Bladder cancer-associated cancer-testis antigen-derived long peptides encompassing both CTL and promiscuous HLA class II-restricted Th cell epitopes induced CD4+ T cells expressing converged T-cell receptor genes in vitro.

DEP domain containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1) are human cancer testis antigens that are frequently overexpressed in urinary bladder cancer. In a phase I/II clinical trial, a DEPDC1- and MPHOSPH1-derived short peptide vaccine demonstrated promising efficacy in preventing bladder cancer recurrence. Here, we aimed to identify long peptides (LPs) derived from DEPDC1 and MPHOSPH1 that induced both T-helper (Th) cells and tumor-reactive cytotoxic T lymphocytes (CTLs). Stimulation of peripheral blood mononuclear cells (PBMCs) from healthy donors with the synthetic DEPDC1- and MPHOSPH1-LPs predicted to bind to promiscuous human leukocyte antigen (HLA) class II molecules by a computer algorithm induced specific CD4+ T cells as revealed by interferon-γ enzyme-linked immunospot assays. Three of six LPs encompassed HLA-A2- or -A24-restricted CTL epitopes or both, and all six LPs stimulated DEPDC1- or MPHOSPH1-specific Th cells restricted by promiscuous and frequently observed HLA class II molecules in the Japanese population. Some LPs are naturally processed from the proteins in DCs, and the capacity of these LPs to cross-prime CTLs was confirmed in vivo using HLA-A2 or -A24 transgenic mice. The LP-specific and HLA class II-restricted T-cell responses were also observed in PBMCs from patients with bladder cancer. Repeated stimulation of PBMCs with DEPDC1-LPs and MPHOSPH1-LPs yielded clonal Th cells expressing specific T-cell receptor (TCR)-α and ß genes. These DEPDC1- or MPHOSPH1-derived LPs may have applications in immunotherapy in patients with bladder cancer, and the TCR genes identified may be useful for monitoring of Th cells specific to LPs in vivo.

Sensitization of transient receptor potential vanilloid 4 and increasing its endogenous ligand 5,6-epoxyeicosatrienoic acid in rats with monoiodoacetate-induced osteoarthritis.

Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.

[Anesthetic Management of a Patient with Langerhans Cell Histiocytosis during Cesarean section].

Langerhans cell histiocytosis is a rare disease, associated with histiocyte increases, and granuloma, in various organs. About 160 patients are reported in Japan. A pregnant patient with a pulmonary Langerhans cell histiocytosis underwent cesarean section under spinal anesthesia. She had repeated pneumothorax with bilateral pulmonary cysts rapidly becoming worse during pregnancy. She was treated with continuous oxygen after 28 weeks of the pregnancy. On 34 weeks of the pregnancy, spinal anesthesia with 0.5% hyperbaric bupivacaine (2 ml) and fentanyl (25 µg) for cesarean section was performed, and provided excellent analgesia without any side-effects.

Optimization of enrichment conditions on TiO2 chromatography using glycerol as an additive reagent for effective phosphoproteomic analysis.

Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.

Spectomycin B1 as a novel SUMOylation inhibitor that directly binds to SUMO E2.

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate; that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers.

Identification of potential prognostic markers for knee osteoarthritis by serum proteomic analysis.

BACKGROUND: As osteoarthritis (OA) is a highly heterogeneous disease in terms of progression, establishment of prognostic biomarkers would be highly beneficial for treatment. The present study was performed to identify novel biomarkers capable of predicting the progression of knee OA. METHODS: A total of 69 plasma samples (OA patients undergoing radiographic progression, n = 25; nonprogression, n = 33; healthy donors, n = 11) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and ion peaks of interest were identified by liquid chromatography and matrix-assisted laser desorption/ionization (MALDI)-TOF MS. The identities of these proteins were further validated by immunoprecipitation combined with SELDI-TOF MS analysis. RESULTS: SELDI-TOF MS analysis indicated that the intensities of 3 ion peaks differed significantly between progressors and nonprogressors. Subsequent analyses indicated that these peaks corresponded to apolipoprotein C-I, C-III, and an N-terminal truncated form of transthyretin, respectively. The identities of these proteins were confirmed by the loss of ion peaks in SELDI-TOF MS spectra by immunoprecipitation using specific antibodies for the respective proteins. CONCLUSIONS: Three potential biomarkers were identified whose serum levels differed significantly between OA progressors and nonprogressors. These biomarkers are expected to be prognostic biomarkers for knee OA and to facilitate the development of novel disease-modifying treatments for OA.

The effect of intravenous glucose solutions on neonatal blood glucose levels after cesarean delivery.

PURPOSE: Intravenous solutions are often administered to the mother on the day of a cesarean delivery to minimize the effect of preoperative fasting or to stabilize the hemodynamics. Different intravenous solutions contain varying amounts of glucose, and rapid administration may lead to hypoglycemia in the neonate. We conducted a study to compare blood glucose levels of the mother and the fetus/neonate after they were rapidly given a Ringer's solution containing 0, 1, or 5 % glucose. The effect of the glucose load that these intravenous solutions impose during cesarean delivery has not been fully reported. Therefore, we compared the effect of 0 % (Group I, n = 15), 1 % (Group II, n = 15), and 5 % (Group III, n = 15) glucose acetated Ringer's solutions on maternal and umbilical blood glucose levels to determine the optimal glucose concentration. METHODS: Once the patients were in the operating room, the intravenous solutions were administered before delivery. The primary endpoint was changes in umbilical blood glucose levels and minimum neonatal blood glucose levels, and the secondary endpoint was the proportion of neonates who received a glucose infusion. RESULTS: Maternal blood glucose levels before and after intravenous infusion were 79.2 ± 12.2 and 74.6 ± 4.6 in Group I, 81.2 ± 12.9 and 103.3 ± 11.2 in Group II (P < 0.001), and 82.3 ± 8.7 and 252.5 ± 41.8 in Group III (P < 0.001). Umbilical blood glucose levels were 53.9 ± 10.2 in Group I, 80.8 ± 13.7 in Group II, and 181.8 ± 22.2 in Group III (P < 0.01: Group I vs. Group II and P < 0.01: Group II vs. Group III) (P < 0.001: Group I vs. Group III). Minimum neonatal blood glucose levels measured up to 8 h after birth were 35.7 ± 9.6 in Group I, 49.8 ± 10.8 in Group II, and 29.2 ± 7.5 in Group III. Neonatal hypoglycemia requiring glucose before the first milk feeding occurred in 6 neonates whose mothers were in Group I, 3 in Group II, and 9 in Group III, indicating a trend towards less neonatal hypoglycemia in Group II. CONCLUSIONS: The use of 1 % glucose acetated Ringer's solution did not induce hyperglycemia in the mother and it was able to maintain appropriate blood glucose levels in the fetus.

Potential plasma biomarkers for progression of knee osteoarthritis using glycoproteomic analysis coupled with a 2D-LC-MALDI system.

BACKGROUND: Although osteoarthritis (OA) is a highly prevalent joint disease, to date, no reliable biomarkers have been found for the disease. In this study, we attempted to identify factors the amounts of which significantly change in association with the progression of knee OA. METHODS: A total of 68 subjects with primary knee OA were enrolled in the study. These subjects were followed up over an 18-month period, and plasma and serum samples were obtained together with knee radiographs every 6 months, i.e., 0, 6, 12 and 18 months after the enrollment. Progressors and non-progressors were determined from the changes on radiographs, and plasma samples from those subjects were subjected to N-glycoproteomic 2D-LC-MALDI analysis. MS peaks were identified, and intensities for respective peaks were compared between the progressors and non-progressors to find the peak intensities of which differed significantly between the two groups of subjects. Proteins represented by the chosen peaks were identified by MS/MS analysis. Expression of the identified proteins was evaluated in synovial tissues from 10 OA knee joints by in situ hybridization, western blotting analysis and ELISA. RESULTS: Among the subjects involved in the study, 3 subjects were determined to be progressors, and 6 plasma and serum samples from these subjects were subjected to the analysis together with another 6 samples from the non-progressors. More than 3000 MS peaks were identified by N-glycoproteomic 2D-LC-MALDI analysis. Among them, 4 peaks were found to have significantly different peak intensities between the progressors and non-progressors. MS/MS analysis revealed that these peaks represented clusterin, hemopexin, alpha-1 acid glycoprotein-2, and macrophage stimulating protein, respectively. The expression of these genes in OA synovium was confirmed by in situ hybridization, and for clusterin and hemopexin, by western blotting analysis and ELISA as well. CONCLUSIONS: In this study, 4 potential biomarkers were identified as potential prognostic markers for knee OA through N-glycoproteomic analysis. To the best of our knowledge, this is the first report for the use of glycoproteomic technology in exploring potential biomarkers for knee OA.

Development of quantitative plasma N-glycoproteomics using label-free 2-D LC-MALDI MS and its applicability for biomarker discovery in hepatocellular carcinoma.

There has been rapid progress in the development of clinical proteomic methodologies with improvements in mass spectrometric technologies and bioinformatics, leading to many new methodologies for biomarker discovery from human plasma. However, it is not easy to find new biomarkers because of the wide dynamic range of plasma proteins and the need for their quantification. Here, we report a new methodology for relative quantitative proteomic analysis combining large-scale glycoproteomics with label-free 2-D LC-MALDI MS. In this method, enrichment of glycopeptides using hydrazide resin enables focusing on plasma proteins with lower abundance corresponding to the tissue leakage region. On quantitative analysis, signal intensities by 2-D LC-MALDI MS were normalized using a peptide internal control, and the values linked to LC data were treated with DeView™ software. Our proteomic method revealed that the quantitative dynamic ranged from 10² to 106 pg/mL of plasma proteins with good reproducibility, and the limit of detection was of the order of a few ng/mL of proteins in biological samples. To evaluate the applicability of our method for biomarker discovery, we performed a feasibility study using plasma samples from patients with hepatocellular carcinoma, and identified biomarker candidates, including ceruloplasmin, alpha-1 antichymotrypsin, and multimerin-1.

[Anesthesia management for cesarean section in a patient with placenta percreta].

Placenta accreta is a placental abnormality with a defect in decidua basalis. Villi tissue invades myometrium and it is difficult to exfoliate the placenta. Placenta percreta is a comparatively rare disorder in placenta accreta. This patient at pregnancy 14 weeks, exploratory laparotomy was performed for acute abdomen. At this time she was diagnosed as placenta percreta, but the pregnancy was continued. Caesarean section was scheduled in 37 weeks 0 days of pregnancy and combined spinal-epidural anesthesia was used. After the operation, we moved the patient to the angiography room to perform uterus artery embolization (UAE). While moving, we monitored maternal hemodynamics using a transportable monitor. During the cesarean section in the operation room, and in moving to the angiography room and in the angiography room, we used the same transportable monitor for secure perioperative management.

[One case of the basilar invagination that prevented tracheal intubation by Airway Scope].

Airway Scope (AWS, Pentax, Tokyo, Japan) is a very useful intubation assistance tool in the case where intubation is difficult with normal Macintosh laryngoscope. In this case, the operation was planned by general anesthesia in a patient with basilar invagination. We decided to intubate the patient using AWS because difficult tracheal intubation was expected. However, glottis was not confirmed even with AWS in the oral cavity, and we could not intubate with AWS. Therefore, we used a fiberscope, and endotracheal intubation was successful.

[Epidural anesthesia for five caesarian sections in patients with placenta previa percreta combined with placenta accreta].

We gave anesthesia for five cases of caesarian section in patients with placenta previa percreta combined with placenta accreta. Five caesarian sections were performed under epidural anesthesia and placentae remained in the uterus. After surgery, they were transferred to the angiography room and received uterine arterial embolization (UAE). They were given the diagnosis of the placenta accreta. However, during anesthesia in patients with placenta accreta, incomplete separation of the placenta causes atonic bleeding. And, uncleanliness of the abrasive degree may lead to difficulty in a prediction of bleeding, and control of hemodynamics is difficult. In the caesarian section of the placenta accreta, UAE prevents excessive bleeding during the operation and FloTrac monitor makes it easy to control hemodynamics.

[Anesthetic management of thoracoscopic mini maze procedure for atrial fibrillation].

We anesthetized three patients with refractory atrial fibrillation undergoing the new thoracoscopic mini Maze procedure. This minimally invasive surgical procedure provides isolation and ablation of the bilateral pulmonary vein without thoracotomy or cardiopulmonary bypass, which was performed first in the United States in 2003. General anesthesia was administrated with a double lumen tracheal tube for bilateral single lung ventilation, and epidural anesthesia was also administrated. We prepared transesophageal echocardiography, a pulmonary artery catheter with pacing wires, and a 20 G arterial line connected to continuous cardiac output monitor. We also prepared a 14 G peripheral intravenous line and two 4 Fr sheaths on the left femoral artery and vein for bleeding and conversion to cardiopulmonary bypass. One patient required massive transfusion and conversion to open procedure with thoracotomy and cardiopulmonary bypass to control sudden and massive bleeding from the pulmonary vein. Another patient was complicated with dextrocardia, but the procedure was successful. One other patient had no complications and the procedure was performed as planned. Certain anesthetic management is essential for this new procedure and we have to prepare for any predictable events.

[Case of caesarean section using Cell Savers5+ in a patient with the placenta accreta associated with massive hemorrhage].

Placenta accreta which occurs as a complication of total placenta previa is comparatively rare. And the diagnosis of placenta accreta before the caesarean section (C-section) is difficult. We experienced an extremely difficult anesthesia management of placenta accrete because of critical hemorrhage during C-section. We used self-blood collection device Cell Saver5+ (Haemonetics Japan Ltd, Tokyo) for the massive bleeding of 25,500 ml. The Cell Saver could be used to treat life threatening bleeding in C-section without any side effects, although it is thought that the use of the self-blood collection device during C-section had the danger of the amniotic fluid embolism and fetal red cell ontamination. It is necessary to consider using the Cell Saver in the obstetrical operation for life-saving medical treatment in response to unexpected massive bleeding.

[Central venous catheterization complication by a guide wire].

Central venous catheterization using the Seldinger technique is a well known and often used method. On the other hand, there are also well known complications by needle puncture or by indwelling catheter, there are few reports about a guide wire which got hung up around the tricuspid valve. We report a case in which a guide wire got hung up to the chordae tendineae of the tricuspid valve. To insert the AVA 3Xi (Edwards life science Co. Iervine) from the right internal jugular vein, we inserted a guide wire without ease. Resistance appeared when we tried to remove the wire for 20 cm from the inserted state. The X-ray and the transesophageal echocardiography, showed the guide wire in the right ventricle. As actions to be taken, we advanced the central vein catheter of the EXCV catheter kit (Nippon Sherwood Medical Industries Co., Ltd.) to the tip, and a the guide wire was easily removed. There are many reports of the complication by the central venepuncture, but there are few reports about the guide wire which was entrapped in the vicinity of a tricuspid valve. The tip of the guide wire in this case was bent excessively, but the cause of the damage did not become clear by investigation. When a guide wire became hard to withdraw, we should never withdraw a guide wire blindly, but should search a cause and we should use the material which was matched with the cause.

Ginkgolic acid inhibits protein SUMOylation by blocking formation of the E1-SUMO intermediate.

Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However, no small-molecule inhibiting protein SUMOylation has been reported so far. Here, we report inhibition of SUMOylation by ginkgolic acid and its analog, anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells, but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation.

Sudden cardiac arrest during cesarean section due to epidural anaesthesia using ropivacaine: a case report.

Sudden cardiac arrest occurred subsequent to epidural anaesthesia in the patient of elective cesarean section. During cardio-pulmonary resuscitation, immediate section saved the infant and the mother recovered completely. Serial ropivacaine concentrations in maternal serum evaluated potential risk of epidural anaesthesia with ropivacaine. A 39-year-old primirapa woman with an 11-year aortitis syndrome (Takayasu disease) was presented. Low dose aspirin of 81 mg per every 3 day and prednisolone of 12 mg per every 2 day maintained her aortitis in good control through pregnancy. An elective cesarean section was planned preventively because of aortitis at 38 weeks of gestation. As an epidural anaesthesia, 20 ml of 1% ropivacaine was injected by one shot from 3/4 lumbar supine through median approach with an appropriate tube testing method in the operation room. At 22 min after injection, a sudden cardiac arrest following to two times of pulse-less VT (Ventricular tachycardia) and systemic convulsion occurred. Immediate defibrillation of 200 J was performed promptly, and cesarean section delivered a healthy boy of 2,655 g with APGAR scores of 10 points at 1 and 5 min, respectively. Both mother and neonate were discharged from hospital at 11th day without sequare. Ropivacaine was proved to be a cardio-toxic agent in the case. However, immediate resuscitation and operative delivery served good prognosis for mother and neonate.