FNAIT pathogenesis determined by serial analysis of three subsequent pregnancies of a woman with severe fetal and neonatal alloimmune thrombocytopenia (FNAIT) with anti-HPA-4b and anti-HPA-5b alloantibodies in the first sibling.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by anti-HPA alloantibody, and anti-HPA-4b is the most common cause in Japanese. Anti-HPA-5b is frequently detected in pregnant women, but it is still controversial whether anti-HPA-5b causes severe FNAIT. CASE PRESENTATION: A Japanese woman with anti-HPA-4b and anti-HPA-5b alloantibodies delivered a baby with severe FNAIT who was both HPA-4b and HPA-5b incompatible. We carefully monitored the patient's following three pregnancies (the second and the fourth siblings were HPA-4b incompatible and HPA-5b compatible; the third sibling was both HPA-4b and HPA-5b compatible). FNAIT was not observed in all three siblings, although a modest decrease in cord blood platelet count was observed in the HPA-4b incompatible siblings compared to the HPA-4b compatible sibling. Serial monitoring of anti-HPA titer showed that anti-HPA-4b markedly decreased in late pregnancy and recovered after delivery of the HPA-4b incompatible siblings, but these decreases were not observed during the mother's pregnancy with the HPA-4b compatible sibling. In contrast, anti-HPA-5b remained at a high titer during pregnancy with all three siblings. CONCLUSION: Our data indicate that dynamic changes of anti-HPA-4b occur during pregnancy and strongly suggest that anti-HPA-5b was mainly responsible for severe FNAIT in this case.

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.

Haptoglobin phenotype is a critical factor in the use of fucosylated haptoglobin for pancreatic cancer diagnosis.

Fucosylation is one of the most important glycosylations involved in cancer and inflammation. Many studies have reported significant increases in serum fucosylated haptoglobin (Fuc-Hpt) in a variety of cancer patients. In this study, we measured Fuc-Hpt using a lectin-antibody enzyme-linked immunosorbent assay (ELISA) or a novel ELISA system that used a glycan antibody for Fuc-Hpt. Hpt is known to be divided into three phenotypes (Hpt1-1, Hpt2-1, and Hpt2-2), depending on its genetic background. Normal levels of serum Hpt are different in each Hpt phenotype and these phenotypes are associated with the incidence of several human diseases. Here, we investigated how Hpt phenotype affected measurements of Fuc-Hpt, using two kinds of ELISA. Interestingly, we found that serum Fuc-Hpt levels were dramatically lower in the Hpt1-1 phenotype for both types of ELISA. For Hpt2-1 and Hpt2-2, we observed significantly increased serum Fuc-Hpt levels in patients with pancreatic cancer. When cases of the Hpt1-1 phenotype were depleted, our receiver operating characteristic (ROC) curve analyses showed that the area under the curve (AUC) value for pancreatic cancer diagnosis increased in each ELISA. Taken together, our results indicate that Hpt phenotype is a critical for the clinical application of Fuc-Hpt as a cancer biomarker.

Distinct effects of daratumumab on indirect and direct antiglobulin tests: a new method employing 0.01 mol/L dithiothreitol for negating the daratumumab interference with preserving K antigenicity (Osaka method).

BACKGROUND: There is an increasing demand for daratumumab (DARA), an immunoglobulin (Ig)G1κ monoclonal antibody (MoAb) that recognizes CD38, to manage relapsed or refractory multiple myeloma (MM) patients. However, DARA leads to positive and panreactive agglutination reactions in indirect antiglobulin tests (IATs) in vitro (the DARA interference). In addition, effects of DARA on red blood cells (RBCs) in vivo remains elusive. STUDY DESIGN AND METHODS: To develop a new method to negate the DARA interference, the effects of various concentrations of dithiothreitol (DTT) on RBC CD38 and Kell antigenicity in combination with an automatic blood cell washing centrifuge were compared with the AABB standard procedure in parallel. Moreover, direct antiglobulin tests (DATs) for RBCs in DARA-treated MM patients were examined. RESULTS: A quantity of 0.01 mol/L DTT as well as the AABB procedure (equivalent to 0.15 mol/L DTT in our procedure) markedly reduced the reactivity of phycoerythrin-mouse anti-CD38 MoAb and DARA with RBCs. In sharp contrast to the AABB procedure, 0.01 mol/L DTT partially preserved K antigenicity and allowed the determination of phenotype of K antigen even in the presence of the DARA interference. In contrast, DAT for RBCs obtained from MM patients showed a weak positive or negative reaction. Immunoblotting further indicated that DARA induced loss of CD38 in vivo. CONCLUSION: A simple and reliable method to negate the DARA interference with partially preserving Kell antigenicity is proposed (Osaka method). CD38 antigenicity is susceptible to 0.01 mol/L DTT treatment even in the presence of DARA. Our data also demonstrate distinct effects of DARA on IAT in vitro and DAT in vivo.

Serum Mac-2 binding protein is a novel biomarker for chronic pancreatitis.

AIM: To determine the efficacy of Mac-2 binding protein (Mac-2bp) for diagnosis of chronic pancreatitis. METHODS: Fifty-nine healthy volunteers (HV), 162 patients with chronic pancreatitis (CP), and 94 patients with pancreatic ductal adenocarcinoma (PDAC) were enrolled in this study. We measured serum Mac-2bp using our developed enzyme-linked immunosorbent assay kit. Additional biochemical variables were measured using an automated analyzer (including aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, triglyceride, C-reactive protein, and amylase levels) or chemiluminescent enzyme immunoassay (carbohydrate antigen 19-9 and carcinoembryonic antigen). The ability of Mac-2bp to predict CP diagnosis accurately was assessed using receiver operating characteristic (ROC) analyses. RESULTS: Serum Mac-2bp levels were significantly increased in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Area under the ROC curve values of Mac-2bp for the discrimination of CP from HV and PDAC were 0.727 and 0.784, respectively. Multivariate analyses demonstrated that serum Mac-2bp levels were independent determinants for CP diagnosis from HV and PDAC patients. Immunohistological staining showed that Mac-2bp was expressed faintly in the pancreas tissues of both CP and PDAC patients. Serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, and triglyceride levels were significantly higher in patients with CP or PDAC. Serum Mac-2bp levels were highly correlated with protein levels of alanine aminotransferase, γ-glutamyltransferase, and C-reactive protein, but not amylase, suggesting that the damaged liver produces Mac-2bp. CONCLUSION: Measurement of serum Mac-2bp may be a novel and useful biomarker for CP diagnosis as well as liver fibrosis in the general population.

Specific increase in serum core-fucosylated haptoglobin in patients with chronic pancreatitis.

BACKGROUND/OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all malignancies, and its diagnosis in early stages is the most important prognostic factor. Chronic pancreatitis (CP), a common background of PDAC occurrence, is morphologically defined as progressive pancreatic fibrosis and inflammation accompanied by pancreatic exocrine cell atrophy. We recently found that inflammation and fibrosis are independent characteristic histological changes in noncancerous lesions in PDAC patients despite the absence of a past history of clinical CP. Subclinical CP is an important background for PDAC occurrence. Therefore, there is an urgent need to develop a noninvasive and reliable biomarker for CP diagnosis. METHODS: Fifty-nine healthy volunteers (HV), 159 patients with CP, and 83 patients with PDAC were enrolled in this study. We measured serum total fucosylated haptoglobin (Fuc-Hpt) and core-Fuc-Hpt levels using lectin-antibody enzyme-linked immunosorbent assay kits that we developed. In these kits, total Fuc-Hpt and core-Fuc-Hpt were measured using Aleuria aurantia lectin and Pholiota squarrosa lectin, respectively. RESULTS: Serum Fuc-Hpt levels were significantly increased in CP patients compared to HV (P < 0.0001) and were further increased in PDAC patients (P < 0.0001). Interestingly, serum core-Fuc-Hpt levels were significantly higher in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Multivariate analyses demonstrated that total serum core-Fuc-Hpt was an independent determinant for CP diagnosis, but Fuc-Hpt was not. CONCLUSIONS: A dramatic change in oligosaccharides was observed in serum haptoglobin between CP and PDAC. Serum core-Fuc-Hpt may be a novel and useful biomarker for CP diagnosis.

Establishment of a novel lectin-antibody ELISA system to determine core-fucosylated haptoglobin.

BACKGROUND: Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker that increases in various pathological conditions. We previously established a Fuc-Hpt lectin-antibody assay using Aleuria aurantia lectin (AAL), and applied this to diagnose several diseases, including various cancers. AAL recognizes both α1-3/1-4 and α1-6 fucosylation on N/O-linked glycans. These fucosylation types differ in biological function, and in regulation by different fucosyltransferases. Recently, we identified a novel lectin, Pholiota squarrosa lectin (PhoSL), which specifically recognizes α1-6 fucosylation (core-fucosylation). METHODS: We developed a lectin-antibody ELISA kit using PhoSL to determine core-Fuc-Hpt levels in sera from colorectal or pancreatic cancer patients. RESULTS: Serum levels of AAL-reactive Hpt are higher in pancreatic cancer patients, whereas those of PhoSL-reactive Hpt are higher in colorectal cancer patients. Mass spectrometry analyses of Hpt fucosylation levels were consistent with lectin-antibody ELISA results. Hpt-transfected colorectal cancer cell lines produced significant amounts of core-Fuc-Hpt, suggesting that colorectal cancer tissues produce core-Fuc-Hpt. CONCLUSIONS: These differences in Fuc-Hpt patterns might depend on cancer cells and the surrounding cells, which produce Hpt.

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes.

AIM: To evaluate/isolate cancer stem cells (CSCs) from tissue or cell lines according to various definitions and cell surface markers. METHODS: Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. RESULTS: Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabine-resistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. CONCLUSION: Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.

A case of neonatal alloimmune thrombocytopenia in the presence of both anti-HPA-4b and anti-HPA-5b antibody: clinical and serological analysis of the subsequent pregnancy.

Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies raised against fetal platelet antigens inherited from the paternal parent. In contrast to Caucasians, in Asians, predominantly in Japanese, most frequently detected antibodies in NAIT are anti-HPA-4b and anti-HPA-5b. In some NAIT cases multiple alloantibodies are detected. In such cases it is very difficult to determine which antibody is the dominant antibody in NAIT. In this case report, we describe a NAIT case (first sibling) with severe thrombocytopenia and cephalhematoma in the presence of both anti-HPA-4b and anti-HPA-5b antibodies in the maternal serum. We carefully examined titers of anti-HPA antibodies during the subsequent pregnancy with HPA-4b-positive and HPA-5b-negative fetus determined by amniocentesis at gestational week 16. We administered IVIG (1 g/kg/w) to the mother from gestational week 32 to 35. The mother subsequently delivered a second sibling with normal platelet count by cesarean section. Although we could not completely rule out the involvement of anti-HPA-4b, our findings suggested that anti-HPA-5b was implicated in the NAIT in the first sibling.

Mutation of GDP-mannose-4,6-dehydratase in colorectal cancer metastasis.

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.

Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem around the world. NAFLD patients with nonalcoholic steatohepatitis (NASH) can develop cirrhosis and hepatocellular carcinoma. The ability to distinguish NASH from simple steatosis would be of great clinical significance. Ballooning hepatocytes are characteristic of typical pathological NASH; here, the polarized secretion of proteins is disrupted due to destruction of the cytoskeleton. We previously reported that fucosylated glycoproteins are secreted into bile, but not into sera in normal liver. Therefore, we hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes, and serum fucosylated glycoproteins would increase in NASH patients. To confirm our hypothesis, we evaluated serum fucosylated haptoglobin (Fuc-Hpt) levels in biopsy-proven NAFLD patients (n = 126) using a lectin-antibody ELISA kit. Fuc-Hpt levels were significantly increased in NASH patients compared with non-NASH (NAFLD patients without NASH) patients. Interestingly, Fuc-Hpt levels showed a significant stepwise increase with increasing hepatocyte ballooning scores. Multiple logistic regression analysis showed that Fuc-Hpt levels were independent and significant determinants of the presence of ballooning hepatocytes. Moreover, Fuc-Hpt levels were useful in monitoring liver fibrosis staging. Next, to investigate the significance of serum Fuc-Hpt in a larger population, we measured Fuc-Hpt levels in ultrasound-diagnosed NAFLD subjects (n = 870) who received a medical health checkup. To evaluate NAFLD disease severity, we used the FIB-4 index (based on age, serum AST and ALT levels, and platelet counts). Fuc-Hpt levels increased stepwise with increasing FIB-4 index. CONCLUSION: Measurement of serum Fuc-Hpt levels can distinguish NASH from non-NASH patients, and predict the presence of ballooning hepatocytes in NAFLD patients with sufficient accuracy. These results support the potential usefulness of measuring Fuc-Hpt levels in clinical practice.