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Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Figure10

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Information

Immune checkpoint inhibitors have shown efficacy in various cancers. Although programmed death ligand 1/2 (PD-L1/L2) expressions have been demonstrated as predictive biomarkers of response to immune checkpoint inhibitors and prognostic markers, whether PD-L1/L2 expression is altered in esophageal squamous cell carcinoma during the therapeutic course is unclear. Whether PD-L1/L2 expression in metastatic or recurrent lesions is consistent with that in primary tumors is also unknown. This study included 561 surgically resected esophageal squamous cell carcinomas and PD-L1/L2 expression was evaluated by immunohistochemistry. We investigated the influence of chemotherapeutic drugs (cisplatin and fluorouracil) on PD-L1/L2 expression and PD-L1/L2-related pathways in vitro. We also examined PD-L1/L2 expression in 18 surgically resected lymph node metastases and 10 recurrent lesions compared with primary lesions. The positive rate of PD-L1 was significantly higher in patients with preoperative chemotherapy than in those without preoperative therapy. The positive rate of PD-L2 expression showed no significant difference between patient groups. Cisplatin increased PD-L1 expression in cancer cell lines in vitro, but decreased PD-L2 in some cell lines. The effects of cisplatin on phosphorylated signal transducer and activator of transcription 1/3 (pSTAT1/3) also differed depending on cell lines. Fluorouracil increased PD-L1 and PD-L2 expression. PD-L1/L2 expression in lymph node metastases and recurrent lesions did not always match expression in primary lesions. PD-L1/L2 expression may be altered by preoperative chemotherapy, and PD-L1 /L2 expression in primary lesions does not always match that of metastatic/recurrent lesions. Thus, one-time evaluation is not sufficient to evaluate PD-L1/L2 expression as a biomarker in esophageal cancer.

Remodeling the tumor microenvironment (TME) to benefit cancer cells is crucial for tumor progression. Although diffuse-type gastric cancer (DGC) preferentially interacts with the TME, the precise mechanism of the complicated network remains unknown. This study aimed to investigate the mutual activation mechanism underlying DGC progression.Mass cytometry analysis of co-cultured macrophages, noncancerous fibroblasts (NFs), and DGC cells was performed. RNA sequencing was applied to examine gene expression in fibroblasts. DGC cells were treated with cytokines to examine their effect on characteristic changes. The TCGA and Kumamoto University cohorts were used to evaluate the clinical relevance of the in vitro findings.Cohort analysis revealed that DGC patients had a poor prognosis. The fibroblasts and macrophages interacted with DGC cells to form a cell cluster in the invasive front of DGC tissue. The original 3D triple co-culture system determined the promotional effects of nonmalignant cells on DGC invasive growth. We notably identified a mixed-polarized macrophage cell type with M1/M2 cell surface markers in a triple co-culture system. IL-1β from mixed-polarized macrophages induced the conversion of NFs to cancer-associated fibroblast-like (CAF-like) cells, promoting the malignant phenotype of DGC cells by inducing the secretion of IL-6, IL-24, and leukemia inhibitory factor (LIF). Moreover, IL-6 and colony stimulating factor 2 (GM-CSF) cooperated to maintain the stable state of mixed-polarized macrophages. Finally, we found that mixed-polarized macrophages were frequently detected in DGC tissues.These findings demonstrated that mixed-polarized macrophages exist as a novel subtype through the reciprocal interaction between DGC cells and nonmalignant cells.

Pancreatic cancer has an extremely poor prognosis, even after curative resection. Treatment options for pancreatic cancer remain limited, therefore new therapeutic targets are urgently needed. We searched for genes predictive of poor prognosis in pancreatic cancer using a public database and validated the survival impact of the selected gene in a patient cohort. We used a public database to search for genes associated with early pancreatic cancer recurrence. As a validation cohort, 201 patients who underwent radical resection in our institution were enrolled. Expression of the target gene was evaluated using immunohistochemistry (IHC). We evaluated growth and invasiveness using small interfering RNAs, then performed pathway analysis using gene set enrichment analysis. We extracted ARHGEF2 from GSE21501 as a gene with a high hazard ratio (HR) for early recurrence within 1 year. The high ARHGEF2 expression group had significantly poorer recurrence-free survival (RFS) and poorer overall survival (OS) than the low ARHGEF2 expression group. Multivariate analysis demonstrated that high ARHGEF2 expression was an independent poor prognostic factor for RFS (HR 1.92) and OS (HR 1.63). In vitro, ARHGEF2 suppression resulted in reduced cell growth and invasiveness. Bioinformatic analysis revealed that ARHGEF2 expression was associated with MYC, G2M, E2F, and CDC25A expression, suggesting that c-Myc and cell cycle genes are associated with high ARHGEF2 expression. IHC revealed a positive correlation between ARHGEF2 and c-Myc expression. High ARHGEF2 expression is associated with cell cycle progression, and predicts early recurrence and poor survival in patients with pancreatic cancer.

The arachidonic acid cascade is a major inflammatory pathway that produces prostaglandin E

Some reports showed the immune tolerance of soluble human leukocyte antigen E (HLA-E), but the role that soluble HLA-E plays in gastric cancer (GC) is unknown. We aimed to clarify the molecular mechanism and clinical significance of soluble HLA-E in GC.We examined the expression of HLA-E on GC cells and soluble HLA-E under co-culture with natural killer (NK) cells in a time-dependent manner. Changes in NK cell activity were investigated using anti-NK group 2 member A (NKG2A) antibodies in the presence of soluble HLA-E. Expression of soluble HLA-E in the serum of GC patients was determined.Whereas HLA-E expression on GC cells peaked with interferon (IFN)-γ secretion by NK cells in a time-dependent manner, soluble HLA-E was upregulated in conditioned medium. Pre-incubation with anti-NKG2A antibodies increased the activation of NKG2AHLA-E expression dynamically changes on GC cells and in conditioned medium. Furthermore, soluble HLA-E can contribute to immune escape in GC cell lines, which may have significance in clinical practice. Moreover, soluble HLA-E may be a potential prognostic biomarker.

Background The predictive significance of programmed death ligand 1 (PD-L1) for programmed death 1 (PD-1) inhibitors remains unclear in gastric cancer (GC) due to the dynamic alteration by treatments. We aimed to elucidate the effects of trastuzumab (Tmab) on PD-L1 expression in GC. Methods PD-L1 expression was evaluated by multicolour flow cytometry analysis after co-culturing GG cell lines and immune cells with Tmab. IFN-γ in the co-culture experiments was quantified. Immunohistochemistry (IHC) for PD-L1 expression using clinical samples was also performed to confirm PD-L1 alteration by Tmab. Results PD-L1 expression was significantly upregulated by Tmab in HER2-amplified GC cell lines co-cultured with peripheral blood mononuclear cells (PBMCs). PD-L1 upregulation by Tmab was also observed in the GC cells co-cultured with NK cells in time-dependent manner, but not with monocytes. IFN-γ concentration in conditioned media from co-cultured PBMCs and NK cells with Tmab was significantly higher and anti-IFN-γ significantly suppress the Tmab-induced PD-L1 upregulation. IHC also suggested PD-L1 upregulation after Tmab treatment. Conclusions Tmab can upregulate PD-L1 expression on GC cells through interaction with NK cells. These results suggest clinical implications in the assessment of the predictive significance of PD-L1 expression for PD-1 inhibitors.

Summary: In the tumor microenvironment, senescent non-malignant cells, including cancer-associated fibroblasts (CAFs), exhibit a secretory profile under stress conditions; this senescence-associated secretory phenotype (SASP) leads to cancer progression and chemoresistance. However, the role of senescent CAFs in metastatic lesions and the molecular mechanism of inflammation-related SASP induction are not well understood. We show that pro-inflammatory cytokine-driven EZH2 downregulation maintains the SASP by demethylating H3K27me3 marks in CAFs and enhances peritoneal tumor formation of gastric cancer (GC) through JAK/STAT3 signaling in a mouse model. A JAK/STAT3 inhibitor blocks the increase in GC cell viability induced by senescent CAFs and peritoneal tumor formation. Single-cell mass cytometry revealed that fibroblasts exist in the ascites of GC patients with peritoneal dissemination, and the fibroblast population shows p16 expression and SASP factors at high levels. These findings provide insights into the inflammation-related SASP maintenance by histone modification and the role of senescent CAFs in GC peritoneal dissemination.

Objectives. To evaluate 24-hour intraocular pressure (IOP) variation in patients with primary open-angle glaucoma (POAG) treated with triple eye drops. Subjects and Methods. The IOP was measured in 74 eyes in 74 POAG patients (seated) on triple therapy (PG analogue, β-blocker, carbonic anhydrase inhibitor) at about every 3 hours. Results. The peak IOP was 13.5 ± 3.1 at 1:00, and the trough IOP was at 12.6 ± 2.4 mmHg at 7:00. The IOP at 7:00 was significantly lower than that at 10:00, 1:00, and 3:00 (p<0.05). Based on the time of the peak IOP, we classified the patients into two groups: diurnal (28 eyes) and nocturnal types (37 eyes). There was significant difference at the spherical equivalent between diurnal and nocturnal types (p=0.014). To assess the influence of reflective error, we conducted subanalysis for two groups: high myopic (26 eyes, ≤−6D) and low/nonmyopic (24 eyes, ≥−2D) groups. In the low/nonmyopia group, the IOP was significantly higher at 1:00 and 3:00 than at 13:00, 16:00, and 7: 00 (p<0.05). Conclusion. The mean of IOP elevated outside of clinic hour in the POAG patients on triple therapy. The low/nonmyopia patient should be carefully treated because the IOP of the patients at night elevated significantly.

Research on heart tissue engineering is an exciting and promising area. Although we previously developed bioengineered myocardium using cell sheet-based tissue engineering technologies, the issue of appropriate cell sources remained unresolved. In the present study, we created cell sheets of mouse embryonic stem (ES) cell-derived cardiomyocytes after expansion in three-dimensional stirred suspension cultures. Serial treatment of the suspension cultures with noggin and granulocyte colony-stimulating factor significantly increased the number of cardiomyocytes by more than fourfold compared with untreated cultures. After drug selection for ES cells expressing the neomycin-resistance gene under the control of the α-myosin heavy chain promoter, almost all of the cells showed spontaneous beating and expressed several cardiac contractive proteins in a fine striated pattern. When ES-derived cardiomyocytes alone were seeded onto temperature-responsive culture dishes, cell sheets were not created, whereas cocultures with cardiac fibroblasts promoted cell sheet formation. The cardiomyocytes in the cell sheets beat spontaneously and synchronously, and expressed connexin 43 at the edge of adjacent cardiomyocytes. Furthermore, when the extracellular action potential was recorded, unidirectional action potential propagation was observed. The present findings suggest that stirred suspension cultures with appropriate growth factors are capable of producing cardiomyocytes effectively and easily, and that ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium.

To evaluate the effects of carteolol hydrochloride (carteolol) on 24-h variations in intraocular pressure (IOP) in patients with normal-tension glaucoma (NTG).Twelve patients with NTG were treated with carteolol 2% solution foror=8 weeks; their pretreatment 24-h IOP variations, blood pressure (BP), and pulse rate (PR) were compared with those measured after the treatment period.Daytime IOP (at 07:00, 10:00, 13:00, and 16:00), maximum IOP, and the mean 24-h IOP were significantly reduced after treatment, as was the 24-h IOP range. Systolic BP in the morning and both systolic and diastolic BP in the afternoon were significantly decreased by the treatment, whereas no significant change of PR was observed.Carteolol had no effect on nocturnal IOP but significantly helped reduce daytime IOP, maximum IOP, mean 24-h IOP, and the 24-h IOP range. The drug exerted no statistically significant effect on the PR.

PURPOSE To study the effect of the concomitant use of latanoprost and brinzolamide on the 24-hour variation in the intraocular pressure (IOP) in patients with normal-tension glaucoma (NTG). METHODS We studied a total of 44 eyes from 22 NTG patients. Mean 24-hour IOP variation was determined after a washout period of > or =4 weeks. Latanoprost monotherapy was continued in both eyes for 8 weeks. Thereafter, patients were randomized to continue latanoprost monotherapy in 1 eye whereas brinzolamide was added as an adjunct to latanoprost therapy in the other eye. Eight weeks after the initiation of brinzolamide treatment, the 24-hour IOP variation was remeasured. IOP was measured in the sitting position 8 times daily using a Goldmann applanation tonometer before and after treatment. RESULTS The eyes treated with latanoprost monotherapy and those treated with latanoprost and brinzolamide showed a significant decrease in IOP at all time points. Percent reductions in the diurnal mean IOP (mean IOP at 10 AM, 1 PM, and 4 PM) and in nocturnal mean IOP (mean IOP at 10 PM, 1 AM, and 3 AM) were significantly greater in the eyes treated with the combination of latanoprost and brinzolamide than those with latanoprost alone (diurnal mean IOP: latanoprost and brinzolamide=19.8%, latanoprost=14.1%, P

Meeting abstracts Although clinical studies have established that antigen-loaded dendritic cells (DCs) vaccines are safe and promising therapy for various kinds of malignancies, their clinical efficacy remains to be established. Issues that limit the clinical efficacy of DC-based immunotherapy, as

Congenital syphilis (CS) is a public health burden in both developing and developed countries. We report two cases of CS in premature neonates with severe clinical manifestations; Patient 1 (gestational age 31 weeks, birth weight 1423 g) had disseminated idiopathic coagulation (DIC) while Patient 2 (gestational age 34 weeks and 6 days, birth weight 2299 g) had refractory syphilitic meningitis. Their mothers were single and had neither received antenatal care nor undergone syphilis screening. Both neonates were delivered via an emergency cesarean section and had birth asphyxia and transient tachypnea of newborn. Physical examination revealed massive hepatosplenomegaly. Laboratory testing of maternal and neonatal blood showed increased rapid plasma reagin (RPR) titer and positive Treponema pallidum hemagglutination assay. Diagnosis of CS was further supported by a positive IgM fluorescent treponemal antibody absorption test and large amounts of T. pallidum spirochetes detected in the placenta. Each neonate was initially treated with ampicillin and cefotaxime for early bacterial sepsis/meningitis that coexisted with CS. Patient 1 received fresh frozen plasma and antithrombin III to treat DIC. Patient 2 experienced a relapse of CS during initial antibiotic treatment, necessitating parenteral penicillin G. Treatment was effective in both neonates, as shown by reductions in RPR. Monitoring of growth and neurological development through to age 4 showed no evidence of apparent delay or complications. Without adequate antenatal care and maternal screening tests for infection, CS is difficult for non-specialists to diagnose at birth, because the clinical manifestations are similar to those of neonatal sepsis and meningitis. Ampicillin was insufficient for treating CS and penicillin G was necessary.

For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45(+) leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 × 10(7) cells (range 4.40 × 10(6)-2.48 × 10(8) cells). From this fraction, 6.39 × 10(6) (range 3.23 × 10(5)-2.53 × 10(7)) CD45(-) cells were collected, and the tumor cell purity was over 80 % defined as CD45(-)CD326(+). A sufficient amount of tumor lysate, average = 2416 μg (range 25-8743 μg), was extracted from CD45(-)CD326(+) tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.

The purpose of this study was to determine whether genetic polymorphisms affecting high-density lipoprotein (HDL)-associated antioxidant enzymes were associated with open-angle glaucoma (OAG). The rationale for this study was that the modification of low-density lipoprotein (LDL) by HDL prevents oxidative modification which can then cause dysfunction of endothelial cells.We studied 284 normal Japanese controls and 555 Japanese patients with OAG, including primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG). The possible associations of polymorphisms of PON1/L55M, PON1/Q192R, PON2/S311C, and PAF-AH/V279F with OAG were investigated. We compared the genotype distributions and allele frequency in controls and patient groups. The age at diagnosis, intraocular pressure (IOP) at diagnosis, and visual field score at diagnosis were examined for association with polymorphisms.The distributions of genotypes and allele frequency for the four polymorphisms were not significantly different between any patient group and controls. In NTG patients, 55M carriers of the PON1 gene were significantly older at diagnosis than 55M non-carriers (P=0.001). The IOP at diagnosis was significantly higher in glaucoma patients carrying 192R in the PON1 gene than in patients not carrying 192R (P=0.006). No significant differences were seen in clinical characteristics of OAG patients in relation to other polymorphisms.PON1 gene polymorphisms may influence the features of Japanese patients with OAG.

Although a large percentage of elementary school children show high interest in science classes, they tend to lose interest and dislike them in junior and senior high schools. This paper aims to clarify the causes that bring about this loss of interest in science. The study was based on a questionnaire that was conducted among the university students who want to become teachers. The result of the investigation reveals that neither their favor/disfavor of science classes in elementary school nor their nature-baaed experiences are related to their favor/disfavor of science classes in high school. The students who disliked science classes in high school claim that they lost interest because teachers demanded rote memorization and lessons were not effectual or inspiring. The authors conclude that science teachers should improve teaching strategies to enhance the students' interest and enthusiasm in the learning of science.

We retrospectively examined intraocular pressure variations after visual field examination in primary open angle glaucoma (POAG), together with its influencing factors and its association with 24-hour intraocular pressure variations.Subjects were 94 eyes (52 POAG patients) subjected to measurements of 24-hour intraocular pressure and of changes in intraocular pressure after visual field examination using a Humphrey Visual Field Analyzer. Subjects were classified into three groups according to the magnitude of variation (large, intermediate and small), and 24-hour intraocular pressure variations were compared among the three groups. Factors influencing intraocular pressure variations after visual field examination and those associated with the large variation group were investigated.Average intraocular pressure variation after visual field examination was -0.28 ± 1.90 (range - 6.0(-) + 5.0) mmHg. No significant influencing factors were identified. The intraocular pressure at 3 a.m. was significantly higher in the large variation group than other two groups (p0.001). Central corneal thickness was correlated with the large variation group (odds ratio = 1.04; 95% confidence interval, 1.01-1.07 ; p = 0.02).No particular tendencies in intraocular pressure variations were found after visual field examination. Increases in intraocular pressure during the night might be associated with large intraocular pressure variations after visual field examination.

We have produced biodegradable or enteric-coated microspheres containing camostat mesylate, a protease inhibitor, using a water-oil-water emulsion solvent evaporation method. The characteristics of the microspheres were determined. When polylactic acid, a biodegradable polymer, was used as a wall material, the optimized microsphere obtained showed a loading efficiency of almost 95% and had a mean diameter of 30 pim. This microsphere showed a sustained-release profile, with nearly 25% of drug being released at seven days in a dissolution test. When hypromellose acetate succinate (AS-HG type, with a high content of succinyl group) was used as an enteric wall material, optimized microspheres showed a loading efficiency of almost 80%. In this case, pH 3.0 citrate buffer was used as an internal aqueous phase, and citrate buffer containing 0.5 % polyvinylalcohol was used as the external aqueous phase. These microspheres showed a rapid release profile in pH 6.8 buffer, whereas the release was extremely slow in pH 1.2 buffer. Hypromellose acetate succinate microspheres were also prepared containing 10% (w/w) N-benzoyl-dl-arg-4-nitroanilide as a model substrate for trypsin, with or without 5% (w/w) camostat. These microspheres were incubated in pH 6 or 7 buffer containing trypsin at 37°C. When camostat was included in the microspheres, the substrate was protected from attack by trypsin, while in the absence of camostat, the released substrate was immediately attacked by trypsin to produce the degradation product N-benzoyl-dl-arginine.

The objectives of this study were to prepare insulin-loaded acrylic hydrogel formulations containing various absorption enhancers, to perform in vitro and in vivo characterization of these formulations, and to evaluate the factors which affecting insulin availability on rectal delivery of insulin using this hydrogel system. The acrylic block copolymer of methacrylic acid and methacrylate, Eudispert, was used to make the hydrogel formulations. As absorption enhancers, 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD), lauric acid (C12), or the sodium salt of C12 (C12Na), were incorporated into the hydrogels. In an in vitro release test, the release rate of insulin from the hydrogels decreased as the polymer concentration of the hydrogel increased. The addition of C12Na to the hydrogel further increased the insulin release rate, which was greater at higher concentrations of the enhancer. A portion of the C12Na was found to remain bound to the acrylic polymer in dissolution medium. Serum insulin levels were determined at various time points after the administration of insulin solution or insulin-loaded (50 units/kg body weight) Eudispert hydrogels containing 5% (w/w) of C12, C12Na, or DM-beta-CyD to in situ loops in various regions of the rat intestine. The most effective enhancement of insulin release was observed with formulations containing C12Na. The bioavailability of insulin from the hydrogels was lower than that from the insulin solutions. Hydrogel formulations containing 7% or 10% Eudispert remained in the rectum for 5 h after rectal administration. However, the 5% (w/w) C12Na solution stained with Evan's-blue had diffused out and the dye had reached the upper intestinal tract within 2 h. Finally, the rectal administration of insulin-loaded hydrogels, containing 4%, 7%, or 10% (w/w) Eudispert and 5% (w/w) of enhancer (C12, C12Na, or DM-beta-CyD) to normal rats was shown to decrease serum glucose concentrations. The greatest effect was found with insulin-loaded 7% (Eudispert) hydrogel containing C12Na which having cosiderable large insulin release rate and bioadhesive characteristics.

The rectal delivery of insulin using a bioadhesive hydrogel preparation containing various absorption enhancers was evaluated in rats. Hydrogels were prepared from acrylic block copolymers of methacrylic acid and methacrylate (Eudispert hv). Sodium salts of various fatty acids were incorporated into the hydrogels as absorption enhancers. Sodium oleic acid and 2,6-di-O-methyl-β-cyclo-dextrin (DM-β-CyD) were used as control enhancers. Rectal administration of 50 units kg−1 insulin-loaded 7% (w/w) Eudispert hydrogels containing 5% (w/w) sodium lauric acid (C12) caused a significant (P < 0.01) decrease in plasma glucose levels compared with gels containing 5% DM-β-CyD or sodium oleic acid (43% vs 26 and 17%, respectively), in normal rats. The optimized gel formulation containing C12 was administered to non-insulin-dependent diabetes mellitus (NIDDM) rats. At a dose of 10 units kg−1 insulin, the decrease in plasma glucose levels was significantly (P < 0.05) greater in NIDDM rats compared with normal rats (35 vs 22%, respectively). Bioavailability was approximately 18% in both NIDDM and normal rats. As an index of local toxicity, the amount of protein released from the rectal membrane after administration of hydrogels was measured in normal rats. The amount of protein released for hydrogels containing C12 was significantly (P < 0.05) lower than that for C12 solution. Eudispert hydrogels containing lauric acid may be useful rectal preparations for the delivery of insulin.

The objective of this study was to prepare and evaluate the possibility of cylindrical xerogel preparations with zero-order release characteristics. As model polymers, polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), and acrylic block copolymers of methacrylic acid and the methacrylate, Eudispert, were selected and formed into xerogel formulations. Tegafur, 5-fluorouracil (5-FU), aspirin, benzoic acid, p-methoxybenzoic acid, theophylline, and salicylamide were employed as model compounds and thereby incorporated into xerogel matrices.In a dissolution test (rotating basket method; pH 7.4), PVA xerogel did not erode nor swell in dissolution medium, and did not exhibit zero-order release, but rather exhibited Fickian's law diffusion followed by the initial burst release profile. In the case of HPMC xerogel, swelling of the polymer was observed to some extent, but the release profile was almost the same as PVA, suggesting the Fickian diffusion of drug from HPMC gel; in contrast, Eudispert gel showed zero-order release in every drug. The polymer was also gradually dissolved into the medium at a zero order release rate.Finally, the Eudispert xerogel containing theophylline was orally administered to beagle dogs, and plasma was withdrawn periodically. The sustained-release characteristics were observed and the possibility of the cylindrical xerogel preparation for oral usage was demonstrated.

The hypoglycemic effect and the alpha-glucosidase activity inhibition of acarbose (AC:alpha-glucosidase inhibitor) were investigated in normal and KK-Ay mice, an animal model of noninsulin-dependent diabetes mellitus (NIDDM). AC improved hyperglycemia after an oral administration of maltose or sucrose, dose dependently in normal mice (1, 10, and 50mg/kg body weight) and in KK-Ay mice (50mg/kg). Furthermore, AC (50mg/kg) significantly inhibited maltase and sucrase activities in the small intestines of normal and KK-Ay mice (inhibitory efficacy: sucrasemaltase). The enzymatic inhibition in KK-Ay mice is stronger than in normal mice. However, AC (50 mg/kg) did not suppress the blood glucose in oral lactose tolerance and did not inhibit the lactase activity in either normal or KK-Ay mice. These findings indicate that the AC effect on the inhibition of alpha-glucosidase activity is selective for sucrase and maltase in normal and NIDDM mice.

The effects of high-fat diets containing fish oil or lard on blood glucose and plasma insulin after oral glucose loading were compared in genetically diabetic (db/db) mice, one of the animal models of non-insulin-dependent diabetes mellitus (NIDDM) with hyperinsulinemia. The blood glucose levels were significantly decreased in 27% of the mice fed high-fat diets containing 20% fish oil 30 and 60 min after the oral administration of glucose (both; p < 0.05). Conversely, the plasma insulin levels were significantly increased 30 min after the glucose loading as compared to 27% of the mice fed high-fat diets containing 20% lard (p < 0.01). In addition, a significant hypoglycemic effect was observed 60 min after the subcutaneous administration of insulin to mice on the fish oil diet (p < 0.05), whereas no effect was demonstrated in the case of those on the lard diet. The average body weight of the fish oil-treated mice was not significantly different from that of the lard-treated mice. The fish oil diet has a beneficial effect on glucose tolerance by increasing the insulin secretory capacity from pancreatic beta cells and also ameliorating insulin resistance.

Although family studies and genome-wide association studies have shown that genetic factors play a role in glaucoma, it has been difficult to identify the specific genetic variants involved. We tested 669 single nucleotide polymorphisms (SNPs) from the region of chromosome 2 that includes the GLC1B glaucoma locus for association with primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG) in the Japanese population. We performed a two-stage case-control study. The first cohort consisted of 123 POAG cases, 121 NTG cases and 120 controls: the second cohort consisted of 187 POAG cases, 286 NTG cases, and 271 controls. Out of six SNPs showing significant association with POAG in the first round screening, seven SNPs were tested in the second round. Rs678350 in the HK2 gene coding sequence showed significant allelic (p = 0.0027 in Stage Two, 2.7XE-4 in meta-analysis) association with POAG, and significant allelic (p = 4.7XE-4 in Stage Two, 1.0XE-5 in meta-analysis) association with NTG. Although alleles in the TMEM182 gene did not show significant association with glaucoma in the second round, subjects with the A/A allele in TMEM182 rs869833 showed worse visual field mean deviation (p = 0.01). Even though rs2033008 in the NCK2 gene coding sequence did not show significant association in the first round, it had previously shown association with NTG so it was tested for association with NTG in round 2 (p = 0.0053 in Stage Two). Immunohistochemistry showed that both HK2 and NCK2 are expressed in the retinal ganglion cell layer. Once multi-testing was taken into account, only HK2 showed significant association with POAG and NTG in Stage Two. Our data also support previous reports of NCK2 association with NTG, and raise questions about what role TMEM182 might play in phenotypic variability. Our data suggest that HK2 may play an important role in NTG in the Japanese population.

This chapter describes the structural chemistry and the biological aspects of carboxypeptidase G 3 . Carboxypeptidase G 3 consists of four subunits of identical molecular mass of 15 kDa. The pI is estimated to be 7.2. The enzyme is suspected to be a zinc-dependent metallopeptidase. The amino acid sequence and gene for carboxypeptidase G 3 are not known yet. The enzyme exists on the cell membrane of Pseudomonas sp. bacteria and seems to be a constitutive enzyme. The enzyme shows in vitro antitumor activity. Carboxypeptidase G 3 very slightly inhibits the growth of murine leukemia, but shows remarkable antitumor activities against human carcinoma KB and PC-3 cells among ten kinds of human carcinoma cell lines. Its antitumor activity mechanism seems to be based on the degradation of folic acid. Unlike carboxypeptidases G, G 1 and G 2 , the carboxypeptidase G 3 acts not only on the L-form of acidic amino acids, but also on the D-form and slightly hydrolyzes Bz-DL-Glu. However, some properties such as C-terminal glutamate-releasing activity and the requirement for Zn 2+ are quite similar to those of the other enzymes.

Determination of volatile components in essential oils from Atractylodis plants was studied by headspace gas chromatography. The crude drug of 0.20 g with 1.0 ml of water in a capped vial was heated at 130 degrees C for 45 min. Then 0.5 ml of vaporized components were collected by gas tightsillinge, and were applied into the injection port of GC or GC/MS. Consequently we could analyze and confirm the following components; hinesol and beta-eudesmol were found to be contained in Atractylodis Lanceae Rhizoma and atractylon in Atractylodis Rhizoma. beta-Eudesmol was analyzed by headspace gas chromatography, and the obtained calibration curve showed good linearity over the range from 2.5 micrograms to 10.0 mg. The result agreed with those obtained using numerical analyses by the steam distillation method. Atractylodis was found to have a wide variety of components depending on the available sources and on the stored conditions. This method was, therefore, more rapid and simpler determination of essential oils in crude drugs using headspace gas chromatography than those used previously. The method was useful means of the analyses of components in crude drugs such as Atractylodis plants and quality control.

Although intraocular pressure (IOP) is the most definitive cause of glaucoma, a subtype of open angle glaucoma (OAG) termed normal tension glaucoma (NTG), which occurs in spite of normal IOP, accounts for a large part of glaucoma cases, especially in Japan. To find common genetic variants contributing to NTG in Japanese patients, we conducted a genome-wide association study (GWAS). We performed the first screening for 531,009 autosomal SNPs with a discovery cohort of 286 cases and 557 controls, and then a second screening for the top 30 suggestive loci in an independent cohort of 183 cases and 514 controls. Our findings identified a significantly associated SNP; rs523096 [combined p-value = 7.40× 10−8, odds ratio (OR) = 2.00 with 95% confidence interval (CI) 1.55–2.58] located 10 kbp upstream of CDKN2B on chromosome 9p21. Moreover, analysis of another independent case-control set successfully replicated the results of the screening studies (combined values of all 3 stages p = 4.96 × 10−11, OR = 2.13 with 95% CI 1.69–2.68). The SNPs near rs523096 were recently reported to be associated with OAG associated with elevated IOP in primary open-angle glaucoma (POAG), the predominant subtype of glaucoma in Caucasian populations. Our results revealed that the 9p21 locus is also associated with NTG in Japanese. In addition, we identified SNPs more strongly associated with NTG.

Purpose To determine whether polymorphisms in the Toll-like receptor 4 ( TLR4 ) gene are associated with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma (XFG) in Japanese individuals. Design Genetic association study. Methods Setting: Multicenter study. Study population: One hundred eighty-four unrelated Japanese patients with POAG, 365 unrelated patients with NTG, and 109 unrelated patients with XFG from 5 hospitals. Procedures: Genomic DNA was extracted from leukocytes of the peripheral blood, and 8 polymorphisms in the TLR4 genes were amplified by polymerase chain reaction (PCR) and directly sequenced. Allele and genotype frequencies and the inferred haplotypes were estimated. Main outcome measures: Differences in allele and genotype frequencies and haplotypes between subjects with POAG, NTG, and XFG. Results The allele frequency of rs2149356 of the TLR4 gene in the POAG, NTG, and XFG groups was the most significantly different from that of the control group (minor allele frequency 0.446, 0.395, 0.404, vs 0.308; P = .000058, P = .0030, and P = .015). The allele frequencies of the 5 TLR4 SNPs were higher in all of the glaucoma groups than that in the control group. The statistics of genotypes of TLR4 were approximately the same for all allele frequencies. The haplotypic frequencies with Tag SNPs studied earlier showed that only POAG was statistically significant. Other haplotypes, such as rs10759930, rs1927914, rs1927911, and rs2149356, had higher statistical significance (overall P = .00078 in POAG, overall P = .018 in NTG, and overall P = .014 in XFG). Conclusions This study demonstrated that TLR4 polymorphisms are associated with NTG in the Japanese, and they also play a role in the pathogenesis of POAG and XFG.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60, 000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (Mr : 15, 000). The enzyme hydrolyzed predominately acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not noly on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.

To evaluate the relationship between difficulty in daily living and binocular visual field in patients with glaucoma.We examined 144 patients with glaucoma who had bilateral visual field disturbances. The mean age was 68 +/- 13 years. Difficulty in daily living was assessed using a questionnaire consisting of 10 questions on daily activities. Each response was rated on a scale of 1 to 4: with a score of 4 for "no difficulty", 3 for "some difficulty", 2 for "great difficulty" and 1 for "cannot manage at all". The binocular visual field was assessed using the Esterman visual field test and the Esterman score was calculated. The relationship between difficulty in daily living and Esterman score was evaluated.The mean total score for difficulty in daily living was 34 +/- 7 (range; 13-40). The mean Esterman score was 79 +/- 24 (range; 0-100). The total score for difficulty in daily living correlates positively with the Esterman score (r2 = 0.48, p0.0001).The present study indicates that the Esterman score is a good predictor of difficulty daily living.

We investigated the correlation of age and intraocular pressure (IOP) with visual field damage in patients with normal-tension glaucoma (NTG) and asymmetric visual field defects.A total of 88 NTG patients (176 eyes) were investigated. Patients in whom visual field defects were more severe in the eye with higher IOP than in the fellow eye were grouped as IOP-visual field-concordant patients, and the others as IOP-visual field-discordant patients. Mean 24-hour IOP was used as an indicator of IOP, and mean deviation (MD) as an indicator of visual field defects. Patients were also compared by age tertile.The IOP-visual field-concordant group comprised 53 of the 88 patients (60.2%), among whom the70-years tertile had significantly greater absolute values for MD asymmetry. In contrast, no significant differences in asymmetry by age tertile were seen in the IOP-visual field-discordant group.Elevated IOP is a cause of visual field defects in many NTG patients. Among IOP-visual field-concordant patients, particular attention is required to visual field changes in the eyes of patients with higher IOP.