Lectin Reactivity Research Articles

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease.

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.

Aberrant Glycosylation in Pancreatic Ductal Adenocarcinoma 3D Organoids Is Mediated by KRAS Mutations.

Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells, to gasp the comprehensive cancer cell surface glycan expression profile using lectin microarray and transcriptomic analyses. Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. We successfully established 16 3D organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS was mutated in 13 (7 G12V, 5 G12D, 1 Q61L) and wild in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Lectin reactivity of AAL (Aleuria aurantia) and AOL (Aspergillus oryzae) with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. Meanwhile, mannose-binding lectin (rRSL [Ralstonia solanacearum] and rBC2LA [Burkholderia cenocepacia]) signals were higher while those of galactose-binding lectins (rGal3C and rCGL2) were lower in the KRAS mutants. We demonstrated here that PDAC 3D-cultured organoids with KRAS mutations were dominantly covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers.

Abstract B099: Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations

Abstract Introduction: Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. Moreover, as glycans constitute the outermost layer of cells, diverse alterations during carcinogenesis affect the multiple processes of invasion and metastasis, making glycosylation a keystone mechanism in cancer progression. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells for comprehensive cell surface glycan expression profile using lectin microarray and transcriptomic analyses and then analyzed the relationship between altered glycans and glycan-related genes, with a particular focus on the status of KRAS mutations. Materials and methods: Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system containing extracellular matrix and specific niche factors. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. Results: We successfully established 16 3D-organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS mutations (7 G12V, 5 G12D, 1 Q61L) were found in 13 organoids, while wild-type KRAS without mutations was detected in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Pairings of a PDAC organoid with a non-PDAC counterpart were available from 2 patients; one was a PDAC-normal MPD pair and the other was PDAC-IPMN pair, eliminating the influence of individual variations in glycan expression status for those pairs. Lectin reactivity of AAL and AOL with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. And also, in the two cases with PDAC and non-PDAC organoids pairs, the reactivity of AAL were significantly elevated in the KRAS mutants, with FUT6 and FUT3 elevated. Conclusion: PDAC 3D-cultured organoids with KRAS mutations were covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers. Citation Format: Hiromitsu Nakahashi, Tatsuya Oda, Osamu Shimomura, Yoshimasa Akashi, Kazuhiro Takahashi, Yoshihiro Miyazaki, Tomoaki Furuta, Yukihito Kuroda, Pakavarin Louphrasitthiphol, Bryan J. Mathis, Hiroaki Tateno. Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B099.

Reduction in disialyl-T antigen levels in mice deficient for both St6galnac3 and St6galnac4 results in blood filling of lymph nodes

Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galβ1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis.

ABO Genotyping finds more A2 to B kidney transplant opportunities than lectin-based subtyping

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A1 (eg, A2) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity.

Profiling the glycome of Cardicola forsteri, a blood fluke parasitic to bluefin tuna

Infections by blood flukes (Cardicola spp.) are considered the most significant health issue for ranched bluefin tuna, a major aquaculture industry in Japan and Australia. The host–parasite interfaces of trematodes, namely their teguments, are particularly rich in carbohydrates, which function both in evasion and modulation of the host immune system, while some are primary antigenic targets. In this study, histochemistry and mass spectrometry techniques were used to profile the glycans of Cardicola forsteri. Fluorescent lectin staining of adult flukes indicates the presence of oligomannose (Concanavalin A-reactive) and fucosylated (Pisum sativum agglutinin-reactive) N-glycans. Additionally, reactivity of succinylated wheat germ agglutinin (s-WGA) was localised to several internal organs of the digestive and monoecious reproductive systems. Glycan structures were further investigated with tandem mass spectrometry, which revealed structures indicated by lectin reactivity. While O-glycans from these adult specimens were not detectable by mass spectrometry, several oligomannose, paucimannosidic, and complex-type N-glycans were identified, including some carrying hexuronic acid and many carrying core xylose. This is, to our knowledge, the first glycomic characterisation of a marine platyhelminth, with broader implications for research into other trematodes.

Glycans unique to the relapse-prone subset within triple-negative breast cancer as revealed by lectin array-based analysis of surgical specimens.

Molecular and cellular characteristics of the relapse-prone subset within triple-negative breast cancer (TNBC) remain unclear. Aberrant glycosylation is involved in the malignant behavior of cancer cells. In the present study, we aimed to reveal glycan profiles unique to relapsed TNBC patients. Thirty TNBC patients who did not undergo neoadjuvant chemotherapy but postoperative standard adjuvant therapy from 2009 through 2016 at Juntendo Hospital were investigated. TNBC cells were resected from primary breast cancer sections of formalin-fixed surgical specimens using laser-assisted microdissection. The binding intensities of the extracted glycoproteins to 45 lectins were quantified using lectin microarray and compared between relapsed and non-relapsed patients. Immunohistochemical staining with TJA-II lectin in specimen sections was performed. Five patients relapsed during the follow-up (range 37-123 months). Lectin microarray analysis revealed that 7 out of 45 lectins showed significant differences in binding intensity between the relapsed and the non-relapsed group. TJA-II, ACA, WFA, and BPL showed stronger binding in the relapsed group. PNGase F treatment of TNBC cell lysates suggested that TJA-II and ACA bind O-glycans. TJA-II staining of tissue sections revealed strong binding to cell surface membranes and to the cytoplasm of TNBC cells, but not to other types of cells. Significantly more TNBC cells were stained in tissue sections from relapsed than non-relapsed patients. TNBC cells from relapsed patients showed a unique lectin reactivity, with higher levels of TJA-II (also WFA and BPL) binding than in non-relapsed patients. The results are potentially useful to develop new prognostic and therapeutic tools.

Species-specific and heterogeneous distribution of sialoglycoconjugates in the primary olfactory center of three species of Asian salamanders (Cynops)

Sialic acids (Sia) are terminal components of glycoconjugates that are involved in molecular and cellular interactions in the olfactory system. Diverse glycoconjugates are expressed in the salamander olfactory projection; however, their sialylation and the linkage of Sia to underlying sugars remain largely unknown. The present study aimed to determine the expression of Sia linked to galactose (Gal)-N-acetylglucosamine and N-acetylgalactosamine (GalNAc) in the olfactory bulbs of three species of salamanders using lectin binding. Abundant distribution of sialoglycoconjugates was observed in the salamander olfactory bulb by lectins, Sambucus sieboldiana (SSA) and Maackia amurensis (MAM). Moreover, SSA and MAM showed heterogeneous bindings in the primary olfactory projection of Cynops pyrrhogaster and C. orientalis. Lectin reactivities obviously decreased in all layers of the olfactory bulb after sialidase digestion, indicating selective binding to sialoglycoconjugates. Next, we examined the expression of the subterminal sugar residues, Gal and GalNAc, after terminal Sia removal. Desialylation in the olfactory bulb enhanced the reactivity of Jacalin and Vicia villosa (VVA) lectins that recognize Gal and GalNAc respectively. Together with the binding of SSA and MAM, Sia linked to Gal and GalNAc might be a major component of sialoglycoconjugates in the salamander olfactory projection.

Pregnant mice submitted to Surgical Embryo Euthanasia (SEE) induce loss of expression of inducible nitric oxide synthase isoform and NO concentration in the maternal-fetal interface

The well-succeeded pregnancy in humans and rodents is the consequence of close interaction between maternal and fetal cells with intervening of cytokines and chemical mediators. In this process a pregnant uterus subset NK cells - uterine Natural Killer cells (uNK cells) play a pivotal modulatory role under the influence of local physiological hypoxia and other alterations. The aim of the present work was to evaluate the expression and commitment of induced form of nitric oxide synthase (iNOS) and NO concentration in the homeostasis of pregnant uterus. It was used normal pregnant mice on gd 10th and those submitted to surgical intervention to induce mechanical lesion in the embryos (SEE). Uterine samples were collected at 0.5, 1, 2 and 6 h after embryo lesion and processed for paraffin embedding and tissue homogenate. The samples destinate for paraffin embedding was performed the Dolichos biflorus (DBA) lectin cytochemistry and anti-iNOS immunocytochemistry. The samples destinate to tissue homogenates were processed for SDS-PAGE and Western-blot using anti-iNOS and evaluate of NO concentration. The embryo-injured uterine segments showed hyperemia and hemorrhage at mesometrial region in which the DBA lectin reaction showed altered uNK cells suggesting the degranulation. Positive reaction with anti-iNOS was seen on uNK cells, trophoblast giant cells, endometrial stromal and decidual cells and smooth muscle cells in the normal pregnant uterus, but 1 and 2 h after embryo lesion, the iNOS labeling decreased or was absent only in uNK cells. The same results was obtained with NO concentration. These results confirm the unique constitutive expression of iNOS in the pregnant mice uterus, being the uNK cells the only one responsive against stress of embryo failure, besides showing that excessive NO produced by quick activation of uNK-iNOS should affect the local vascular permeability.

Altered N-glycan profile of IgG-depleted serum proteins in Hashimoto's thyroiditis

BackgroundHashimoto's thyroiditis (HT) is an autoimmune disease characterized by chronic inflammation of thyroid gland. Although HT is the most common cause of hypothyroidism, the pathogenesis of this disease is not fully understood. Glycosylation of serum proteins was examined in HT only to a limited extent. The study was designed to determine the glycosylation pattern of IgG-depleted sera from HT patients. MethodsSerum N-glycans released by N-glycosidase F (PNGase F) digestion were analyzed by normal-phase high-performance liquid chromatography (NP-HPLC). N-glycan structures in each collected HPLC fraction were determined by liquid chromatography-mass spectrometry (LC-MS) and exoglycosidase digestion. Fucosylation and sialylation was also analyzed by lectin blotting. ResultsThe results showed an increase of monosialylated tri-antennary structure (A3G3S1) and disialylated diantennary N-glycan with antennary fucose (FA2G2S2). Subsequently, we analyzed the serum N-glycan profile by lectin blotting using lectins specific for fucose and sialic acid. We found a significant decrease of Lens culinaris agglutinin (LCA) staining in HT samples, which resulted from the reduction of α1,6-linked core fucose in HT serum. We also observed an increase of Maackia amurensis II lectin (MAL-II) reaction in HT due to the elevated level of α2,3-sialylation in HT sera. ConclusionsThe detected alterations of serum protein sialylation might be caused by chronic inflammation in HT. The obtained results complete our previous IgG N-glycosylation analysis in autoimmune thyroid patients and show that the altered N-glycosylation of serum proteins is characteristic for autoimmunity process in HT.General SignificanceThyroid autoimmunity is accompanied by changes of serum protein sialylation.

Vitronectins produced by human cirrhotic liver and CCl4-treated rats differ in their glycosylation pattern and tissue remodeling activity.

Liver cirrhosis (LC) is a disease characterized by pathological accumulation and alteration of extracellular matrix (ECM) proteins; the interaction between two such proteins, collagen and vitronectin (VN), is considered to be the key to controlling ECM remodeling in liver cirrhosis. If it is possible to control the modification of oligosaccharides on VN, it may be possible to retard progression of liver cirrhosis. In this study, we examined the relationship between changes in VN glycosylation and activity related to the remodeling of hepatic tissue in human LC and a rat model of LC generated using carbon tetrachloride (CCl4). Plasma concentrations of VN in human LC declined to approximately two‐thirds that in normal plasma, but the ratio of active VN, which has collagen‐binding activities, increased 2.8 times in LC plasma. In contrast, purified LC‐VN exhibited similar binding activities toward type I, IV, and V collagens to those of normal VN. Lectin reactivities and carbohydrate analyses of LC‐VN revealed that branching, fucosylation, and sialylation of N‐glycans were higher than those of normal VN. On the other hand, the plasma level of rat CCl4‐VN increased and the ratio of active molecules to collagen in plasma decreased. Increased fucosylation of LC‐VN was not detected in carbohydrates of CCl4‐VN. The changes in rat VN due to CCl4 treatment did not correspond to the changes in plasma levels of human VN caused by LC, the ratio of active molecules, or carbohydrate composition, thereby indicating that CCl4‐treated rats are not an appropriate model for studying VNs in human LC. Glycosidase treatment of VNs supported the hypothesis that the collagen‐binding activity of VN is modulated by alterations of glycosylation during LC, which may contribute to (a) the matrix incorporation of VN and (b) tissue fibrosis.

Reduced fucosylation in the distal intestinal epithelium of mice subjected to chronic social defeat stress

Psychological stress can cause dysfunction of the gastrointestinal tract by regulating its interaction with central nervous system (brain-gut axis). Chronic social defeat stress (CSDS) is widely used to produce a rodent model of stress-induced human mood disorders and depression. We previously showed that CSDS significantly affects the intestinal ecosystem including cecal and fecal microbiota, intestinal gene expression profiles and cecal metabolite profiles. Here, we investigated whether the glycosylation pattern in the intestinal epithelium was affected in C57BL/6 mice exposed to CSDS (hereinafter referred to as CSDS mice). A lectin microarray analysis revealed that CSDS significantly reduced the reactivity of fucose-specific lectins (rAOL, TJA-II, rAAL, rGC2, AOL, AAL, rPAIIL and rRSIIL) with distal intestinal mucosa, but not with mucosa from proximal intestine and colon. Flow cytometric analysis confirmed the reduced TJA-II reactivity with intestinal epithelial cells in CSDS mice. In addition, distal intestine expression levels of the genes encoding fucosyltransferase 1 and 2 (Fut1 and Fut2) were downregulated in CSDS mice. These findings suggest that CSDS alters the fucosylation pattern in the distal intestinal epithelium, which could be used as a sensitive marker for CSDS exposure.

Application of lectin microarrays for the analysis of seminal plasma glycome.

We report application of lectin microarrays, exploiting simultaneous interaction of seminal plasma samples with multiple lectins of different sugar specificities to compare the glycomes of fertile and infertile men. The results indicate reduced lectin reactivity associated with decreased fertility, especially affecting oligozoospermic subjects and probably O-glycosylation. Lectin microarrays may become a potent tool for semen analysis in search of the association of glycosylation and male fertility.

Lectin Histochemistry Showed a Heterogeneous Population of Cells Among Human Mesenchymal Stem Cells Isolated From Adipose Tissue

Objectives: Adipose tissue as an appropriate source of Mesenchymal Stem Cells (MSCs) has the potential to differentiate into multiple lineages. Glycoconjugates content of the MSCs can be considered as biomarkers in self-renewal, pluripotency and differentiation processes. In this study, the lectin profile of MSCs isolated from adipose tissue was detected and according to that, a subpopulation was determined. Materials & Methods: MSCs were isolated from adipose tissue by explanting of the tissue pieces. The FITC-conjugated lectins, WGA, UEA, PNA, BSA and PWM were used to detect the terminal sugar residues. The cells were then counterstained with DAPI. The intensity of the reaction was evaluated by ImageJ software. The cells were also stained with PAS method. Results: MSCs were reacted with all lectins with different intensity of the reactions. The cells reacted with WGA, UEA, and BSA “strongly” and with PWM “moderately” and with PNA with “weak” intensity. The morphological analysis of the isolated MSCs revealed the existence of the two different cell types in the cultures. Two types of cells were detected according to nucleus size and lectin reactivity. The cells with large nuclei constitute 20.62% of the total cells and stained significant more intensity by UEA and less intense with PWM (both P=0.014) and PNA (P=0.044). Flow cytometry with CD34 shows that these large cells were not endothelial cells. Conclusion: The MSCs derived from adipose tissue seem to be a heterogeneous populations and lectin profile of the cells showed that they are different in the expression of the glycoconjugates.

Abstract 5231: Undifferentiated cell maker rBC2LC-N lectin have high affinity to pancreatic cancer cells and residual cancer cells

Abstract The glycosylation in cancer cells remains still unclear despite the whole human genome understanding. Recent technical advances give us great awareness about the consequence of the glycosylation. The glycomes of cancer cell surfaces are often unique with aberrant glycans, including sialylation, fucosylation, O-glycan truncation, and N-and O-linked glycan branching. The sugar binding proteins named ‘lectins’ are helpful materials to recognize the glycan structure and characterization. Recent study suggested lectins are more widely accepted that involved in many biological phenomena inside mammalian or as chemical research tools. Despite years of effort to develop cancer therapies, there were no effective molecular targeting drugs for pancreatic adenocarcinoma (PDAC). One possible reason for this difficulties, PDAC cells are densely covered with glycans which may cause the recalcitrance to approach molecular targeting drugs. Our group developed a high-density lectin microarray using 96 kinds of lectins, in which a panel well-defined lectins is immobilized onto a slide, has been successfully used for high-throughput analysis of complex carbohydrates included in serum glycoproteins and whole cells. Using this approach, we demonstrated that a recombinant lectin probe, rBC2LC-N, specifically bound to undifferentiated pluripotent stem cells (ES/iPS cells), but not differentiated somatic cells; this has now been developed as a useful undifferentiation marker. In this study, we employed this lectin microarray to investigate the specific glycan that covered pancreatic cancer cells with stem cell features. Firstly, we selected a cell line which reflect clinical PDAC cells among 6 pancreatic cancer cell lines by morphological analysis and cancer stem cell marker expressions. As previously reported, we regarded Capan-1 cell line as a closest one which maintains both self-renewal and differentiation capacity. As a result, we successfully identified top 10 highlighted lectins which have significant specificity to pancreatic cancer cells. rBC2LC-N lectin was the most significant one (P value = 9.44E-17), followed by ADA (P= 7.05E-9), TJA2 (P= 1.4E-8), ACG (P= 1.11E-6). We reconfirmed the specific reactivity of these lectins to Capan-1 by FACS and lectin histochemistry. rBC2LC-N lectin reactivity in 70 clinical cases of PDAC revealed that all cases were confirmed to be positive. Furthermore, we investigated this rBC2LC-N lectin have a high affinity to residual cancer cells after gemcitabine treatment in patient derived xenograft models. These reactions well agreed with those of undifferentiated iPS/ES cells. These coincidental match of glycan expression between on iPS/ES cells and on pancreatic cancer cells may suggest a great benefit to understand the ambiguous stem cell recognition in cancer community and novel therapeutic strategy. Citation Format: Osamu Shimomura, Tastsuya Oda, Hiroaki Tateno, Sho Tachino, Junji Matsui, Yuusuke Ozawa, Jun Hirabayashi, Nobuhiro Ohkohchi. Undifferentiated cell maker rBC2LC-N lectin have high affinity to pancreatic cancer cells and residual cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5231. doi:10.1158/1538-7445.AM2017-5231

MP28-07 AN AUTOMATED-MICROCAPILLARY ELECTROPHORESIS-BASED IMMUNOASSAY SYSTEM MAY IMPROVE DIAGNOSTIC ACCURACY OF PROSTATE CANCER AND BE A GOOD INDICATOR OF GLEASON SCORE

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2017MP28-07 AN AUTOMATED-MICROCAPILLARY ELECTROPHORESIS-BASED IMMUNOASSAY SYSTEM MAY IMPROVE DIAGNOSTIC ACCURACY OF PROSTATE CANCER AND BE A GOOD INDICATOR OF GLEASON SCORE Tomokazu Ishikawa, Tohru Yoneyama, Yuki Tobisawa, Shingo Hatakeyama, Tatsuo Kurosawa, Kenji Nakamura, Takuya Koie, Yasuhiro Hashimoto, and Chikara Ohyama Tomokazu IshikawaTomokazu Ishikawa More articles by this author , Tohru YoneyamaTohru Yoneyama More articles by this author , Yuki TobisawaYuki Tobisawa More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Tatsuo KurosawaTatsuo Kurosawa More articles by this author , Kenji NakamuraKenji Nakamura More articles by this author , Takuya KoieTakuya Koie More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.820AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Lack of specificity in PSA test for early detection of prostate cancer (PCa) is one of the major issues all over the world. Therefore, the development of novel assay system with improved specificity is urgently needed. We identified PCa-associated aberrant glycosylation of PSA (S2,3PSA) and developed an automated-microcapillary electrophoresis-based immunoassay system (μTAS method). Furthermore, we successfully applied this assay system for clinical setting. METHODS S2,3PSA was clearly differentiated by the lectin reactivity to recognize aberrant glycosylation on free PSA (fPSA) and calculate the ratio (%) in the microfluidic separation channel with fPSA antibody-label conjugates. We measured %S2,3PSA by utilizing μTAS method with extremely shortened assay time (9 min) and evaluated the cutoff values in biopsy proven patient samples (103 with PCa and 50 with non-PCa) including PSA less than 20.0 ng/ml. Receiver operating characteristic (ROC) curves were used to evaluate diagnostic accuracy for %S2,3PSA in comparison with other methods. RESULTS %S2,3PSA of PCa was significantly higher than those of non-PCa (p < 0.0001) and the appropriate cutoff value was determined to be 40% of S2,3PSA ratio in terms of diagnostic accuracy. The area under the curve (AUC) for the detection of PCa with %S2,3PSA ratio was 0.851, which was significantly more effective than that with total PSA (AUC; 0.658). And also, good correlation in this assay with Gleason score after radical prostatectomy (pGS) was suggested for the discrimination of high risk PCa. Especially, high-risk PCa patients with GS≥4+3 could be separated by the S2,3PSA percentage cut off of 50%, achieving a high performance (AUC; 0.921). CONCLUSIONS Although the present study is still preliminary, these results suggest that the newly developed serum %S2,3PSA test may contribute improved diagnostic accuracy having good correlation with prostatectomy Gleason score. Additional validation studies are warranted. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e340 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Tomokazu Ishikawa More articles by this author Tohru Yoneyama More articles by this author Yuki Tobisawa More articles by this author Shingo Hatakeyama More articles by this author Tatsuo Kurosawa More articles by this author Kenji Nakamura More articles by this author Takuya Koie More articles by this author Yasuhiro Hashimoto More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Hidden IgG Antibodies to the Tumor-Associated Thomsen-Friedenreich Antigen in Gastric Cancer Patients: Lectin Reactivity, Avidity, and Clinical Relevance.

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

Glycoconjugates of the Rat Eyeball Structural Components under Experimental Hypothyroidism according to Lectin Histochemistry Studies

AbstractObjective: Thyroid disorders are currently among the most widespread endocrine pathologies, influencing multiple organs including the eyeballs. Lectins are used for the histochemical investigation of cell and tissue carbohydrates, which are involved in a wide range of physiological and pathological conditions, e.g. manifestations of cellular metabolism disorders, malignant transformation of cells, agglutination of viruses and microorganisms etc.Aim: of present investigation was to use a set of lectins with different carbohydrate specificities to study the rearrangement of carbohydrate determinants of rat eye structural components under the influences of experimental mercazolil-induced hypothyroidism.Methods: Experiments were carried out on 35 adult male Wistar rats weighing 180-240 g. Control group included 10 animals; experimental group of 25 rats during 14 days received 5 mg/kg of mercazolil with daily food allowance. Samples of thyroid glands and eyeballs were fixed in Bouin’s fluid and embedded in paraffin. The lectin panel included PNA, HPA, SNA, LABA, WGA and CNFA, conjugated to horseradish peroxidase. Lectin receptor sites were visualized with 3,3’-diaminobenzidine in the presence of H2O2. For specificity control of histochemical reactions the exclusion of lectin-peroxidase conjugates from staining protocol was used. For routine histological examination sections were stained haematoxylin and eosin.Results: Morphological investigations revealed negative influence of experimental hypothyroidism on corneal epithelium and retina, as well as on the eyeball associated Garder’s gland. By means of lectin histochemistry methods it was identified increased reactivity of rod and cone layer with WGA, CNFA and LABA, encompassingthe accumulation of DGlcNАc, GalNAc(β1-4)GlcNAc and αLFuc carbohydrate determinants. These changes were accompanied with reduced HPA and SNA reactivity (αDGalNАc and NeuNAc(α2-6) DGal residues). These modifications in rods and cones lectin labeling were supplemented with the increased CNFA binding in the areas of synaptic contacts in the outer and inner plexiform layers, as well as with enhanced exposure of LABA receptor sites (αLFuc) in perikarya of amacrine and ganglion neurons of retina. In the cornea under experimental hypothyroidism it was detected reduction of HPA and CNFA labeling (αDGalNАc and GalNAc (β1-4) GlcNAc determinants) in the anterior epithelium, which was accompanied with the increased reactivity of these same lectins with stromal keratocytes and collagen fibers.Conclusion: Reported drift in corneal lectin binding apparently indicates negative influence of hypothyroidism on epithelial adhesion to Bowman’s membrane and corneal transparency conditions, the latters depending on crystallins and keratan sulphates produced by keratocytes. Detected rearrangements in carbohydrate determinants of retinal compartments presumably reflect alterations in the functional activity of neurons and subsequent disturbancies in nerve impulses transduction. Lectins WGA, CNFA, LABA and HPA can be recommended for selective histochemical labeling of structural components of the rat eyeball.

Postnatal morphological and lectin histochemical observation of the submucosal glands of rat nasopharynx

The development of submucosal glands of rat nasopharynx was studied with respect to their morphological maturation and glycoprotein alterations during the postnatal period. This study examined the histological morphology with hematoxylin–eosin and the binding pattern of lectins, soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin-I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and succinylated WGA (sucWGA) on frozen sections from newborn into adulthood. At birth, nasopharyngeal glands consisted of rudimentary secretory units which by postnatal day 3 (PN3) showed the characteristic features of salivary glands comprised of mixed mucous and serous cells. With maturation, serous cells increased in number and were arranged in clusters. Lectin reactivity at birth was detected at the acinar cell basal membranes for DBA, SBA, VVA, UEA-1 and PNA. At PN3, lectins labeled the apical cytoplasm and basolateral membranes of mucous cells and progressively with maturation, extended from the apical to basal portions of the cytoplasm with variable reactivity of VVA, PNA and sucWGA. Serous cells were labeled by UEA-1 starting from PN10 and also by PNA in adults. Ducts showed variable lectin reaction on the luminal membrane with strong reactivity of DBA and UEA-1 at PN21. Taken together, lectin histochemistry indicated the transitional occurrence of glycoproteins depending on the stage of maturation of the glands. Moreover, these results emphasize the difference in the morphology and lectin histochemistry between the nasopharyngeal and palatine glands.

Total and Lectin-Binding Proteome of Spherulin from Coccidioides posadasii.

Coccidioides is a virulent dimorphic fungus that causes coccidioidomycosis (valley fever) in mammals, including humans. Although the genome has been sequenced, a proteomic analysis does not exist. To address this gap in proteomic knowledge, we generated the proteome of spherulin (a well-studied lysate of fungal spherules) and identified 1390 proteins. Some of the proteins included glycosylation enzymes, which led us to hypothesize that fungal glycosylation patterns may be different from those of mammals and could be exploited to detect Coccidioides in tissues. We performed lectin-based immunohistochemistry on formalin-fixed paraffin-embedded human patients' lung tissues. GSL-II (Griffonia simplificonia lectin II) and sWGA (succinylated wheat germ agglutinin) lectins bound specifically to endospores and spherules in infected lungs. To identify lectin-binding glycoproteins in spherulin, we performed lectin-affinity chromatography, followed by LC-MS/MS. A total of 195 glycoproteins from spherulin bound to GSL-II, 224 glycoproteins bound to sWGA, and 145 glycoproteins bound to both lectins. This is the first report of the specific reactivity of GSL-II and sWGA lectins to Coccidioides endospores and spherules in infected human tissues and the first listing of the Coccidioidal proteome from spherulin using sequences present in three Coccidioides databases: RefSeq, SwissProt, and The Broad Institute's Coccidioides Genome project.