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.

rBC2LCN-reactive SERPINA3 is a glycobiomarker candidate for pancreatic ductal adenocarcinoma.

Early detection is urgently needed to improve the patient's pancreatic ductal adenocarcinoma (PDAC) survival. Previously, we identified a novel tumor-associated glycan, H-type3, which is expressed on PDAC cells and is detected by rBC2LCN (recombinant N-terminal domain of BC2L-C identified from Burkholderia cenocepacia) lectin. Here, we identified that SERPINA3 is an rBC2LCN-reactive glycoprotein (BC2-S3) secreted from PDAC cells into the blood in patients with PDAC by liquid chromatography-tandem mass spectrometry analysis and lectin blotting. In immune staining, BC2-S3 was detected specifically in the tumor but not in normal tissues of PDAC. Lectin-ELISA was then developed to measure the serum level of BC2-S3 in healthy control (HC, n = 99) and patients with PDAC (n = 88). BC2-S3 exhibited higher in patients with PDAC than in those with HC. BC2-S3 showed similar diagnostic performance in all stages of PDAC (stages IA-IV, true positive rate = 76.1%, true negative rate = 81.8%) to CA19-9 (72.7%, 75.8%). Remarkably, BC2-S3 showed a significantly higher detection rate (89.7%) for early stage PDAC (IA-IIA) than CA19-9 (62.1%, P = 0.029). The combination of BC2-S3 and CA19-9 further improved the diagnostic ability for all stages of PDAC (81.8%, 87.9%). In conclusion, BC2-S3 is a glycobiomarker candidate for PDAC.

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.

Induction of specific adaptive immune responses by immunization with newly designed artificial glycosphingolipids.

We previously found that artificial glycosphingolipids (artGSLs) containing very-long-chain fatty acids behave as strong immunogens in mice and promote the production of antibodies recognizing the oligosaccharide portion of artGSLs as the epitope. Here, we report that the oligosaccharide structure of artGSLs influences these immunogenic properties. We evaluated the antibody-inducing activity of artGSLs with different oligosaccharide structures in mice and found strong IgG-inducing activity only with an artGSL containing a core-fucosylated tetraoligosaccharide (Manß1,4GlcNAcß1,4[Fucα1,6]GlcNAc). To characterize the immunogenic properties of this artGSL, we analyzed various derivatives and found that the non-reducing terminal mannose structure was critical for the antibody-inducing activity. These artGSLs also exhibited IgG-inducing activity dependent on co-administration of lipid A adjuvant, but no cytokine-inducing activity similar to α-galactosylceramide was detected. Furthermore, repetitive immunization with the artGSL promoted the production of antibodies against a core-fucosylated α-fetoprotein isoform (AFP-L3) known as a hepatocellular carcinoma-specific antigen. These results indicate that the newly designed artGSLs specifically induce adaptive immune responses and promote antibody production by B cells, which can be utilized to develop anti-glycoconjugate antibodies and cancer vaccines targeting tumor-associated carbohydrate antigens.

Rectal mucosal prolapse syndrome as an unusual gastrointestinal manifestation of Sjögren's syndrome: a case report.

INTRODUCTION: Rectal mucosal prolapse syndrome, histologically characterized by fibromuscular obliteration in the lamina propria, hyperplastic glands and thickened muscularis mucosa, causes rectal bleeding. Sjögren's syndrome is an autoimmune exocrinopathy that chiefly destroys the salivary and lacrimal glands by lympho-plasmacytic infiltration. Although various gastrointestinal manifestations have been reported in patients with Sjögren's syndrome, there have not been to our knowledge any case reports to date of rectal mucosal prolapse syndrome in association with Sjögren's syndrome. CASE PRESENTATION: A 68-year-old Japanese woman with Sjögren's syndrome and long-term constipation consulted our hospital because of rectal bleeding. Because of dysphagia and xerostomia, she had consistently refused recommendations to take oral medicines including cathartics. Therefore, she frequently strained excessively during defecation. Colonoscopy and radiological examinations disclosed eroded flat protrusions of the rectum. Microscopic examination demonstrated inflamed mucosa with elongated tortuous glands and fibromuscular obliteration. Based on these findings, a diagnosis of rectal mucosal prolapse syndrome was made. Prohibition of straining during defecation and sulfasalazine suppository use were effective. CONCLUSION: This case highlights the importance of defecation control in patients with Sjögren's syndrome. In the case presented, rectal mucosal prolapse syndrome following long-term excessive straining during defecation caused rectal bleeding. Clinicians should consider rectal mucosal prolapse syndrome as a gastrointestinal manifestation of Sjögren's syndrome.

Evaluation of a new method for the diagnosis of alterations of Lens culinaris agglutinin binding of thyroglobulin molecules in thyroid carcinoma.

BACKGROUND: The measurement of serum thyroglobulin (Tg) is widely used as a marker for recurrence of thyroid carcinoma following total thyroidectomy. However, this method cannot differentiate between benign and malignant disease. We focused on the sugar chain in the Tg molecule and investigated the usefulness of Lens culinaris agglutinin (LCA)-reactive Tg ratios in sera and wash fluids obtained during fine-needle aspiration (FNA) for the detection of thyroid carcinoma. METHODS: The study was performed using 203 serum samples (115 from patients with benign thyroid disease and 88 from patients with thyroid carcinomas) and 176 wash fluid samples (143 benign, 21 malignant, and 12 inconclusive). LCA-reactive Tg ratios were determined using an enzyme-linked immunosorbent assay, and a comparison was made between malignant and benign lesions. RESULTS: In serum, the ratio in patients with malignancy was 79.5+/-6.0 [mean+/-standard deviation (SD)], significantly lower than in patients with benign lesions (84.9+/-3.5). The ratios in wash fluid from malignant lesions (75.8+/-18.9) were also significantly lower than those from benign lesions (85.6+/-3.9). CONCLUSIONS: These results suggest that this method could distinguish between benign and malignant lesions and may be useful for screening serum and wash samples.

An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

Trend of drug-resistant HIV type 1 emergence among therapy-naive patients in Nagoya, Japan: an 8-year surveillance from 1999 to 2006.

We studied the emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) with major amino acid mutations in 402 therapy-naive patients at Nagoya Medical Center, Japan, between 1999 and 2006. The mean prevalence of drug-resistant HIV-1 was 6.7% (range, 2.3-10.0%; n = 27). HIV-1 variants with protease inhibitor (PI)-resistant mutations alone were most frequently found (3.5%, n = 14), followed by those with nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutations alone (1.7%, n = 7). Variants with nucleoside reverse transcriptase inhibitor (NRTI)-resistant mutations alone were sporadically found (1.0%, n = 4). A variant possessing both NRTI- and PI-resistant mutations was detected in one patient (0.2%) and a variant possessing both NNRTI- and PI-resistant mutations was identified in another patient (0.2%). In addition, another 17 variants (4.2%, n = 17) with only 215-revertant mutations (T215C/D/G/L/S) that can easily reconvert to the nucleoside analogue-associated mutation of T215Y/F were found. The 402 viruses were phylogenetically analyzed, revealing three independent clusters comprising PI-resistant variants with the M46I or L90M mutation, NNRTI-resistant variants with the K103N mutation, and 215-revertant variants. The PI-resistant and 215-revertant strains have been spreading since 2000, and the NNRTI-resistant strain has started spreading since 2003. The nature of the epidemic and information for successfully blocking the spread of drug-resistant HIV-1 were clarified in this study.

Discrimination of thyroglobulin from thyroid carcinoma tissue and that from benign thyroid tissues with use of competitive assay between lectin and anti-thyroglobulin antibody.

Thyroglobulin is produced only by thyroid follicular cells, and has a molecular weight of 660,000 and carbohydrate content of approximately 10%. The composition of carbohydrate chains on thyroglobulin from thyroid carcinoma has been reported to differ from that in normal thyroid tissue. In this study, heterogeneities of carbohydrate chains on thyroglobulin obtained from thyroid tissues were investigated by competitive reaction between lectin and anti-thyroglobulin monoclonal antibody. Concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin-120 and Datura stramonium agglutinin were compared. The ratio of Lens culinaris agglutinin-reactive thyroglobulin to thyroglobulin was significantly lower in thyroid carcinoma than in normal thyroid tissue, Graves' disease and benign thyroid tumor. However, no differences between malignant and benign tissues were observed with the other lectins tested. Differences in carbohydrate chain on thyroglobulin were observed between malignant and benign thyroid tissues.

Microcirculatory changes in experimental mesenteric longitudinal ulcers of the small intestine in rats.

Rats receiving intracolonic administration of indomethacin develop longitudinal ulcers on the mesenteric side of the small intestine that are similar to those seen in the acute phase of Crohn's disease. To investigate the causative role of microcirculatory disturbances and to elucidate the therapeutic effect of antioxidants on this enteropathy in rats, we serially evaluated changes in regional blood flow of the small intestine using laser Doppler perfusion imaging and the colored microsphere injection method. Both methods disclosed stepwise hyperperfusion limited to the mesenteric side of the small intestine following transient ischemia during the initial 30-60 minutes. In addition, both a radical scavenger and a radical production inhibitor significantly ameliorated the mesenteric longitudinal ulcers. We concluded that ischemia-reperfusion on the mesenteric side accompanying excessive production of radicals might be strongly involved in indomethacin-induced longitudinal ulcers of the small intestine in rats.

The clinical utility of Lens culinaris agglutinin-reactive thyroglobulin ratio in serum for distinguishing benign from malignant conditions of the thyroid.

BACKGROUND: Traditionally, the follow-up of differentiated thyroid carcinoma consists of periodic withdrawal from L-T4-suppressive therapy to allow performance of a highly sensitive serum Tg measurement to detect recurrences. We investigated Lens culinaris agglutinin-reactive thyroglobulin ratios in serum to evaluate in usefulness for detection of thyroid carcinoma. METHODS: The study was conducted on 93 serum sample from 23 healthy volunteers, 32 patients with benign thyroid tumor, 28 patients with thyroid carcinoma without metastasis, and 10 patients with thyroid carcinoma with lymph node metastasis. RESULTS: The Lens culinaris Agglutinin reactive thyroglobulin ratio in patients with thyroid carcinoma was significantly lower than in patients with benign thyroid tumor with serum thyroglobulin concentration >200 ng/ml. Among cases of thyroid carcinoma with lymph node metastasis, Lens culinaris Agglutinin reactive thyroglobulin ratios were significantly lower than in patient with thyroid carcinoma without metastasis and those with benign tumor regardless of serum thyroglobulin concentration. CONCLUSION: Measurement of Lens culinaris Agglutinin reactive thyroglobulin ratio in serum may be useful for distinguishing between thyroid carcinoma and benign thyroid tumor.

A study of oligosaccharide variants of alpha-fetoproteins produced by normal fetuses and fetuses with trisomy 21.

BACKGROUND: The mechanisms of the increase in the percentage of alpha-fetoproteins (AFPs) that strongly binds to Lens culinaris agglutinin (AFP-L3) in pregnancies with a trisomy 21 fetus have not been analyzed. To investigate the oligosaccharide variants of AFP produced by normal fetuses and fetuses with trisomy 21, the lectin reactivity of AFP was analyzed. METHODS: Fetal liver tissue, amniotic fluid, and maternal serum were obtained from five normal pregnancies and five pregnancies with a trisomy 21 fetus. The percentages of AFP reactive to lectins were determined by lectin-affinity electrophoresis coupled with antibody-affinity blotting. RESULTS: The percentages of AFP-L3 in the fetal liver and the maternal serum were 23.9 and 27.0%, respectively, in normal pregnancies, and 28.7 and 38.5%, respectively, in pregnancies with a trisomy 21 fetus. There was no statistically significant difference between the percentage in the fetal liver and the percentage in the maternal serum in normal pregnancies; however, a significant difference (P < 0.01) was found in pregnancies with a trisomy 21 fetus. In regard to the percentage of AFP-L3 in the fetal liver, there was no significant difference; however, a significant difference (P < 0.05) was found in the maternal serum between normal pregnancies and pregnancies with a trisomy 21 fetus. CONCLUSIONS: The transference of the AFP-L3 fraction might be relatively high in the placentas of women carrying a trisomy 21 fetus, and this could be the one of the reasons for the increase in the percentage of AFP-L3 in the maternal serum in pregnancies with a trisomy 21 fetus.

A study on the microheterogeneity of alpha-fetoproteins produced by yolk sac and germ cell tumors.

BACKGROUND: It is generally believed that the lower the grade of differentiation of glycoprotein-producing cells, the more often modification by bisecting N-acetylglucosamine (GlcNAc) or fucose (Fuc) at the sugar chain of the glycoprotein or increase in branching of side chains occurs. We examined the characteristics of the alpha-fetoprotein (AFP) sugar chain stored in amniotic and exocoelomic fluid during 5-9 weeks of gestation and analyzed serum-derived AFP of patients with germ cell tumors. METHODS: Total AFP concentrations in embryonic fluid at 5-9 weeks of gestation (n = 11) and serum of patients with germ cell tumors (n = 7) were measured using a radioimmunoassay (RIA) method. The percentages of AFPs reactive with Lens culinaris agglutinin (LCA), concanavalin A (Con A), erythroagglutinating phytohemagglutinin-E4 (E-PHA) and Ricinus communis agglutinin-120 (RCA 120) were obtained by lectin-affinity electrophoresis coupled with antibody-affinity blotting. RESULTS: It was revealed that at 5-9 weeks of gestation, AFP variants that had been modified by the Fuc residue, which bound to the GlcNAc residue at the reducing end of the sugar chain, and bisecting GlcNAc residues gradually decreased as pregnancy advanced; however, the presence of N-acetylneuraminic acid (Neu5Ac) at the nonreducing ends changed little. CONCLUSIONS: It appears very likely that the changes in the relative amounts of AFP variants in the embryonic fluid during early pregnancy were due to differentiation of the yolk sac. The grade of differentiation of yolk sac tumors was very similar to that of the normal yolk sac at around 6 weeks of gestation.

Simultaneous determination of percentage of Lens culinaris agglutinin-reactive alpha-fetoprotein and alpha-fetoprotein concentration using the LiBASys clinical auto-analyzer.

BACKGROUND: Lens culinaris agglutinin (LCA)-reactive alpha-fetoprotein (AFP-L3) percentage of total AFP concentration [(AFP-L3/total AFP)x100] has been used as an effective marker for earlier diagnosis, for assessment of therapeutic effects and for predicting the prognosis of hepatocellular carcinoma (HCC). METHODS: A new clinical automatic analyzer, the "LiBASys", and an assay kit for the simultaneous determination of AFP-L3 percentage and concentration of AFP in human serum were developed. LiBASys performed automatic re-measurement and also met stat samples. Both AFP-L3 percentage and AFP concentration were calculated simultaneously and printed out continuously every 3.4 min per test. RESULTS: A linear dose-response relationship was observed up to 1000 ng/ml AFP concentration. The detection limit and functional sensitivity of LCA-nonreactive AFP (AFP-L1) and AFP-L3 concentrations were 0.4 and 0.8 ng/ml, and 0.4 and 1.0 ng/ml, respectively. Interassay CVs of AFP-L3 percentage and AFP concentration ranged from 2.8% to 13.4% and 2.6% to 4.6%, respectively. Assay results exhibited good correlations with those of commercially available lectin-affinity electrophoresis (r=0.984) for AFP-L3 percentage and with the RIA method (r=0.999) for AFP concentration. CONCLUSIONS: This method simultaneously yields both qualitative and quantitative results for AFP-L3 percentage and AFP concentration.