Prohaptoglobin is a possible prognostic biomarker for colorectal cancer.

BACKGROUND AND AIMS: Fucosylated haptoglobin is a novel glycan biomarker for colorectal and other cancers, while the significance of its precursor, prohaptoglobin (proHp), remains to be elucidated. In this study, we investigated whether proHp can be a colorectal cancer (CRC) biomarker and the biological functions of proHp in CRC using 10-7G, a monoclonal antibody recently developed in our laboratory. MATERIALS AND METHODS: Serum proHp level in 74 patients with CRC was semi-quantified by western blotting, and 5-year recurrence-free survival and overall survival were analyzed for groups stratified by proHp status (high vs. low). We also performed immunohistochemical analyses of 17 CRC tissue sections using 10-7G mAb. The biological functions of proHp were evaluated by overexpressing proHp in CRC cell lines. RESULTS: Serum proHp correlated with the clinical stage and poorer prognosis of CRC. In the primary CRC sections, immune cells were stained positive for 10-7G in ∼50% of the cases. Overexpression of proHp in HCT116 human CRC cells induced epithelial-mesenchymal transition-like changes and promoted cell migration in CRC cells. CONCLUSION: We provide evidence for the first time that proHp has potential as a prognostic biomarker for CRC and demonstrated specific biological activities of proHp.

The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro.

Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

Establishment of a novel 70K Mac-2 binding protein antibody through screening of fucosylation-related antibodies.

Mac-2 binding protein (Mac-2bp) is a serum glycoprotein that contains seven N-glycans, and Mac-2bp serum levels are increased in patients with several types of cancer and liver disease. Mac-2bp glycosylation isomer has been applied as a clinical biomarker of several diseases, including liver fibrosis. In the present study, we identified fucosylated Mac-2bp in the conditioned medium of cancer cells resistant to anticancer therapies using glycoproteomic analyses. Fucosylation is one of the most important types of glycosylation involved in carcinogenesis and cancer stemness. To establish a next-generation glycan antibody for fucosylated Mac-2bp, we used fucosylation-deficient HEK293T cells to prepare reference Mac-2bp antigens and performed antibody screening. Unexpectedly, the 19-8H mAb obtained with our screen recognized 70K Mac-2bp, which is C-terminus-truncated product, rather than specifically recognizing fucosylated Mac-2bp. We performed immunocytochemistry using our novel 19-8H mAb, which resulted in strong cell surface staining of anticancer drug-resistant cancer cells. Therefore, our novel 19-8H mAb represents a valuable tool for cancer biology research that can help elucidate the biological function of 70K Mac-2bp.

Twin research shows glycan changes are more susceptible to environmental factors than their carrier glycoproteins.

Changes in protein glycosylation are clinically used as biomarkers. In the present study, we employed a twin cohort to investigate the contributions of genetic and environmental factors to glycan modifications of glycoproteins. Mac-2 binding protein (Mac-2 bp), haptoglobin (Hp), and their glycosylated forms are liver fibrosis and cancer biomarkers. Sera from 107 twin pairs without clinical information were used as a training cohort for the Mac-2 bp and Mac-2 bp glycosylation isomer (M2BPGi) assay. As a validation cohort, 22 twin pairs were enrolled in the study. For each twin pair, one twin was diagnosed with liver or pancreatic disease. For the training cohort, the correlation ratios of serum Mac-2 bp and M2BPGi levels in twin sera with random sequences were 0.30 and 0.018, respectively. The correlation ratios between twin pairs in the validation cohort for serum Mac-2 bp and M2BPGi levels were 0.75 and 0.35, respectively. In contrast, correlation ratios of serum Hp and fucosylated haptoglobin (Fuc-Hp) levels between twin sera with liver and pancreatic disease were 0.49 and 0.16, respectively. Although serum protein levels of glycoproteins are susceptible to genetic factors, characteristic glycan changes of these glycoproteins are more susceptible to environmental factors, including liver and pancreatic disease.

Transcription factor SP1 regulates haptoglobin fucosylation via induction of GDP-fucose transporter 1 in the hepatoma cell line HepG2.

Fucosylation is involved in cancer and inflammation, and several fucosylated proteins, such as AFP-L3 for hepatocellular carcinoma, are used as cancer biomarkers. We previously reported an increase in serum fucosylated haptoglobin (Fuc-Hp) as a biomarker for several cancers, including pancreatic and colon cancer and hepatocellular carcinoma. The regulation of fucosylated protein production is a complex cellular process involving various fucosylation regulatory genes. In this report, we investigated the molecular mechanisms regulating Fuc-Hp production in cytokine-treated hepatoma cells using a partial least squares (PLS) regression model. We found that SLC35C1, which encodes GDP-fucose transporter 1 (GFT1), is the most responsible factor for Fuc-Hp production among various fucosylation regulatory genes. Furthermore, the transcription factor SP1 was essential in regulating SLC35C1 expression. We also found that an SP1 inhibitor was able to suppress Fuc-Hp production without affecting total Hp levels. Taken together, Fuc-Hp production was regulated by SP1 via induction of GFT1 in the hepatoma cell line HepG2.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1ß converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.

Enterococcus spp. have higher fitness for survival, in a pH-dependent manner, in pancreatic juice among duodenal bacterial flora.

BACKGROUND AND AIM: Bacterial infection is involved in the progression of many gastrointestinal diseases, including those of pancreas; however, how and which bacteria colonize in pancreatic juice and tissue have yet to be elucidated. Recently, we reported that Enterococcus faecalis exists in the pancreatic juice and tissues of patients with chronic pancreatic disease. Here, we investigated the survival of E. faecalis in duodenal juice with different pH conditions. METHODS: Pancreatic juice samples from 62 patients with cancers of the duodeno-pancreato-biliary region were evaluated for the presence of E. faecalis. 16S ribosomal RNA polymerase chain reaction and 16S-based metagenome analyses were performed to determine the bacterial composition. The survival of E. faecalis in various pancreatic juice conditions was evaluated. RESULTS: Of 62 samples, 27% (17/62) were positive for Enterococcus spp., among which 71% (12/17) contained E. faecalis. Enterococcus spp. showed the highest fitness for survival in alkaline pancreatic juice among various bacterial species. The microbiome of pancreatic juice from patients with pancreatic and bile duct cancer showed diversity, but Enterococcus spp. were enriched among duodenal tumors and intraductal papillary mucinous neoplasms. CONCLUSIONS: Alkalinity is one of the important factors for the selective survival of E. faecalis among microbiota. E. faecalis can colonize the pancreatic duct when the pancreatic juice condition is altered.

Challenges in the Application of Glyco-Technology to Hepatitis B Virus Therapy and Diagnosis.

Hepatitis B virus (HBV) is a major pathogen that causes acute/chronic hepatitis. Continuous HBV infection can lead to the development of hepatocellular carcinoma (HCC). Although several different anti-HBV treatments are available for chronic hepatitis B patients, discontinuing these medications is difficult. Patients with chronic hepatitis B at high risk for HCC therefore require close observation. However, no suitable biomarkers for detecting high-risk groups for HCC exist, except for serum HBV-DNA, but a number of HCC biomarkers are used clinically, such as alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II). Glycosylation is an important post-translational protein modification involved in many human pathologic conditions. HBV surface proteins contain various oligosaccharides, and several reports have described their biological functions. Inhibition of HBV glycosylation represents a potential novel anti-HBV therapy. It is thought that glycosylation of hepatocytes/hepatoma cells is also important for HBV infection, as it prevents HBV from infecting cells other than hepatocytes, even if the cells express the HBV receptor. In this review, we summarize considerable research regarding the relationship between HBV and glycosylation as it relates to the development of novel diagnostic tests and therapies for HBV.

Branched-chain amino acids protect the liver from cirrhotic injury via suppression of activation of lipopolysaccharide-binding protein, toll-like receptor 4, and signal transducer and activator of transcription 3, as well as Enterococcus faecalis translocation.

OBJECTIVES: Branched-chain amino acids (BCAAs) are used as nutritional support and for improving prognosis in liver cirrhosis. Here we investigate the molecular mechanisms of BCAA treatment and liver damage focused on pathways related to lipopolysaccharide-binding protein (LBP). METHODS: Serum LBP levels were measured in cirrhotic patients and in cirrhotic rats treated with BCAA to examine the correlation between liver function and survival. In cirrhotic rats, liver damage, Enterococcus faecalis translocation, serum capsular polysaccharide, and intestinal tight junction levels were assessed. Damaged HepG2 cells were cultured with BCAA-supplemented, BCAA-deficient, or control amino acid medium, followed by examination of LBP expression. RESULTS: Serum LBP levels were significantly increased in deceased patients individuals with liver cirrhosis. The survival rate in patients with lower serum LBP (<3.48 µg/mL) was significantly improved. In BCAA-treated rat liver samples, protein expression of LBP, toll-like receptor 4 (TLR4), and phosphorylated signal transduction and activator of transcription 3 (STAT3) were significantly reduced. Also in BCAA-treated rats, intestinal zonula occludens gene expression was increased, whereas hepatic translocation of E. faecalis and serum capsular polysaccharide levels were reduced. In damaged HepG2 cells, lipopolysaccharide-induced elevation of LBP expression was rapidly and strongly repressed in BCAA-enriched medium. CONCLUSIONS: Serum LBP level is a prognostic biomarker in liver cirrhosis. BCAA treatment reduced translocation of E. faecalis through intestinal tight junction recovery and reduced LBP expression in the liver, which repressed activation of LBP, toll-like receptor 4, and signal transduction and activator of transcription 3. Our findings suggest that BCAA supplementation protects the liver from damage via multiple pathways.

Identification of fucosylated haptoglobin-producing cells in pancreatic cancer tissue and its molecular mechanism.

Fucosylated haptoglobin is a well-established glyco-biomarker of pancreatic cancer. We recently established a novel anti-glycan antibody (10-7G mAb) that specifically recognizes fucosylated haptoglobins, including prohaptoglobin (proHpt). Serum concentrations of the 10-7G value, as measured by ELISA, were increased in patients with pancreatic cancer relative to the healthy controls. However, it is currently unknown which specific tissue or cell type produces fucosylated haptoglobins or proHpt. In the present study, we performed immunohistochemical (IHC) and ELISA analyses of pancreatic cancer tissue samples using 10-7G mAb. Among 21 pancreatic tissue sections, only 1 showed direct staining of pancreatic cells with the 10-7G mAb. However, 12 of the 21 sections stained positively for immune cells. Although there was no significant difference in the 10-7G expression between the positive and negative staining IHC groups, the median value of serum 10-7G was slightly higher in IHC-positive cases. Among many assayed leukemic cell lines, differentiated THP-1 cells (a human acute monocytic leukemia cell line) were found to have the highest levels of proHpt, per Western blot using 10-7G mAb. Interestingly, production of proHpt in vitro was dramatically increased under either hypoxic conditions or after IL-6 treatment. These results suggest that immune cells, including macrophages, in the pancreatic tissue microenvironment produce fucosylated haptoglobin and proHpt. Thus, fucosylated haptoglobins can be detected by the 10-7G mAb and may be a promising biomarker for pancreatic cancer.

Detection of fucosylated haptoglobin using the 10-7G antibody as a biomarker for evaluating endoscopic remission in ulcerative colitis.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammation of the digestive tract. Although fecal and serum biomarkers have been extremely important and supportive for monitoring of IBD, their low sensitivity and high variability characteristics limit clinical efficacy. Thus, the establishment of better biomarkers is expected. Fucosylation is one of the most important glycosylation modifications of proteins. Fucosylated haptoglobin (Fuc-Hpt) is used as a biomarker for several cancers and inflammation-related diseases. We recently established a novel glycan monoclonal antibody (mAb), designated 10-7G, which recognizes Fuc-Hpt. We developed an enzyme-linked immunosorbent assay (ELISA) to measure serum levels of Fuc-Hpt (10-7G values). AIM: To investigate the usefulness of the serum 10-7G values as a potential biomarker for monitoring disease activity in IBD. METHODS: This was a case control study. Intestinal tissues of IBD patients (n = 10) were examined immunohistochemically using the 10-7G mAb. We determined 10-7G values using serum from patients with ulcerative colitis (UC, n = 110), Crohn's disease (n = 45), acute enteritis (AE, n = 11), and healthy volunteers (HVs) who exhibited normal (n = 20) or high (n = 79) C-reactive protein (CRP) levels at medical check-up. We investigated the correlation between the 10-7G value and various clinical parameters of IBD patients by correlation analysis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the usefulness of the 10-7G values as a biomarker for clinical and endoscopic remission of UC compared to conventional serum biomarkers. RESULTS: In the immunohistochemical analysis, positive 10-7G mAb staining was observed in lymphocytes infiltrating into inflammatory sites of the mucosal layer and lymphoid follicles. The 10-7G values were significantly higher in patients with IBD (P < 0.001) and AE (P < 0.05) compared with HVs. In addition, 10-7G values were correlated with clinical examination parameters related to inflammation in patients with UC, particularly the CRP level (rs = 0.525, P = 0.003) and clinical activity index score (rs = 0.435, P = 0.038). However, there was no correlation between 10-7G values and CRP in HVs with high CRP levels, suggesting that the 10-7G values is not the same as a general inflammation biomarker. ROC curve analysis showed that area under the curve (AUC) value of 10-7G values for the diagnosis of endoscopic remission was higher than other biomarkers (AUC value = 0.699). CONCLUSION: The serum 10-7G value is a novel biomarker for evaluating intestinal inflammation and endoscopic mucosal healing in UC.

Serum Mac-2 Binding Protein Levels Associate with Metabolic Parameters and Predict Liver Fibrosis Progression in Subjects with Fatty Liver Disease: A 7-Year Longitudinal Study.

Background: Mac-2 binding protein (M2BP) is a highly glycosylated secreted glycoprotein that is involved in immune defense and regulation. Our cross-sectional studies indicated that serum M2BP was a useful liver fibrosis biomarker for nonalcoholic fatty liver disease (NAFLD). In this study, we conducted a 7-year longitudinal study to investigate the significance of serum M2BP levels (baseline and at 7-year follow-up) and their relationships with other metabolic parameters of fatty liver disease. Methods: We enrolled 715 study subjects (521 male and 194 female) during health examinations. Study subjects received blood sampling tests and abdominal ultrasound tests at baseline and follow-up. Results: Univariate analyses demonstrated that serum M2BP levels were significantly correlated with various parameters related to metabolic risk (body mass index (BMI), systolic blood pressure, triglyceride, high density lipoprotein (HDL)-cholesterol) and metabolic syndrome diseases (obesity, hypertension, dyslipidemia, diabetes mellitus, fatty liver (FL)). Multiple logistic regression analyses demonstrated that BMI and FL were independent determinants for serum M2BP levels. Baseline serum M2BP levels were significant independent determinants for changes in platelet count, Fibrosis-4 (FIB4) index, and NAFLD fibrosis score. Higher serum M2BP levels (>1.80 µg/mL) strongly correlated with changes in the FIB4-index. Conclusions: The results of this study suggest that changes in serum M2BP levels reflect changes in specific metabolic disease-related parameters, and baseline serum M2BP levels could predict changes in liver fibrosis.

Loss of core fucosylation reduces low-density lipoprotein receptor expression in hepatocytes by inducing PCSK9 production.

Fucosylation is a type of glycosylation, a form of post-transcriptional regulation of proteins, involved in cancer and inflammation. It involves the attachment of a fucose residue to N-glycans, O-glycans, and glycolipids, which is catalyzed by a family of enzymes called fucosyltransferases (Futs). Among the many Futs, α-1,6-fucosyltransferase (Fut8) is the only enzyme that produces α-1,6-fucosylated oligosaccharides (core fucose). In the human liver, the expression and activity of Fut8 are frequently elevated during progression of chronic liver diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-known negative regulator of the low-density lipoprotein receptor (LDLR). Here, we found that loss of core fucose in immortalized hepatocytes led to LDLR downregulation through a dramatic induction of PCSK9. We used the immortalized hepatocytes derived from Fut8 knockout mice or a Fut8 knockdown AML12 hepatocyte cell line. Using these cells, we investigated the effects of Fut8 on hepatocyte cholesterol influx. Both cell lines had reduced LDLR protein levels, resulting from marked increases in PCSK9 expression. Intracellular cholesterol levels were significantly lower and LDL cholesterol uptake was suppressed in Fut8-KO cells. Hepatocyte nuclear factor 1α accumulated in nuclei of Fut8-KO hepatocytes, which mediated increases in PCSK9 mRNA expression. Our findings demonstrated that loss of core fucosylation promoted degradation of LDLR and impaired cholesterol uptake, which is a novel mechanism that regulates cholesterol influx, suggesting that Fut8 might be a novel causative gene for familial hypercholesterolemia.

Identification of the epitope of 10-7G glycan antibody to recognize cancer-associated haptoglobin.

We previously identified fucosylated haptoglobin (Fuc-Hpt) as a clinical serum biomarker of pancreatic cancer and established the novel glycan monoclonal antibody (mAb) 10-7G. This antibody recognizes cancer-associated haptoglobin including Fuc-Hpt and the precursor of haptoglobin. Interestingly, Western blot analysis showed that the 10-7G mAb reacts with the haptoglobin α chain, which has no N-glycan potential sites; haptoglobin ß chain has four N-glycan sites. In this study, we identified the epitope for the 10-7G mAb using haptoglobin deletion mutants, as well as inhibition ELISA with recombinant peptides. We illustrated molecular graphics to show a relationship between the epitope and the ß chain. Furthermore, we hypothesized that the 10-7G mAb minimally recognizes normal haptoglobin, but aberrant glycosylation on the ß chain causes conformational changes, enabling the 10-7G mAb to easily access the epitope within the α chain. Because 10-7G values, an enzyme-linked immunosorbent assay-immobilized 10-7G mAb, in patients with pancreatic cancer varied by haptoglobin phenotype, the amount of aberrant glycosylation needed to affect haptoglobin conformation probably depends on haptoglobin phenotype. Taken together, the 10-7G mAb recognized characteristic peptides on the haptoglobin α chain as a result of conformational changes and is a promising tool for diagnosing pancreatic cancer by haptoglobin phenotype.

Development of α-Gal-Antibody Conjugates to Increase Immune Response by Recruiting Natural Antibodies.

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.

Core fucose is essential glycosylation for CD14-dependent Toll-like receptor 4 and Toll-like receptor 2 signalling in macrophages.

Core fucosylation, catalysed by α-1, 6 fucosyltransferase (FUT8), regulates growth factor receptors in immune function. Although core fucose regulates many immune cell types, few reports confront the association between core fucose activity and an innate immune reaction. Here, we have investigated the function of core fucose in macrophages in vivo and in vitro using Fut8-deficient mice and cells. Following lipopolysaccharide (LPS) stimulation, inflammatory cytokine production in Fut8-deficient (Fut8-/-) macrophages was suppressed in both in vivo and in vitro experiments. Because LPS is recognized by Toll-like receptor 4 (TLR4), which induces the signalling cascade, TLR4 signalling was assumed to be impaired in Fut8-/- cells. Flow cytometry analyses revealed, however, that a lack of core fucose reduced the expression of, not TLR4, but CD14, which is necessary for TLR4 endocytosis. Because CD14 is necessary for TLR2 signalling, the immune response of TLR2 was also impaired in Fut8-/- macrophages. Moreover, in the dextran sodium sulphate (DSS)-induced murine colitis model, the mice grafted with Fut8-/- bone marrow cells exhibited higher resistance to inflammation than those grafted with Fut8+/+ bone marrow cells. These findings indicate that core fucose is essential for CD14-dependent TLR4 and TLR2 signalling in murine macrophage activity, leading to DSS-induced experimental colitis.

Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer.

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.