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.

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.

Very low frequency component of heart rate variability as a marker for therapeutic efficacy in patients with obstructive sleep apnea: Preliminary study.

BACKGROUND: Although positive airway pressure (PAP) therapy is effective for treating obstructive sleep apnea (OSA), some patients with severe OSA are intolerable to this treatment, which may lead to an increase in the mortality and morbidity of cardiovascular diseases. We investigated the relationship between heart rate variability (HRV) and sleep parameters during natural sleep and treatment of patients with OSA. MATERIALS AND METHODS: This was the cross-sectional observation study. Patients were 17 males with severe OSA who were unable to accept continuous PAP. Standard polysomnography was performed for two consecutive nights, i.e., during natural sleep and following night with bilevel PAP (BiPAP) treatment. Time-dependent responses of the amplitudes of low frequency (LF), very low frequency (VLF), and high frequency components of HRV were assessed with the technique of complex demodulation. RESULTS: Apnea-hypopnea index, oxygen desaturation time, and percentage of stage 1 sleep were significantly reduced, whereas the percentages of rapid eye movement and stages 3 + 4 sleep were increased, by BiPAP treatment. Therapy also reduced the amplitudes of VLF and LF components of HRV. Difference in amplitudes of VLF during natural sleep and treatment with BiPAP was significantly correlated with difference in percentages of stage 1 and stages 3 + 4 sleep. CONCLUSION: Therapy-induced amelioration of OSA and sleep quality was accompanied by decrease in the amplitudes of VLF components of HRV. The VLF component may thus reflect physiological changes in both autonomic activity and sleep structure and serve as an objective marker for therapeutic efficacy in patients with severe OSA.

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.

Pars plana vitrectomy and panretinal photocoagulation combined with trabeculectomy for successful treatment of neovascular glaucoma.

BACKGROUND: Treatment of neovascular glaucoma (NVG) must be focused on the reduction of intraocular pressure (IOP) and prompt application of pan retinal photocoagulation (PRP). A combination of complete PRP during vitrectomy with trabeculectomy should theoretically be a better method to lower the IOP rapidly in eyes with NVG. The purpose of our study is to assess the efficacy of combining pars plana vitrectomy and PRP with trabeculectomy assisted by mitomycin C (MMC) on NVG eyes secondary to diabetic retinopathy. METHODS: Twenty-five eyes with NVG associated with diabetic retinopathy had pars plana vitrectomy, followed by PRP and trabeculectomy with MMC. The eyes were divided into two groups: nine eyes with vitreous hemorrhage, fibrovascular membrane and/ or retinal detachment were placed in the Proliferation group; and 16 eyes without vitreous hemorrhage, fibrovascular membrane, or retinal detachment were placed in the PC (photocoagulation) group. These eyes had vitrectomy performed so that PRP could be safely performed from ora to ora. The surgical outcome in the two groups was assessed by Kaplan-Meier survival analysis. The criteria for success were a postoperative intraocular pressure (IOP) < or =21 mmHg and a preservation of light perception. RESULTS: In the Proliferation group, Kaplan-Meier life-table analysis showed that the success rate was 55.6% after 1 year and 18.5% after 2 years. The success rate in the PC group was 81.2% from 1 to 3 years after surgery. The surgical outcome was significantly better in the PC group than in the Proliferation group (P=0.009). In the Proliferation group, four eyes had preoperative vitreous hemorrhage, three eyes had a fibrovascular membrane, and two eyes had a retinal detachment. Three of four eyes with vitreous hemorrhage achieved good IOP control. On the other hand, the IOP of all eyes with retinal detachment and fibrovascular membrane were not lowered significantly. CONCLUSIONS: Complete PRP combined with trabeculectomy with MMC can effectively reduce the elevated IOP in eyes with NVG. However, this combined treatment is not effective in eyes with proliferative membranes and retinal detachments.

REM sleep is impaired by a small amount of alcohol in young women sensitive to alcohol.

OBJECTIVE: The effect of a small amount of alcohol on the sleep structure in relation to alcohol sensitivity was examined using polysomnography (PSG). METHODS: Alcohol sensitivity was evaluated using alcohol patch test for all subjects. PSGs were performed on three nights after one night for acclimation, and subjects consumed no alcohol, 0.28 or 0.69 g ethanol/kg body weight, respectively, before going to bed. The percentages of sleep time in each sleep stage of 1, 2, 3+4 and rapid eye movement (REM), REM latency, and REM cycle were calculated. SUBJECTS: Thirteen healthy female students (age 21.1 +/- 0.7 years) were enrolled in this study. RESULTS: In all subjects, there were no significant differences in any of the sleep parameters between baseline night and alcohol nights. Six of the 13 subjects were sensitive to alcohol, in whom %stage REM was significantly decreased by alcohol consumption (baseline night: 18.3 +/- 6.2%, alcohol night I: 9.8 +/- 5.1% and alcohol night II: 11.0 +/- 2.8%), and the REM latency was significantly prolonged. The standard deviation of REM cycle was significantly greater on alcohol nights I and II than baseline night. There were no significant differences in other sleep parameters. In the other seven subjects who were insensitive to alcohol, none of the sleep parameters were significantly affected by alcohol consumption. CONCLUSION: REM sleep was adversely affected by a small amount of alcohol in alcohol-sensitive healthy young women. Alcohol sensitivity might play some important role in impaired REM sleep by an ingestion of a small amount of alcohol.

Binding affinity of bunazosin, dorzolamide, and timolol to synthetic melanin.

PURPOSE: The purpose of this study was to compare the binding affinity of bunazosin and dorzolamide to synthetic melanin relative to that of timolol. METHODS: Synthetic melanin was prepared from dopa by the action of tyrosinase. Timolol, dorzolamide, and bunazosin were incubated separately at a concentration of 10(-4) M in 2 ml of 0.066 M phosphate buffer containing 5 mg of synthetic melanin. After centrifugation, the absorbance of each free drug in the supernatant was measured at its optimum wavelength. The percentage of each drug bound to melanin was calculated directly from the change in absorbance relative to the initial value. RESULTS: The increase in the binding rates of all three drugs seemed to reach a plateau after 30 min. After incubating for 60 min, the binding rate of timolol was 22.2% +/- 4.9%, bunazosin 36.3% +/- 2.5%, and dorzolamide 8.5% +/- 1.9%. There were statistically significant differences between the binding rates of each drug. CONCLUSIONS: Under our study conditions, the order of binding affinity of these ocular hypotensive agents to synthetic melanin seems to be as follows: bunazosin>timolol>dorzolamide.

Effect of a single drop of latanoprost on intraocular pressure and blood-aqueous barrier permeability in patients with uveitis.

The purpose of our study is to evaluate the effect of a single drop of latanoprost on the intraocular pressure and blood-aqueous barrier permeability in 8 patients with uveitis. The degree of inflammation was determined by the intensity of aqueous flare measured with a laser flare cell meter every 2 hours from 11:00 to 17:00 hours for 2 days. Intraocular pressure was measured at 11:00 and 17:00 hours with a Goldmann applanation tonometer on both days. Patients were given one drop of 0.005% latanoprost at 11:00 hours on day 2 and results were compared with day 1 when latanoprost was not administered. There was no significant difference in the intensity of aqueous flare or intraocular pressure between day 1 and day 2. A single drop of latanoprost had little effect on intraocular pressure and aqueous flare intensity in patients with uveitis.