The aryl hydrocarbon receptor instructs the immunomodulatory profile of a subset of Clec4a4+ eosinophils unique to the small intestine.

C-type lectin domain family 4, member a4 (Clec4a4) is a C-type lectin inhibitory receptor specific for glycans thought to be exclusively expressed on murine CD8α− conventional dendritic cells. Using newly generated Clec4a4-mCherry knock-in mice, we identify a subset of Clec4a4-expressing eosinophils uniquely localized in the small intestine lamina propria. Clec4a4+ eosinophils evinced an immunomodulatory signature, whereas Clec4a4− eosinophils manifested a proinflammatory profile. Clec4a4+ eosinophils expressed high levels of aryl hydrocarbon receptor (Ahr), which drove the expression of Clec4a4 as well as other immunomodulatory features, such as PD-L1. The abundance of Clec4a4+ eosinophils was dependent on dietary AHR ligands, increased with aging, and declined in inflammatory conditions. Mice lacking AHR in eosinophils expanded innate lymphoid cells of type 2 and cleared Nippostrongylus brasiliensis infection more effectively than did wild-type mice. These results highlight the heterogeneity of eosinophils in response to tissue cues and identify a unique AHR-dependent subset of eosinophils in the small intestine with an immunomodulatory profile.

Expression of low-sulfated keratan sulfate in non-mucinous ovarian carcinoma.

Keratan sulfate glycosaminoglycan is composed of repeating N-acetyllactosamine (LacNAc) disaccharide units consisting of galactose (Gal) and N-acetylglucosamine (GlcNAc), both often 6-O-sulfated. Sulfate contents of keratan sulfate are heterogeneous depending upon the origins. In this study, keratan sulfate is classified as either highly sulfated (in which both GlcNAc and Gal residues are 6-O-sulfated) or low-sulfated (in which only GlcNAc residues are 6-O-sulfated). It is reported that highly sulfated keratan sulfate detected by the 5D4 monoclonal antibody is preferentially expressed in normal epithelial cells lining the female genital tract and in their neoplastic counterparts; however, expression of low-sulfated keratan sulfate in either has not been characterized. In the present study, we generated the 294-1B1 monoclonal antibody, which selectively recognizes low-sulfated keratan sulfate, and performed precise glycan analysis of sulfated glycans expressed on human serous ovarian carcinoma OVCAR-3 cells. We found that OVCAR-3 cells do not express highly sulfated keratan sulfate but rather express low-sulfated form, which was heterogeneous in 294-1B1 reactivity. Comparison of mass spectrometry spectra of sulfated glycans in 294-1B1-positive versus -negative OVCAR-3 cells indicated that the 294-1B1 epitope is likely at least 2, and possibly 3 or more, tandem GlcNAc-6-O-sulfated LacNAc units. Then, using the 294-1B1 antibody, we performed quantitative immunohistochemical analysis of 40 specimens from patients with ovarian cancer, consisting of 10 each of serous, endometrioid, clear cell, and mucinous carcinomas, and found that among them low-sulfated keratan sulfate was widely expressed in all but mucinous ovarian carcinoma.

Structural Elucidation and Prognostic Relevance of 297-11A-Sulfated Glycans in Ovarian Carcinoma.

Ovarian carcinoma is usually diagnosed at an advanced stage with peritoneal dissemination and/or lymph node metastasis, and the prognosis for such advanced carcinoma is very poor. Therefore, new biomarkers to predict patient prognosis are needed. Miyamoto et al. previously showed that keratan sulfate (KS) detected by the 5D4 monoclonal antibody was expressed in ovarian carcinoma. However, the detailed structure of such KS was not determined, and the biological significance of this finding remained to be clarified. We previously generated the 297-11A monoclonal antibody, which recognizes galactose (Gal)-6-O-sulfated N-acetyllactosamine (LacNAc) located at the nonreducing terminus. Because the 297-11A epitope overlaps with that of 5D4, here we chose to use the 297-11A antibody as a tool to analyze KS and related structures. We conducted immunohistochemical analysis of 98 ovarian carcinoma cases with 297-11A antibody combined with a series of glycosidases and performed mass spectrometry analysis of the human serous ovarian carcinoma cell line OVCAR-3 to deduce the glycan structure of 297-11A-sulfated glycans. We also performed western blot analysis to assess a potential association of 297-11A-sulfated glycans with the mucin core protein mucin 16 (MUC16; also known as cancer antigen 125 (CA125)). Finally, we examined the relationship between 297-11A expression and patient prognosis. Consequently, 297-11A-sulfated glycans were primarily expressed in serous and endometrioid carcinomas and poorly expressed in mucinous and clear cell carcinomas. We reveal that structurally, 297-11A-sulfated glycans expressed in ovarian carcinoma are O-glycans carrying partially sialylated, Gal-6-O-sulfated LacNAc and that these glycans are likely displayed on MUC16 mucin core proteins. Of clinical importance is that expression of 297-11A-sulfated glycans correlated with shorter progression-free survival in patients. Thus, 297-11A-sulfated glycans may serve as a predictor of ovarian carcinoma recurrence.

Sulfated Glycans Recognized by S1 Monoclonal Antibody can Serve as a Diagnostic Marker for Malignant Pleural Mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a malignant neoplasm of the pleura caused by asbestos exposure. For diagnosis of MPM, immunohistochemistry using multiple markers is recommended to rule out differential diagnoses, such as pulmonary adenocarcinoma. However, the specificity of currently used markers is not fully satisfactory. We previously developed a monoclonal antibody named S1, which recognizes 6-sulfo sialyl Lewis x, an L-selectin ligand expressed on high endothelial venules. During the screening process, we discovered that this antibody stained normal pleural mesothelium. This finding prompted us to hypothesize that the epitope recognized by S1 might serve as a new diagnostic marker for MPM. METHODS: To test this hypothesis, we immunostained human MPM (n = 22) and lung adenocarcinoma (n = 25) tissues using S1 antibody. RESULTS: 77.3% of MPM were S1 positive, and if limited to epithelioid type, the positivity rate was 100%, while that of lung adenocarcinoma was only 36.0%. Statistical analysis revealed a significant difference in the S1 positivity rate between each disease. Furthermore, immunohistochemistry using a series of anti-carbohydrate antibodies combined with glycosidase digestion revealed the structure of sulfated glycans expressed in MPM to be 6-sulfo sialyl N-acetyllactosamine attached to core 2-branched O-glycans. CONCLUSION: We propose that the S1 glycoepitope could serve as a new diagnostic marker for MPM.

Preferential expression of sialyl 6'-sulfo N-acetyllactosamine-capped O-glycans on high endothelial venules in human peripheral lymph nodes.

Lymphocyte "homing", the physiologic trafficking of lymphocytes from the circulation to secondary lymphoid organs, is regulated by sequential adhesive interactions between lymphocytes and endothelial cells that constitute high endothelial venules (HEVs). Initial lymphocyte "rolling" is mediated by relatively weak, transient adhesive interactions between L-selectin expressed on lymphocytes and sulfated mucin-type O-glycans expressed on HEVs. Keratan sulfate galactose (Gal)-6-O-sulfotransferase (KSGal6ST) catalyzes 6-O-sulfation of Gal in keratan sulfate glycosaminoglycan chains but also transfers sulfate to Gal in much shorter glycan chains, such as sialylated N-acetyllactosamine (LacNAc)-capped O-glycans. In mice, KSGal6ST is reportedly expressed in HEVs and functions in synthesizing 6-sulfo Gal-containing O-glycans on HEVs. However, in humans, the presence of 6-sulfo Gal-containing O-glycans on HEVs is not reported. Employing the newly developed monoclonal antibody 297-11A, which recognizes non-sialylated terminal 6'-sulfo LacNAc, we demonstrate that sialyl 6'-sulfo (and/or 6,6'-disulfo) LacNAc-capped O-glycans are preferentially displayed on HEVs in human peripheral lymph nodes (PLNs) and to a lesser extent in mesenteric LNs (MLNs) but not in Peyer's patches (PPs). We also found that the scaffold protein mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed on HEVs in PPs and MLNs but not PLNs, was modified by 297-11A-positive sulfated glycans less efficiently than was CD34. Moreover, 297-11A-positive sulfated glycans were also displayed on HEV-like vessels induced in tumor-infiltrating lymphocyte (TIL) aggregates formed in various cancers. These findings collectively indicate that 297-11A-positive sulfated glycans potentially play a role in physiologic lymphocyte homing as well as in lymphocyte recruitment under pathologic conditions.

Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae ST258 isolated from a Japanese patient without a history of foreign travel - a new public health concern in Japan: a case report.

BACKGROUND: Thus far, studies on Klebsiella pneumoniae carbapenemase (KPC)-producing organisms have only been reported in those with a history of foreign travel, and a specific Japanese KPC-producing isolate has not yet been reported. CASE PRESENTATION: We describe a Japanese patient, with no history of travel to foreign countries, admitted due to aspiration pneumonia, and a KPC-producing isolate detected in his sputum. Fortunately, his pneumonia resolved. His close contacts did not have a history of foreign travel, and the isolate was not detected in other patients. CONCLUSIONS: The potential for KPC-producing organisms to become endemic in Japan is currently of great concern.

Appearance of High Endothelial Venule-Like Vessels in Benign Prostatic Hyperplasia is Associated With Lower Urinary tract Symptoms.

BACKGROUND: Chronic prostatic inflammation is implicated in the pathogenesis of benign prostatic hyperplasia (BPH)-associated lower urinary tract symptoms (LUTS). Previous studies evaluated the degree of chronic prostatic inflammation based on histological scores, which may contain subjective factors. We previously demonstrated that the number of high endothelial venule (HEV)-like vessels correlates positively with the magnitude of inflammation in chronic inflammatory gastrointestinal diseases. Here, we evaluated the degree of BPH-associated chronic prostate inflammation based on appearance of HEV-like vessels and determined whether the extent of inflammation correlated with LUTS severity, as evaluated by a urodynamic study. METHODS: Eighty-six BPH tissue specimens derived from patients who had undergone urodynamic analysis were immunostained for CD34 and MECA-79 to determine HEV-like vessel number. Triple immunohistochemistry for either CD3 and CD20 or CD4 and CD8, together with MECA-79, was conducted to identify lymphocyte subsets associated with HEV-like vessels. We also determined whether the magnitude of chronic prostatic inflammation, as assessed by HEV-like vessel number, correlated with the degree of LUTS. RESULTS: HEV-like vessels were induced in lymphoid aggregates seen frequently in BPH. The number of HEV-like vessels positively correlated not only with the magnitude of chronic prostatic inflammation but also with the degree of LUTS, particularly with symptoms associated with voiding function, which was measured objectively in a pressure flow study. CONCLUSIONS: Chronic prostate inflammation may promote BPH and resulting voiding dysfunction. Assessment of the number of HEV-like vessels could be a surrogate for identifying the degree of chronic prostatic inflammation. Prostate 77:794-802, 2017. © 2017 Wiley Periodicals, Inc.

Role of sialyl 6-sulfo Lewis X in antitumor immunity against oral squamous cell carcinoma.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) reportedly play a pivotal role in antitumor immunity against oral squamous cell carcinoma (OSCC); however, mechanisms governing TIL recruitment to OSCC tissues remain to be clarified. This study was undertaken to assess a potential association between TILs and high endothelial venule (HEV)-like vessels that express sialyl 6-sulfo Lewis X (LeX). METHODS: OSCC tissue sections (n=41) were subjected to immunohistochemistry for sialyl 6-sulfo LeX and CD34 to allow quantitation of HEV-like vessels. Triple immunohistochemistry for sialyl 6-sulfo LeX and either CD3 and CD20 or CD4 and CD8 was conducted to determine which lymphocyte subset is more closely associated with HEV-like vessels. RESULTS: HEV-like vessels expressing sialyl 6-sulfo LeX were detected in 27 of 41 (65.9%) OSCC cases, and these vessels were more frequently found in early disease (T1/T2 stages) compared with advanced (T3/T4) stages. The number of T cells attached to the inner surface of these HEV-like vessels was significantly greater than that of B cells, while the number of CD4+ helper T cells and CD8+ cytotoxic T cells did not differ significantly. Interestingly, sialyl 6-sulfo LeX was also expressed on the membrane of a fraction of OSCC cells, and CD8+ cytotoxic T cells were almost exclusively found attached to these carcinoma cells. CONCLUSIONS: Sialyl 6-sulfo LeX is displayed not only on HEV-like vessels but also on OSCC cells and may potentially function in antitumor immunity against OSCC.

Apical membrane expression of distinct sulfated glycans represents a novel marker of cholangiolocellular carcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver neoplasm, followed by hepatocellular carcinoma. ICC can be further subclassified as (i) perihilar and (ii) peripheral types, the latter histologically resembling small-sized intrahepatic bile ducts, such as interlobular bile ducts, cholangioles/ductules and the canals of Hering. Cholangiolocellular carcinoma (CoCC), now classified by the World Health Organization as a subtype of combined hepatocellular-cholangiocarcinoma, is currently regarded as a subtype of peripheral-type ICC. The present study was undertaken to determine whether sulfated glycans recognized by the MECA-79 monoclonal antibody could serve as a CoCC marker. Using immunohistochemistry, we show that MECA-79 sulfated glycans are preferentially expressed at the apical membrane of cholangiocytes found in small-sized intrahepatic bile ducts in normal liver and in canalicular structures formed in CoCC. We also report that apical membrane MECA-79 sulfated glycan expression colocalizes with that of mucin 1 (MUC1) core proteins. We also present immunoblotting of Chinese hamster ovary cells overexpressing FLAG-tagged MUC1 to show that MUC1 serves as a MECA-79 scaffold. Furthermore, we report that SSP-25 human ICC cells overexpressing N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), but not GlcNAc6ST-1, exhibit membrane expression of MECA-79 sulfated glycans, suggesting that GlcNAc6ST-2 catalyzes MECA-79 epitope biosynthesis in cholangiocytes. Moreover, both wild-type and GlcNAc6ST-1 knockout mice exhibit apical membrane MECA-79 expression in small-sized intrahepatic bile ducts, namely interlobular bile ducts, whereas MECA-79 expression was completely absent in comparable tissues from GlcNAc6ST-1 and GlcNAc6ST-2 double knockout mice. These data collectively indicate that apical membrane localization of MUC1 proteins decorated with GlcNAc6ST-2-dependent MECA-79 sulfated glycans may mark cholangiocytes with cholangiolar/ductular differentiation and could serve as a useful CoCC marker.

Neuronal-specific overexpression of a mutant valosin-containing protein associated with IBMPFD promotes aberrant ubiquitin and TDP-43 accumulation and cognitive dysfunction in transgenic mice.

Mutations in valosin-containing protein (VCP) cause a rare, autosomal dominant disease called inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). One-third of patients with IBMPFD develop frontotemporal dementia, characterized by an extensive neurodegeneration in the frontal and temporal lobes. Neuropathologic hallmarks include nuclear and cytosolic inclusions positive to ubiquitin and transactive response DNA-binding protein 43 (TDP-43) in neurons and glial activation in affected regions. However, the pathogenic mechanisms by which mutant VCP triggers neurodegeneration remain unknown. Herein, we generated a mouse model selectively overexpressing a human mutant VCP in neurons to study pathogenic mechanisms of mutant VCP-mediated neurodegeneration and cognitive impairment. The overexpression of VCPA232E mutation in forebrain regions produced significant progressive impairments of cognitive function, including deficits in spatial memory, object recognition, and fear conditioning. Although overexpressed or endogenous VCP did not seem to focally aggregate inside neurons, TDP-43 and ubiquitin accumulated with age in transgenic mouse brains. TDP-43 was also found to co-localize with stress granules in the cytosolic compartment. Together with the appearance of high-molecular-weight TDP-43 in cytosolic fractions, these findings demonstrate the mislocalization and accumulation of abnormal TDP-43 in the cytosol of transgenic mice, which likely lead to an increase in cellular stress and cognitive impairment. Taken together, these results highlight an important pathologic link between VCP and cognition.

Expression of Podocalyxin Potentially Decorated With Low-sulfated Keratan Sulfate in Human Testicular Embryonal Carcinoma.

SummaryWe previously demonstrated that among various histological types of human testicular germinal cell tumors (GCTs), embryonal carcinoma (EC) preferentially expresses low-sulfated keratan sulfate (KS) consisting of repeating N-acetyllactosamine (LacNAc) disaccharide units composed of galactose and 6-O-sulfated N-acetylglucosamine (GlcNAc), which is recognized by the R-10G antibody. Recently, we generated another anti-low-sulfated KS monoclonal antibody, 294-1B1. Immunohistochemical analysis of testicular GCTs (n=83) revealed that the low-sulfated KS recognized by 294-1B1 is also preferentially expressed in EC but minimally in other GCT histological types. Moreover, immunolabeling with R-10G and 294-1B1 antibodies was resistant to peptide-N-glycosidase F digestion, and EC was not stained with the MECA-79 antibody, indicating that low-sulfated KS expressed in EC contains mucin-type core 2 O-glycans carrying GlcNAc-6-O-sulfated oligo-LacNAc. Double immunofluorescence staining showed that R-10G and 294-1B1 antibody signals colocalized with those for podocalyxin (PODXL). Furthermore, western blot analysis of recombinant human PODXL•IgG fusion proteins secreted from low-sulfated KS-expressing human embryonic kidney 293T cells revealed that PODXL functions as a core protein for low-sulfated KS. Taken together, these findings strongly suggest that the PODXL glycoform decorated with low-sulfated KS is preferentially expressed in human testicular EC and may therefore serve as a diagnostic marker for this malignancy.

Establishment of a human ovarian clear cell carcinoma cell line mutant in PIK3CB but not PIK3CA.

A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient's tumour tissue. Notably, MTC-22 cells, and the patient's carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations-one in ARID1A, which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase-were seen in the patient's tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy.

Heparan sulfate subdomains that are degraded by Sulf accumulate in cerebral amyloid ß plaques of Alzheimer's disease: evidence from mouse models and patients.

Alzheimer's disease (AD) is characterized by extracellular cerebral accumulation of amyloid ß peptide (Aß). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aß aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains.

Distinct sulfated glycans expressed in intrahepatic cholangiocarcinoma: a potential target for new therapy.

Cholangiocytes exhibit morphological and functional heterogeneity, depending on their anatomical localization; however, like other ductal organs, their mucosal surface is covered with mucin, which functions to prevent the entry of foreign substances, lubricate and prevent clogging by bile. Recently, the authors discovered that distinct sulfated glycans recognized by a series of antisulfated glycan antibodies are expressed not only in normal intrahepatic bile ducts but also in intrahepatic cholangiocarcinoma (iCCA). In this review, the authors first describe the anatomy of bile ducts and the biochemical characteristics of bile-duct-associated mucins, and then describe differences in structure and expression patterns of these sulfated glycans in physiological and pathological conditions. Finally, potential therapeutic strategies for iCCA using antisulfated glycan antibodies are discussed.

Paradoxical Expression of R-10G-reactive Antigen in Human Testicular Embryonal Carcinoma.

Thus far, several monoclonal antibodies directed against cell-surface carbohydrate antigens have been generated. Among them, R-10G reportedly reacts selectively with human embryonic stem and induced pluripotent stem cells, but not with embryonal carcinoma (EC) cells. However, EC cells derived from patients' EC tumors may exhibit varying levels of R-10G-reactive antigen expression. Thus, we asked whether human EC tissues or germ cell tumor (GCT) tissues other than EC express R-10G-reactive antigen. To do so, we quantitatively analyzed R-10G-reactive antigen expression in 83 testicular GCT surgical specimens containing a total of 125 various GCT components. Accordingly, in all EC components examined, the EC cell plasma membrane was immunolabeled with R-10G, while most seminoma components were R-10G-negative. In non-seminomatous GCT (NSGCT) other than EC (non-EC NSGCT), R-10G-reactive antigen expression was variable, but signal distribution was focal, and the average intensity was weaker than that seen in EC. The percentages of R-10G-positive cells in these three groups varied with high statistical significance (p<0.001 for all combinations). These findings indicate that the R-10G-reactive antigen is preferentially expressed in human testicular EC tissues and, thus, could be used as a diagnostic marker for this malignancy.

Prominent expression of sialyl Lewis X-capped core 2-branched O-glycans on high endothelial venule-like vessels in gastric MALT lymphoma.

High endothelial venule (HEV)-like vessels have been observed in gastric B cell lymphoma of mucosa-associated lymphoid tissue type (MALT lymphoma), as well as in its preceding lesion, chronic Helicobacter pylori gastritis. Previously we reported that glycans on HEV-like vessels in the latter lesion served as L-selectin ligands, although their function is unclear. We have investigated sialyl Lewis X (sLeX)-related glycoepitopes and found that MECA-79(-) /HECA-452(+) /NCC-ST-439(+) HEV-like vessels preferentially mark gastric MALT lymphoma compared to chronic H. pylori gastritis. We then constructed CHO cell lines expressing potential MECA-79(-) /HECA-452(+) /NCC-ST-439(+) glycans, as well as other sLeX-type glycans, on CD34 and evaluated L-selectin binding to those cells, using L-selectin-IgM chimera binding and lymphocyte adhesion assays. L-selectin-IgM chimeras bound to CHO cells expressing 6-sulpho-sLeX attached to core 2-branched O-glycans with or without 6-sulpho-sLeX attached to extended core 1 O-glycans, but only marginally to other CHO cell lines. By contrast, CHO cells expressing 6-sulpho-sLeX attached to extended core 1 and/or core 2-branched O-glycans, as well as non-sulphated sLeX attached to core 2-branched O-glycans, showed substantial lymphocyte binding, while binding was negligible on lines expressing 6-sulpho- and non-sulphated sLeX attached to N-glycans and non-sulphated sLeX attached to extended core 1 O-glycans. These results indicate that MECA-79(-) /HECA-452(+) /NCC-ST-439(+) glycans, specifically, 6-sulpho- and non-sulphated sLeXs attached to core 2-branched O-glycans, expressed on HEV-like vessels in gastric MALT lymphoma function as L-selectin ligands and likely contribute to H. pylori-specific T cell recruitment in the progression of gastric MALT lymphoma.

Expression and Clinical Significance of Spi-B in B-cell Acute Lymphoblastic Leukemia.

Spi-B, a member of the E26 transformation-specific (ETS) family of transcription factors, plays an important role in B cell differentiation. Spi-B also functions in development of diffuse large B-cell lymphoma; thus, we hypothesized that it may participate in leukemogenesis of B-cell acute lymphoblastic leukemia (B-ALL). To test this hypothesis, we first generated an anti-Spi-B monoclonal antibody that recognized Spi-B on formalin-fixed, paraffin-embedded tissue sections. This antibody, designated S28-5, selectively stained B cell nuclei at the pre-plasma cell stage (including centrocytes and centroblasts in germinal centers) and nuclei of plasmacytoid dendritic cells, but not fully differentiated plasma cells, T cells, macrophages, or follicular dendritic cells. Employing S28-5, we then performed immunohistochemical staining of bone marrow aspiration biopsy specimens obtained from B-ALL patients (n=62). Cases that showed stronger nuclear S28-5 signals than T-cell ALL were scored positive. In 26 (42%) of 62 specimens, leukemic cells showed nuclear Spi-B expression, and positivity was associated with patient age at diagnosis, and serum uric acid and creatinine levels. Moreover, Spi-B-positive patients demonstrated significantly shorter overall survival than did Spi-B-negative patients. These results suggest that Spi-B expression may serve as a prognostic indicator of B-ALL.

Vascular E-selectin Expression Detected in Formalin-fixed, Paraffin-embedded Sections With an E-selectin Monoclonal Antibody Correlates With Ulcerative Colitis Activity.

It is widely accepted that E-selectin, an inducible endothelial cell adhesion molecule, plays a critical role in the initial step of neutrophil recruitment to sites of acute inflammation. However, immunohistological analysis of E-selectin has been hampered by lack of E-selectin-specific monoclonal antibodies that can stain formalin-fixed, paraffin-embedded (FFPE) tissue sections. Here, we employed E-selectin•IgM (a soluble form of E-selectin) as immunogen, and then, after negative selection with L-selectin•IgM and P-selectin•IgM and screening of FFPE sections of both COS-1 cells overexpressing E-selectin and acute appendicitis tissues, we successfully generated an E-selectin-specific monoclonal antibody capable of staining FFPE tissue sections. We used this antibody, designated U12-12, to perform quantitative immunohistological analysis of 390 colonic mucosal biopsy specimens representing ulcerative colitis. We found that the higher the histological disease activity, the greater the number of vessels expressing E-selectin, an observation consistent with previous analyses of frozen tissue sections. Furthermore, in active ulcerative colitis, E-selectin-expressing vessels contained neutrophils attached to endothelial cells, presumably in the process of extravasation, which eventually could cause epithelial damage. These results overall indicate that U12-12 is effective for E-selectin immunohistochemistry in archived FFPE samples representing various human diseases.

Expression of functional E-selectin ligands on the plasma membrane of carcinoma cells correlates with poor prognosis in clear cell renal cell carcinoma.

OBJECTIVES: Given the relatively high frequency of metastatic recurrence of clear cell renal cell carcinoma (ccRCC), reliable prognostic markers of ccRCC, particularly those associated with metastasis, are needed. Here, in search of those factors, we assessed the contribution of sialyl Lewis x (sLex) and sialyl Lewis a (sLea), as well as functional E-selectin ligand carbohydrates expressed on carcinoma cells, to metastasis and consequent poor prognosis in ccRCC. MATERIALS AND METHODS: Patients who underwent surgical resection (curative nephrectomy) for RCC, and whose post-operative pathological diagnosis was ccRCC (n = 117) were enrolled in this study. Expression of sLex/sLea carbohydrate antigens in ccRCC was evaluated by immunohistochemistry with an anti-sLex/sLea monoclonal antibody HECA-452. To evaluate membrane expression of sLex/sLea carbohydrate antigens quantitatively, we employed a histological scoring system used to evaluate membrane expression of human epidermal growth factor receptor 2 (HER2) in breast cancer. We also conducted an E-selectin•IgM chimera in situ binding assay to assess expression of functional E-selectin ligand carbohydrates in ccRCC. We then carried out statistical analysis to determine whether membrane expression of HECA-452-reactive sLex/sLea glycans as well as of E-selectin•IgM-binding functional E-selectin ligand carbohydrates correlates with progression-free, overall, or cancer-specific survival. RESULTS: Based on HECA-452 immunochemistry, 106 of 117 ccRCC specimens expressed detectable levels of sLex/sLea glycans, primarily on the plasma membrane, and of those, 31 that showed robust membrane expression were judged as HECA-452-positive. Membrane expression of HECA-452-positive sLex/sLea glycans correlated with shortened progression-free and overall survival. Moreover, in in situ analysis, these HECA-452-positive ccRCC tissues were decorated with E-selectin•IgM chimeric proteins, calcium-dependently. Comparable analysis in normal kidney showed both HECA-452 positivity and chimera binding almost exclusively in epithelial cells that constitute proximal tubules. Membrane expression of functional E-selectin ligand carbohydrates, as detected by the E-selectin•IgM chimera, correlated more significantly with poor prognosis of patients, namely, shortened progression-free, overall and cancer-specific survival, than did HECA-452 positivity. CONCLUSIONS: Expression of E-selectin•IgM-binding functional E-selectin ligand carbohydrates can serve as a reliable and potentially superior prognostic biomarker of patients with ccRCC.

The Conspicuousness of High Endothelial Venules in Angioimmunoblastic T-cell Lymphoma Is Due to Increased Cross-sectional Area, Not Increased Distribution Density.

Angioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma of follicular helper T-cell origin. Histologically, neoplastic T-cells proliferate to form clusters adjacent to or between arborizing high endothelial venules (HEVs). HEVs in normal lymph nodes express sulfated glycans called peripheral lymph node addressin (PNAd); however, it remains unclear whether PNAd is also expressed on HEVs in AITL. Furthermore, although it is widely accepted that HEVs are conspicuous in AITL due to their proliferation, quantitative histological support for this concept is lacking. To investigate these issues, we employed monoclonal antibodies recognizing PNAd, namely, MECA-79, HECA-452, and 297-11A, and performed quantitative immunohistochemical analysis of HEVs in 36 AITL-affected and 67 normal lymph nodes. Staining with all three antibodies confirmed that AITL HEVs express PNAd. Moreover, AITL HEVs were bound calcium-dependently by L-selectin-IgM fusion proteins, indicating that they function in the recruitment of L-selectin-expressing lymphocytes. Unexpectedly, HEV distribution density was not increased but rather decreased in AITL compared with normal lymph nodes, but HEV cross-sectional area in AITL was significantly greater than that seen in normal lymph nodes. Overall, these results indicate that the prominence of AITL HEVs is likely due to increased cross-sectional area rather than increased distribution density.