypoxia (HIF-1α) markers in patients with peri-implantitis suggests an active participation of neutrophils and hypoxia in the pathogenesis of this disease. Since the present study was the first to evaluate the expression of CD15, CD57 and HIF-1α in peri-implant tissues, further studies should be performed to better understand the role of these molecules in peri-implantitis., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Introduction: The aim of the work was to validate the CD15 focus score for the infection pathology of periprosthetic joint infection in a large group and to clarify whether a stratification into low-virulence and high-virulence microbial pathogens is possible by means of the CD15 focus score (quantification of CD15 positive granulocytes)., Methods: The histopathology of 275 synovial tissue samples taken intraoperatively during revision operations (n=127 hip, n=141 knee, n=2 shoulder, n=5 ankle) was evaluated according to the SLIM consensus classification (SLIM=synovial-like interface membrane). Neutrophilic granulocytes (NG) were quantified by the CD15 focus score on the basis of the principle of focal maximum infiltration (focus) with evaluation of one field of vision (about 0.3mm 2 ). The quantification values were compared with the microbiological diagnoses taking into consideration the virulence groups of low-virulence and high-virulence microbial pathogens and mixed infection., Results: The patients with positive microbiological findings (n=160) had significantly (p<0.001, Mann-Whitney U test) higher CD15 focus score values than patients with negative microbiological findings (n=115), the cut-off value being 39 cells per high power field (HPF). The CD15 focus score values of low-virulence microbial pathogens (n=94) were significantly lower (p<0.001, Mann-Whitney U test) than the values of high-virulence microbial pathogens (n=55), the cut-off value being 106 cells per HPF. Based on the microbiological diagnosis the sensitivity with respect to a microbial infection is 0.91, the specificity 0.92 (PPV=0.94; NPV=0.88; accuracy: 0.92; AUC=0.95). Based on the differentiation of the CD15 focus score values between low-virulence and high-virulence microbes the sensitivity is 0.70 and the specificity 0.77 (PPV=0.63; NPV=0.81; accuracy=0.74; AUC=0.74)., Conclusion: As a result of the high sensitivity and specificity, the easy to use CD15 focus score is a diagnostically valid score for microbial periprosthetic infection. A differentiation between low-virulence and high-virulence microorganism of sufficiently high diagnostic quality is additionally possible as a result of the defined quantification of CD15 positive granulocytes (the CD15 focus score) histopathological diagnosis of microbial infections is possible, which on the one hand supports the microbiological diagnosis and on the other hand by the stratification into low-virulence and high-virulence microbial pathogens could represent an additional basis for a pathogen-specific antibiotic treatment in the event of unclear constellations of findings., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80 %) and conventional papillary thyroid carcinoma (PTC) (60.7 %) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6 %) and follicular carcinoma (FCA) (32.1 %). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.

Background and Aims: CD15 is expressed by various cancer types; among these are intrahepatic and perihilar cholangiocarcinoma (CCA). The aim of this study was to elucidate CD15 expression in distal CCA as well as in dysplastic biliary tissue and to determine its prognostic significance., Methods and Results: Tissue samples from patients with intrahepatic (iCCA, n = 22), perihilar (pCCA, n = 7) and distal CCA (dCCA, n = 15), who underwent surgical resection in the period from 2010 to 2015 were evaluated for CD15 expression. Tissue of synchronous lymph node metastasis (n = 13), CCA-associated dysplasia (n = 20), dysplasia in intraductal biopsies (n = 10) and benign proliferations (n = 12), as well as inflammatory biliary lesions (n = 28) and non-inflammatory bile ducts (n = 23), were evaluated equally for CD15 expression. CD15 was found to be expressed highly in iCCA (81.8%), pCCA (85.7%), dCCA (73.3%), CCA-associated dysplasia (70.0%), dysplasia in intraductal biopsies (100%) and metastatic tissue (84.6%). CD15 expression was negative in 58 of 64 benign bile duct alterations resulting in an overall sensitivity and specificity of CD15 in CCA of 80.7 and 90.6% patients, respectively. CD15 expression was correlated significantly with a decreased overall survival in patients with CD15-positive CCA associated dysplasia (P = 0.003). However, CD15 expression in the invasive tumour component was not correlated with clinical outcome., Conclusion: CD15 is a sensitive and specific marker for intraepithelial and invasive neoplasias of the bile duct. Therefore, it can be helpful in the delineation of dysplastic and neoplastic biliary cells from non-neoplastic tissue, which frequently causes a diagnostic problem in indeterminate biliary stricture., (© 2016 John Wiley & Sons Ltd.)

The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (p<0.001). The percent of FUT8 staining in breast cancer tissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients.

Aims: The aim of the present study was to immunohistochemically investigate the expression and prognostic significance of putative cancer stem cell markers CD117 (c-kit), CD34, CD20 and CD15 in a cohort of patients with primary choroidal and ciliary body melanoma., Methods: The immunohistochemical expression of these markers was evaluated using 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 3-amino-9-ethylcarbazole (AEC) chromogens on paraffin-embedded tissue samples from 40 patients who underwent enucleation in the period from 1985 through 2000. Thirty-one patients had adequate tissue specimens for the analysis., Results: CD117 overexpression was observed in 12 of the 31 samples (39%) when AEC chromogen was used and in 14 of 26 (54%) samples when DAB was used. CD15 positivity was seen in three out of 30 (10%) samples with AEC and in six out of 26 (23%) samples with DAB. CD20 and CD34 exhibited no positivity in the tested samples. During the average follow-up time of 8.7 years (range 0.5-22 years), 17 patients (55%) died due to metastatic disease. The Kaplan-Meier plots showed a significantly shorter overall and disease-free survival in CD117-positive patients when the AEC chromogen was used. CD15 expression was not associated with patients' survival. In multivariate analysis, patients expressing the CD117 AEC had 4.13 times higher risk of lethal outcome in comparison with CD117 AEC negative patients., Conclusions: Our retrospective cohort study has for the first time demonstrated a small proportion of CD15-positive uveal melanomas. CD117 AEC overexpression was associated with a worse outcome in patients with choroidal and ciliary body melanoma. Further studies should confirm the validity of these observations and their potential for targeted treatment modalities., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Aim: To explore the relationship of clinicopathological features and the distribution of neutrophils in the tumor microenvironment with the prognosis of cholangiocarcinoma., Methods: Two hundred and fifty-four formalin-fixed and paraffin embedded tissue blocks were analyzed, including tissues from cholangiocarcinoma (n = 254), and tumor adjacent tissues (n = 238). Tissue sections were stained for CD15 using immunohistochemical staining. CD15 expression was detected to identify the distribution of neutrophils in the local tumor microenvironment. The neutrophil density of the tumor tissues and the adjacent tumor tissues was detected to reflect their inflammatory status. Clinical data and follow-up information of cholangiocarcinoma patients who underwent surgery from January 2004 to December 2010 were analyzed retrospectively. The relationship between clinicopathological features and the distribution of neutrophils with prognosis of the patients were analyzed., Results: The positive expression level of CD15 was only significantly related to the TNM stage. CD15 expression was higher in tumor tissues than in adjacent tissues (73.6% vs 54.6%), with significant differences. Patients with high expression of CD15 had significantly shorter overall survival (OS) than those with low expression of CD15 (median overall survival time 39.77 mo vs 16.87 mo, P = 0.008). Patients with high CD15 expression had significantly shorter disease free survival time (DFS) than those with low expression of CD15 (median DFS 38.27 mo vs 16.83 mo, P = 0.029). COX multivariate analysis indicated that high CD15 expression in tumor tissues was an independent risk factor for predicting OS for patients with cholangiocarcinoma [P = 0.012, relative risk (RR) = 1.601], but it was not an independent risk factor for predicting DFS (P = 0.073, RR = 1.462)., Conclusion: Patients with high CD15 expression in cancer tissues had shorter DFS and OS. High expression of CD15 is an independent risk factor for OS.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma entity. We performed a matched-pair analysis to evaluate the prognostic impact of several histopathological features in this distinct Hodgkin lymphoma subtype. Lymph node samples of NLPHL patients were tested for CD15, IgD, phosphorylated STAT6, ICOS and Epstein-Barr virus status of the malignant lymphocyte-predominant cells as well as epithelioid cell clusters and activated T cells in the microenvironment. None of these features was associated with a particular clinical outcome. However, patients presenting with epithelioid cell clusters showed a non-significant trend towards a lower relapse rate, justifying further evaluation of this marker., (© 2014 John Wiley & Sons Ltd.)

Recently, the presence of CEBPA mutation was identified as an important prognostic factor for normal karyotype (NK) acute myeloid leukemia (AML). Because AML with CEBPA mutation is closely associated with CD7, CD15, CD34, and HLA-DR expression, we investigated the prognostic implications of CD7+ CD15+ CD34+ HLA-DR + immunophenotype in NK-AML. We analyzed the immunophenotype of 329 patients with NK-AML from the Japan Adult Leukemia Study Group (JALSG) AML97 population. NK-AML with the CD7+ CD15+ CD34+ HLA-DR + immunophenotype was classified as the CEBPA type, and NK-AML that did not meet this criterion was considered as the non-CEBPA type. The influence of the CEBPA status on event-free survival (EFS) and overall survival (OS) was assessed using log-rank test and a multivariate Cox proportional hazard regression model. Furthermore, the surface antigen expression profile in AML according to the CEBPA mutation status (monoallelic or biallelic) was also investigated. Of the 329 NK-AML patients that were studied, 39 and 243 were classified as having CEBPA and non-CEBPA type NK-AML, respectively. Patients with CEBPA type NK-AML had significantly better EFS and OS than those with non-CEBPA type NK-AML. Multivariate analysis showed that the CEBPA type and white blood cell (WBC) counts of >20 × 10(9)/L were independent prognostic factors for EFS and OS. Moreover, NK-AML with the biallelic CEBPA mutation was more closely associated with CD34 positivity than that with the monoallelic CEBPA mutation. NK-AML with the CD7+ CD15+ CD34+ HLA-DR + immunophenotype is a clinically discrete entity, and this may have a possible role in risk stratification.

Background: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH., Methods: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages., Results: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples., Conclusions: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs., (© 2013 Clinical Cytometry Society.)

Iatrogenic immunodeficiency-associated lymphoproliferative disorders are rare. A small subset of these lesions resembles classical Hodgkin lymphoma (CHL), but there are few data in the literature about these lesions. We describe 10 patients with autoimmune diseases treated with immunomodulator therapeutic agents who developed CHL. The autoimmune diseases included rheumatoid arthritis (n=5), systemic lupus erythematosus (n=2), dermatomyositis (n=1), autoimmune hepatitis (n=1), and Crohn disease (n=1), and the immunomodulatory therapies were methotrexate, azathioprine, tumor necrosis factor-α inhibitors, and thalidomide alone or in various combinations. The study group included 9 women and 1 man with a median age of 50 years (range, 25 to 77 y). The histologic features supported CHL in all cases with Reed-Sternberg (RS) and Hodgkin (H) cells in an inflammatory cell background, although the neoplasm could only be subclassified in 3 patients: 2 nodular sclerosis and 1 mixed cellularity. Immunohistochemical analysis supported the diagnosis of CHL. In all cases the RS-H cells were CD30. Nine of 10 cases were CD15, whereas CD20 was expressed variably in 4/10 cases. CD45/LCA was negative in 8 cases assessed. In situ hybridization for Epstein-Barr virus-encoded RNA was positive in the RS-H cells in 8/10 cases. The microenvironment of these lesions depicted a predominance of T-regulatory cells and M2 histiocytes. Clinical follow-up data were available for 7 patients, with a median posttreatment period of 27 months (range, 12 mo to 7 y). In all 7 patients immunomodulatory drug therapy was discontinued, and chemotherapy for CHL was administered; 2 patients also received local radiation. All 7 patients achieved complete remission and are alive. We conclude that iatrogenic immunodeficiency-associated CHL is highly associated with Epstein-Barr virus infection, and patients usually have a good outcome after discontinuation of immunomodulatory agents and chemotherapy for CHL.

Lymphomas account for less than 5% of thyroid malignant lesions. Vast majority of them are B-cell non-Hodgkin lymphomas (NHL), while Hodgkin lymphoma (HL) is extremely rare. Here we present two cases of HL, at baseline manifesting as a thyroid lesion. First patient, 29-year-old pregnant female, initially suspected for metastatic medullary thyroid cancer, was eventually diagnosed with mixed cellularity type of thyroid HL. Second patient, 22-year-old woman with suspicion of advanced thyroid cancer, was in the end diagnosed with an extra-lymphatic classical HL of the thyroid. In both cases, despite repeated fine-needle aspiration biopsy, cytological examination gave inconclusive or misleading results. On histopathological examination, thyroid tumor cells were positive for CD15 and CD30 antigen, which is typical for Reed-Sternberg cells. In the report authors also discuss difficulties in management as well as potential importance of novel methods such as FISH, PCR and other molecular techniques in diagnostics of thyroid lymphomas., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2896947559559648.

Since TH17 cells could play a role in the pathogenesis of allograft nephropathy, we investigated them in peripheral blood and kidney allograft infiltrates. We compared percentages of TH17 cells and IL17A in peripheral blood of 14 kidney allograft recipients and 8 healthy volunteers. Allograft recipients experiencing graft dysfunction and kidney biopsy specimens showing chronic allograft nephropathy (CAN) were distinguished from a "control group," both of which were tested for TH17 and CD15+ staining. Allograft recipients displayed a significantly lower percentage of TH17 cells and IL17A blood levels than healthy volunteers, suggesting effects of the immunosuppressive regimen. No difference in these values was observed between the CAN group and the control group. On kidney allograft biopsies, CD15+ infiltrate was significantly higher in the CAN group than in the control group. In CAN, IL17 secretion might play a chemoattractant role for neutrophils. However, these preliminary data have to be confirmed in larger studies., (Copyright © 2012. Published by Elsevier Inc.)

Acute promyelocytic leukaemia (APL) is a unique clinicobiologic entity that can be successfully treated with All-trans Retinoic Acid ATRA-based regimens. Some cases of acute myeloid leukaemia (AML) with nucleophosmin (NPM) mutations have an immunophenotype that is similar to APL. The objective of the study is to compare antigenic expression in a group of APL patients with that in AML patients with NPM mutations and an APL-like immunophenotype (CD15- CD34- HLA-DR-). A consecutive series of 40 APL and 12 NPM patients with an APL-like phenotype were included in the study. Immunophenotypic patterns were investigated by multiparametric flow cytometry. Promyelocytic leukaemia-retinoic acid receptor-α transcript type, NPM and FLT3 mutations were investigated using conventional methods. Statistically significant differences were found between APL and NPM-mutated AML in CD33, CD13, CD2 and CD110 reactivity. CD2 expression was absent in every patient with NPM-mutated AML. In addition, mean fluorescence intensity and the coefficient of variation (cv) of CD33 and CD13 showed statistical differences between the two groups for CD33 (p = 0.007) and a trend to significance for CD13 (p = 0.05). Furthermore, among 45 evaluable patients, CD110 expression statistically differentiates between the two groups: [2/33 (6%) in the APL group and 8/12 (66.6%) in the NPM-mutated AML (p = 0.014)]. However, these traits were subtle, raising the possibility of practical diagnostic challenges. In conclusion, CD110 and CD33 reactivity may be useful to distinguish APL from NPM-mutated AML with CD15, CD34 and HLA-DR negativity. Nevertheless, cytogenetic and molecular characterization is necessary to establish the accurate diagnosis of AML., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: The aim of this work was to evaluate the expression of FUT2 gene in saliva and histo ABH antigens of patients with oral lesions., Study Design: In total 178 subjects were examined, half of whom suffered from oral pre-cancerous and cancerous lesions, while the other half were the healthy control group We analyzed the FUT 2 polymorphism by ASO-PCR (allele specific oligonucleotid - polymerase chain reaction) with specific primers for G428 allele and the wild type allele of FUT2 gene. To reveal A, B and H antigens in tissue sections of the patients (n= 89) we used a modified specific red cell adherence technique., Results: We found a high intensity of oral disease in the non-secretor group (OR = 2.43). A total of 58% of the patients with oral pre-cancerous and cancerous lesions was non secretors (se&#95;/&#95;), in contrast with the healthy population (21.5%). A strongly positive reaction was defined as a sheet of indicator erythrocytes adhered to the epithelial cells. In 31 of the 54 samples analyzed the test showed slightly positive results on atypical areas, and there was a complete antigen deletion in areas affected by neoplasia. Nineteen samples showed a total absence of ABH antigens in both histologically normal and pathological areas. Blood group antigens were expressed at a high level in benign and highly differentiated malignant tumors. In poorly differentiated malignant tumors, they were mostly absent., Conclusion: Considering these results we suggest the use of this method to monitor probable preneoplastic lesions in risk population, especially in those with no secretor status (absence of FUT2 gene).

Glioblastoma multiforme (GBM), as many other solid tumours, contains a subpopulation of cells termed cancer stem-like cells responsible for the initiation and propagation of tumour growth. However, a unique immunophenotype/surface antigen composition for the clear identification of brain tumour stem cells (BTSC) has not yet been found. Here we report a novel code of cell surface markers for the identification of different cell subpopulations in neurospheres derived from a GBM with a primitive neuroectodermal tumour (PNET)-like component (GBM-PNET). These subgroups differ in their CD133/CD15 expression pattern and resemble cells with different stem-like genotype and developmental pathway activation levels. Strikingly, clonogenic analysis of cultures differentially expressing the investigated markers enabled the identification of distinct subpopulations of cells endowed with stem cell characteristics. High clonogenicity could be found in CD133(-)/CD15(-) and CD133(+)/CD15(+) but not in CD133(-)/CD15(+) cells. Moreover, cell subpopulations with pronounced clonogenic growth were characterized by high expression of stem cell-related genes. Interestingly, these observations were unique for GBM-PNET and differed from ordinary GBM cultures derived from tumours lacking a PNET component. This work elucidates the complex molecular heterogeneity of in vitro propagated glioblastoma-derived cells and potentially contributes to the development of novel diagnostic modalities aiming at the identification of the brain tumour stem-like cell population in a subgroup of GBMs.

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer's patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Introduction: This study aims to investigate the evidence of neutrophil infiltration in renal tissue from patients with antineutrophil cytoplasmic antibodies (ANCA)-negative pauci-immune crescentic glomerulonephritis (CrGN), and a comparison with their ANCA-positive counterparts was performed., Methods: Renal biopsy specimens from 31 patients with pauci-immune CrGN were collected. Twelve patients were ANCA negative, and 19 patients were ANCA positive. Neutrophil infiltration was investigated by staining of 2 markers, CD15 and myeloperoxidase (MPO), using immunohistochemistry., Results: Positive-stained cells of CD15 (CD15) and MPO (MPO+) were mainly located in glomeruli with cellular crescents and segmental fibrinoid necrotic lesions and in periglomerular area of cellular crescents, especially at the area of ruptured Bowman capsule. Scanty positive staining was found in normal renal tissue. The number of CD15 cells in the glomeruli and periglomerular area was significantly higher in ANCA-negative group than in ANCA-positive group (in glomeruli: 2.70 ± 1.61 versus 1.38 ± 0.85, P = 0.019; in periglomerular area: 4.35 ± 4.36 versus 1.48 ± 1.67, P = 0.047). The number of MPO+ cells in the periglomerular area was also significantly higher in ANCA-negative group than that in ANCA-positive group., Conclusions: Renal neutrophil infiltration might play a pathogenic role in ANCA-negative pauci-immune CrGN, and the neutrophil infiltration might be more severe in ANCA-negative pauci-immune-CrGN than in ANCA-positive one.

Purpose: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non-small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed, paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials., Experimental Design: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines, normal tissue, and tumor tissue., Results: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues, suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004, respectively) and drozitumab (P = 0.03 and P < 0.0001, respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely, with approximately 45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125)., Conclusions: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection.

Granulocytic sarcoma is an uncommon tumor composed of myeloid blasts and/or immature myeloid cells in an extramedullary site which is usually associated with acute or chronic myeloid leukemia. The tumor may also be the initial manifestation of leukemia. The histomorphological diagnosis of granulocytic sarcoma can be challenging to pathologists, especially in the absence of a known hematological disorder. In this case, differentiation of granulocytic sarcoma from malignant lymphomas and other small round cell tumors is very critical. Seven cases of granulocytic sarcoma are reported in this paper. One patient had granulocytic sarcomas at two different sites. Hematoxylin-eosin-stained sections were reexamined. Blastic, poorly differentiated, and well differentiated histopathological variants were found in two, five and one cases, respectively. Immunohistochemical studies were performed on formalin-fixed tissue from all cases using a avidin-biotin-peroxidase complex technique. The panel included antibodies against LCA, CD43, CD34, c-kit, myeloperoxidase, CD68 KP1, CD15, and CD99. All cases stained positively with LCA, CD43, CD34, myeloperoxidase, and CD68. Five cases were positive for c-kit, three cases were positive for CD15, and two cases were positive for CD99. An immunohistochemical panel including at least myeloperoxidase, CD68 and CD34 can be used for detection of myeloid differentiation. It is also important that granulocytic sarcoma be considered in the differential diagnosis of CD99-positive round cell tumors.

It has been suggested that selectin ligands expressed by the endometrial epithelium are essential for the initial adhesion of the blastocyst to the luminal epithelium of human endometrium. One of the enzymes responsible for the production of selectin ligands is fucosyltransferase 4 (FUT4), a member of alpha1,3 fucosyltransferases. The aims of the present study were to characterize FUT4 mRNA and protein in human endometrium during the menstrual cycle and to investigate the hormonal regulation of FUT4 whose mRNA expression was quantified by real-time PCR in fresh endometrial tissue from cycling women and protein expression was analyzed by immunohistochemistry and Western blotting. Hormonal regulation of FUT4 transcription was investigated using an endometrial explant system. FUT4 mRNA was significantly upregulated in fresh tissues during early and mid-secretory phases when compared with other phases of the menstrual cycle. FUT4 protein was localized to glandular and luminal epithelium and the expression levels followed the same pattern as for FUT4 mRNA. Our data also show that, in proliferative explants, progesterone significantly increased FUT4 transcription and translation after 24 h in culture. The inductive effect of progesterone on FUT4 transcription was lost after 48 h of treatment. Estrogen did not have any significant effects. These data suggest that the upregulation of selectin ligands in the human endometrium at the time of implantation may be mediated, at least in part, by the regulation of FUT4 expression.

O-Fucosylation is a post-translational glycosylation in which an O-fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X(4-5)(S/T)C3 present on epidermal growth factor-like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O-fucose to epidermal growth factor-like repeats is protein O-fucosyltransferase 1. Protein O-fucosyltransferase 1-mediated O-fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O-fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N-glycans. By comparing protein O-fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O-fucosyltransferase 1 enzymes possess two putative N-glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N-glycans on protein O-fucosyltransferase 1, we substituted, by site-directed mutagenesis, bovine protein O-fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N-glycan on N163 caused a slight decrease in protein O-fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O-fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O-fucosyltransferase 1, suggesting that N-glycosylation at this site is essential for proper folding of the enzyme.

Soluble ABO blood group substance (SAS) in fresh-frozen plasma (FFP) and its cognate alloantibody titer reduction capacity (TRC) are not considered when prescribing this product for plasma exchange (PEX) therapy of ABO incompatible transplant recipients. SAS was quantified in 250 single FFPs using ELISA. Total and IgG class-specific anti-A TRCs of FFPs were measured using a microhemagglutination inhibition assay. SAS level depended not only on the A subtype (p < 0.0001) and the Secretor status (p < 0.0001), but also on the expression of ALe(b) in A1 secretors (p < 0.0001). The variation was as great as 137.6 arbitrary units (aU) for 14 A1 Le(a-b-) secretors and 1.2 aU for 6 A2 non-secretors. Homozygous expression of the A1, A2 and Secretor alleles did not increase SAS levels. Only total anti-A TRC, but not IgG class-specific TRC depended on the detected SAS level (r = 0.566, p = 0.0003).

LADII (leukocyte adhesion deficiency type II)/CDGIIc (congenital disorder of glycosylation type IIc) is a rare autosomal recessive disease characterized by leukocyte adhesion deficiency as well as severe neurological and developmental abnormalities. It is caused by mutations in the Golgi GDP-fucose transporter, resulting in a reduction of fucosylated antigens on the cell surface. A recent study using fibroblasts from LADII/CDGIIc patients suggested that although terminal fucosylation of N-glycans is reduced severely, protein O-fucosylation is generally unaffected (Sturla, L., Rampal, R., Haltiwanger, R. S., Fruscione, F., Etzioni, A., and Tonetti, M. (2003) J. Biol. Chem. 278, 26727-26733). A potential explanation for this phenomenon is that enzymes adding O-fucose to proteins localize to cell organelles other than the Golgi apparatus. In this study, we investigated the subcellular localization of protein O-fucosyltransferase 1 (O-FucT-1), which is responsible for adding O-fucose to epidermal growth factor-like repeats. Our analysis reveals that, unlike all other known fucosyltransferases, O-FucT-1 is a soluble protein that localizes to the endoplasmic reticulum (ER). In addition, it appears that O-FucT-1 is retained in the ER by a KDEL-like sequence at its C terminus. Our results also suggest that enzymatic addition of O-fucose to proteins occurs in the ER, suggesting that a novel, ER-localized GDP-fucose transporter may exist. The fact that O-FucT-1 recognizes properly folded epidermal growth factor-like repeats, together with this unique localization, suggests that it may play a role in quality control.

Transfection of the mouse Fut1 and Fut2, and human FUT1 genes into human ovarian carcinoma-derived RMG-1 cells resulted in 20-30-fold increases in cellular alpha1,2-fucosyltransferase activity, and in alteration of the glycolipid composition, including not only fucosylated products, but also precursor glycolipids. Although globo-series glycolipids were not significantly affected by the transfection, the major glycolipids belonging to the lacto-series type 1 chain family in RMG-1 cells and the transfectants were the Lc4Cer, Lewis a (Le)a and Leb, and H-1 glycolipids, respectively, suggesting that fucosylation of Lc4Cer to the H-1 glycolipid prevents the further modification of Lc4Cer to Lea and Leb in the transfectants. Also, the lacto-series type 2 chains in RMG-1 cells were LeX, NeuAc-nLc4Cer and NeuAc-LeX, and those in the transfectants were LeX and LeY, indicating that the sialylation of nLc4Cer and LeX is restricted by increased fucosylation of LeX. As a result, the amount of sialic acid released by sialidase from the transfectants decreased to 70% of that from RMG-1 cells, and several membrane-mediated phenomena, such as the cell-to-cell interaction between cancer cells and mesothelial cells, and the cell viability in the presence of an anticancer drug, 5-fluorouracil, for the transfectants was found to be increased in comparison to that for RMG-1 cells. These findings indicate that cell surface carbohydrates are involved in the biological properties, including cell-to-cell adhesion and drug resistance, of cancer cells.

The recent draft sequencing of the rice (Oryza sativa) genome has enabled a genetic analysis of the glycosylation capabilities of an agroeconomically important group of plants, the monocotyledons. In this study, we have not only identified genes putatively encoding enzymes involved in N-glycosylation, but have examined by MALDI-TOF MS the structures of the N-glycans of rice and other monocotyledons (maize, wheat and dates; Zea mays, Triticum aestivum and Phoenix dactylifera); these data show that within the plant kingdom the types of N-glycans found are very similar between monocotyledons, dicotyledons and gymnosperms. Subsequently, we constructed expression vectors for the key enzymes forming plant-typical structures in rice, N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), core alpha1,3-fucosyltransferase (FucTA; EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) and successfully expressed them in Pichia pastoris. Rice GlcNAc-TI, FucTA and xylosyltransferase are therefore the first monocotyledon glycosyltransferases involved in N-glycan biosynthesis to be characterised in a recombinant form.

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).

Objectives: To evaluate the relationship of Lewis genotypes with major cardiovascular risk factors and the intima-media thickness (IMT) of carotid arteries. Lewis genotyping included four major mutations of the Lewis (FUT3) gene at nucleotide positions 59, 1067, 202 and 314., Design: Two complementary population-based cross-sectional studies., Setting: The Atherosclerosis Risk in Communities (ARIC) Study., Subjects: The relationship between Lewis genotype and major cardiovascular risk factors was studied in 761 men and women aged 45-64 years without known clinical atherosclerotic disease; 577 were Caucasians and 184 were African-Americans. The association of Lewis genotype and subclinical carotid atherosclerosis was studied in 419 individuals with, and 819 controls without carotid IMT of >1.0 mm, measured by B-mode ultrasound., Main Outcome Measures: Mean values of cardiovascular risk factors by Lewis genotype. Lewis genotype frequencies in subclinical carotid atherosclerosis cases and controls., Results: Individuals with Lewis genotypes consistent with lack of alpha(1,3/1,4)-fucosyltransferase activity (i.e. Lewis-negative genotype) had statistically significantly lower fasting glucose, factor VIIIc, von Willebrand factor and diastolic blood pressure compared with their counterparts with Lewis-positive genotypes. The distribution of Lewis genotypes and haplotypes was not significantly different between individuals with carotid IMT of >1.0 mm (cases) and their controls. The odds of carotid atherosclerosis in carriers of the Lewis-negative genotype was 1.23 (95% confidence interval 0.70-2.16) compared to individuals with Lewis-positive genotype, controlling for age, gender and race/ARIC field centre., Conclusion: The lack of a statistically significant association between Lewis 'genotype' and subclinical atherosclerosis in our data suggests that earlier studies reporting associations at the 'phenotypic' level may reflect aspects of the biology of the Lewis system other than an inherent genetic property.

This paper describes the setup and the use of a flow cytometric method for monitoring Sf9 insect cell infection by a recombinant baculovirus expressing the human alpha1,3/4 fucosyltransferase Fuc-TIII. Using side scattered light coupled to green fluorescence detection after immunolabeling of the recombinant protein, this method made it possible to monitor baculovirus infection of Sf9 cells grown in batch cultures and infected at different cell densities and multiplicities of infection. The method was able to precisely assess the extent of infection of the insect cells from 60 h postinfection. In asynchronously infected Sf9 cell cultures, the two-step infection process (primary and secondary infection) was well-characterized using this technique. Finally, a reduced sensitivity to baculovirus infection was observed for cells infected at the end of the growth phase compared to the cells infected during exponential growth phase.

To elucidate the clinical significance of phenotypic alterations of Lewis antigen in gastric cancer patients, we investigated Lewis antigens by analyzing the genotypes of the Le and Se genes and by comparing the results obtained with the phenotypic expression of Lewis antigen in gastric cancer tissue and blood cells. One hundred and twenty gastric cancer patients were examined and compared with respect to Lewis blood phenotype and genotype. The expression of Le(a), Le(b), sialylated Le(a), and sialylated Le(x) antigens was immunohistochemically examined in uninvolved gastric mucosa, intestinal metaplasia, and cancerous tissue. We also analyzed the significance of Lewis antigen expression by analyzing patient survival. The frequencies of the Lewis phenotypes of RBCs corresponding to Le(a+b-), Le(a-b+), and Le(a-b-) were 16%, 58%, and 26%, respectively. The Le and le allele gene frequencies calculated from genotyping in gastric cancer patients were 0.623 and 0.377, respectively. The frequency for Le(a-b-) of the RBC phenotype had a tendency to be higher in cancer patients than in normal healthy Koreans. However, no difference in the Lewis gene frequency was found between these gastric cancer patients and healthy persons. The phenotype of Le(a-b+) was most prevalent in uninvolved gastric mucosal tissue, whereas the most prevalent form in tumor tissue was Le(a-b-). Sialyl-Le(a) and sialyl-Le(x) antigens were hardly detectable in uninvolved gastric mucosa, whereas the two antigens were expressed highly in intestinal metaplastic mucosa and tumor cells. In conclusion, the loss of Lewis antigen expression in tissue and on RBCs in gastric cancer patients is not a result of genetic influences, but rather a result of sialylation in tissue. We also confirm that poor prognosis is associated with dimeric sialyl-Le(x) and vascular spread.

Background: Two types of transitional bladder carcinoma have been distinguished based on macroscopic morphology: type A papillary carcinomas, with papillomatous surface outgrowth without infiltration into muscular layer, and type B nodular carcinomas, with a nonpapillomatous surface appearance, most of which display infiltrative growth through muscular layer, and some of which display lymphatic or blood-borne metastasis. However, there is no specific predictor at early stages for later invasive and metastatic clinical outcome of patients with type B tumors., Methods: The study included 1) glycosphingolipid (GSL) composition of type A and B tumors; 2) histologic and immunohistologic patterns of nodular (type B) bladder carcinoma from 44 patients based on a special sampling procedure termed whole-layer core biopsy (WLCB) using the antisialosyl-Le(x) (anti-SLe(x); SLe(x): NeuAcalpha3Galbeta4[Fucalpha3]GlcNAcbeta3Galbeta4GlcCer) SNH3 antibody or other antibodies; 3) comparison of the incidence of metastasis in patients with SNH3 positive versus SNH3 negative primary tumors and of 5-year survival curves; 4) comparison of bladder carcinoma cell lines from tumors with high versus low malignancy in terms of expression patterns of SLe(x), SLe(a), and other carbohydrates, E-selectin dependent adhesion, and transcript levels of five fucosyltransferases., Results: Anti-SLe(x) monoclonal antibody (mAb) SNH3 staining of WLCB samples from 44 type B tumors showed that the majority of tumors (n = 31 patients) were SNH3 positive and the minority (n = 13 patients) were SNH3 negative. SNH3 positive patients had more lymph node or blood-borne metastasis and lower 5-year and 7-year survival rates, as indicated by Kaplan-Meier curves (P = 0.001). Staining of samples with other antibodies, including FH6 and CA19-9, was not correlated with long-term survival. Determination of GSL composition in extracts showed that SLe(x) ganglioside was present in all three patients with nodular tumors but absent in all three patients with papillary tumors tested. Bladder carcinoma cell lines from invasive tumors that maintained their metastatic properties were SNH3 positive, showed high levels of alpha1,3-fucosyltransferase VI (FT-VI) and FT-VII, and displayed E-selectin dependent adhesion. Cell lines from noninvasive tumors or normal bladder epithelia were negative for SNH3 reactivity, FT-VI, and FT-VII, and E-selectin dependent adhesion., Conclusions: SLe(x) expression in primary bladder carcinoma, defined by the mAb SNH3, is a predictor of invasive and metastatic outcome. No other carbohydrate epitope examined to date has equal prognostic value., (Copyright 2002 American Cancer Society. DOI 10.1002/cncr.10268)

Although expression of cutaneous lymphocyte-associated antigen (CLA) is thought to be a specific marker of skin "homing" T cells, it has become clear that CLA is a good marker for high levels of fucosyltransferase VII (Fuc-TVII) activity but does not necessarily represent the epitope required for binding to E-selectin (Wagers et al, 1996, 1997). Therefore, expression of Fuc-TVII is an attractive candidate for identifying skin "homing" T cells. However, analyses of Fuc-TVII expression in human T cells have been performed only at the mRNA level (Nakayama et al, 2000) because of the lack of mAB: In this study Fuc-TVII was for the first time visualized in individual cells by using a novel mAb that we developed. Double immunofluorescence and immunohistochemistry demonstrated the coexpression of Fuc-TVII and CLA in cell lines in which Fuc-TVII mRNA was shown to be expressed at high levels: whereas CLA expression was seen in the cell membrane, Fuc-TVII was identified in a supra- or perinuclear location. Cytoplasmic Fuc-TVII expression was also detectable in both CD4(+) and CD8(+) T cells purified from peripheral blood. Fuc-TVII was also expressed at high levels in many CLA(+) T cells infiltrating the skin. In these peripheral T cells, unlike in cell lines, cytoplasmic expression of Fuc-TVII was not always associated with surface CLA expression. This mAb would serve as a valuable tool for selectively identifying a novel subset of skin "homing" T cells that are not detected by the conventional method because of the lack of CLA expression.

Most in vitro investigations of endothelial cell function have been based on the behaviour of human umbilical vein endothelial cells (HUVEC) in primary tissue culture. However, it is becoming apparent that there is a marked degree of heterogeneity among endothelial cells derived from different vascular beds. We have studied primary HUVEC from 45 umbilical cords. Contrary to previously published reports, we were unable to detect ABO antigens on the surface of cultured HUVEC from group A or B cords. This was not due to an absence of precursor H substance which was uniformly expressed on HUVEC in primary tissue culture. Further investigation revealed an absence of A enzyme activity in A type HUVEC and a level of mRNA only just detectable by RT-PCR. The absence of functional A enzyme activity was confirmed by demonstrating the absence of A substance on von Willebrand factor secreted by A type cells. HUVEC appear to be the only vascular endothelial cells that do not express ABO blood group antigens. We speculate that this may help protect the cord from maternal antibodies during gestation. The lack of ABO blood group antigens on HUVEC may significantly affect their surface function, and therefore care should be taken when extrapolating conclusions from results obtained with these cells (the most widely used human endothelial cell model in vitro) to the properties of adult endothelial cells., (Copyright 2000 S. Karger AG, Basel)

Transgenic animals are a promising source of pharmaceutically-relevant proteins or as a source of organs for xenotransplantation. Beside other posttranslational modifications, glycosylation has been shown to be a critical parameter for the correct function of several glycoproteins. To analyse the contribution of alpha 1,6-fucosylation to N-glycan variability, we partly purified alpha 1,6-fucosyltransferase (alpha 1,6-Fuc-T) activities from various tissues (brain, lung, heart, liver) of agriculturally-relevant animals (porcine, sheep, bovine, rabbit, chicken) and compared some of their biochemical properties. All tissues displayed alpha1,6-Fuc-T activity, although at different levels. No differences were observed in their stability against chemicals, temperature or time, whereas the activities were distinguishable by their pH-optima and their cation preferences. Similarities were found for tissues between species. Lung and heart enzymes showed a narrow pH-optimum around pH 6.0 and an enhanced activity in the presence of divalent cations. alpha 1,6-Fuc-T activities in brain and liver were characterised by a broad pH-optimum from 5.5 to 8.0. Some activities of these tissues were decreased by the addition of EDTA, while others did not show any influence of EDTA or divalent cations. From the significant differences of the alpha 1,6-Fuc-T activities in the tissues, it is possible to hypothesise the presence of more than one single alpha 1, 6-Fuc-T in mammalian tissues.

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked alpha1-->3 to N-acetylglucosamine. A family of alpha1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription-PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with beta4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel-Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel-Palade bodies suggests a function independent of Golgi-associated glycosylation.

The amino acid sequence of Fuc-TIX is very highly conserved between mouse and human. The number of non-synonymous nucleotide substitutions of the Fuc-TIX gene between human and mouse was strikingly low, and almost equivalent to that of the alpha-actin gene. This indicates that Fuc-TIX is under a strong selective pressure of preservation during evolution. The human Fuc-TIX (hFuc-TIX) showed a unique characteristics, i.e. hFuc-TIX was not activated by Mn2+ and Co2+, whereas hFuc-TIV and hFuc-TVI were activated by the cations. The hFuc-TIX transcripts were abundantly expressed in brain and stomach, and interestingly were detected in spleen and peripheral blood leukocytes.

Mammalian cells often contain an enzyme which transfers fucose onto the reducing terminal GlcNAc (GlcNAc-1) of N-glycans with an alpha1,6-linkage. In plants, on the other hand, the fucose is transferred to GlcNAc-1 with an alpha1,3-inkage. Insect cells can exhibit both enzymatic activities. Hitherto, the activity of these fucosyltransferases has been determined by the incorporation of radioactively labelled fucose into an acceptor glycopeptide. This assay, however, cannot discriminate these two activities. Here we report on the use of dansylated glycoasparagine for the specific determination of 1,3- and 1,6-fucosyltransferases. The two possible products and the substrate are separated on a reversed phase column and detected by fluorescence.

This paper reports for the first time the presence of the human Lewis(a) type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L. by HPLC/ESI-MS. The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis. The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewis(a) [structure in text]. A corresponding fucosyltransferase activity catalyzing the formation of Lewis(a) type structures in vitro was identified in cellular extracts of the suspension cultures.

A novel bioluminescence-based solid-phase assay is described for the enzyme GDPFuc:Gal(beta)1-3GlcNAc (Fuc to GlcNAc) alpha1,4-fucosyltransferase (alpha1,4FT), which generates the Lewis a blood group antigen (Le(a)) (Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc-R). Lacto-N-tetraose (LNT,Ga ta)1-3GlcNAc(beta)1-3Gal(beta)1-4Glc) was chemically conjugated to bovine serum albumin (BSA) to generate the acceptor neoglycoprotein LNT-BSA. The Le(a) product of the reaction made in the presence of the donor GDPFuc is detected with a primary monoclonal IgG antibody to Le(a) and a secondary antibody coupled to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Recombinant human GDPFuc:Gal(beta)1-3(4)GlcNAc (Fuc to GlcNAc) alpha1,4/alpha1,3-fucosyltransferase, which exhibits alpha1,4FT activity, was used to optimize the assay. With this assay alpha1,4FT activity is measurable in human serum, in human saliva, and in extracts of the human colon carcinoma cell line SW1116. Activity is absent, however, in extracts of human HL-60 and murine F9 cells, neither of which synthesize Le(a) antigen. Among 10 human donors tested, soluble alpha1,4FT activity, was measurable in serum and saliva of some, but not all donors. However, the presence of enzyme activity in sera does not correlate with Lewis blood group phenotype of erythrocytes. The saliva of one donor, which contained Le(a) antigens, exhibited no alpha1,4FT activity. That saliva was found to contain a heat-stable factor(s) capable of inhibiting the alpha1,4FT activity when mixed with donor saliva containing alpha1,4FT activity. This new assay should be useful in assessing the Lewis enzyme activity in body fluids and its relationship to the Lewis blood group status on cells and secreted glycoconjugates in normal and diseased states.

A fucosyltransferase activity has been detected using lacto-N-biose I as acceptor in the lower eukaryote Dictyostelium discoideum. This transferase requires divalent cations and is inhibited by N-ethylmaleimide and detergent treatment. Apparent calculated Km values for GDP-Fuc and lacto-N-biose I are 1.27 microM and 2.80 mM, respectively. The activity is quantitatively recovered in the supernatant after centrifugation at 100000 x g for 1 h. The reaction product, as determined by gel permeation chromatography, sensitivity to fucosidases, and analysis of partially methylated derivatives, is Fucalpha1-2Galbeta1-3GlcNAc (H type 1 trisaccharide).

An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.

Several pathogenic agents of pediatric gastroenteritis are inhibited by fucosylated oligosaccharides of human milk. Biosynthesis and degradation of fucosyloligosaccharides is controlled by fucosyltransferase and alpha-L-fucosidase. The activity of these enzymes varies reciprocally over the course of lactation. We hypothesized that differences in the specific organization of these enzymes in compartments of human milk might contribute to such differences in activity. Therefore, the distribution of these enzymes in various compartments of human milk was investigated. After ultracentrifugation at 120,000g for 2h, the fucosyltransferase activity distributes evenly between the supernatant and the membranous pellet. Ultracentrifugation at 180,000g for 17 h further fractionated the milk into a clear supernatant, a fluff layer from the supernatant, and a pellet. The fluff was visualized by electron microscopy. The distribution of fucosyltransferase activity in colostrum was compared with that of mature milk from the same donor. In mature milk from Day 30 of lactation, most fucosyltransferase activity was in the membranous fluff fraction, while in colostrum from Day 5 of lactation, most of the fucosyltransferase activity was in the supernatant. In contrast to fucosyltransferase, fucosidase activity was found only in the soluble milk fraction; upon prolonged ultracentrifugation, most of this was membrane associated. The nature of human milk fucosidase was studied. This enzyme is glycosylated and exhibits several characteristics common to other fucosidases. Under the conditions found in human milk, it exhibits almost full activity. The variation in compartmentalization of fucosyltransferase activity during lactation may reflect variations in metabolism of the fucosyloligosaccharides of human milk that protect against disease.