Detection and molecular phylogenetic analysis of chicken astrovirus in Saga prefecture, Japan.

We detected three chicken astrovirus strains in 4-day-old broiler chickens with high mortality and visceral gout and one strain from 150-day-old hens without clinical symptoms in Saga prefecture, Japan. Phylogenetic analysis based on ORF2 amino acid sequences revealed that the strains from the visceral gout cases belonged to subgroup Bi, and the strain from hens without clinical symptoms belonged to subgroup Aiii. Our study showed that CAstV had infiltrated into Saga prefecture, Japan. This is the first report of CAstV in Japan.

[A case report of adult-onset IgA vasculitis with repeated migrating abdominal lesions and manifestations].

A 68-year-old woman was referred to our hospital due to widespread purpura on her legs. A diagnosis of IgA vasculitis was made based on the findings of a skin biopsy. However, after being admitted to our hospital, abdominal pain and lower gastrointestinal hemorrhaging developed. The purpura disappeared gradually, whereas the abdominal pain migrated and persisted. Treatment with prednisolone was initiated, and the clinical course improved temporarily. However, her severe abdominal symptoms recurred while, in addition, the intestinal tract lesions migrated after the prednisolone dosage was tapered. Therefore, intravenous high-dose methylprednisolone was administered followed by oral steroids. The dose was thereafter carefully tapered, and the steroid dose reduction was successful with this treatment. We herein report the clinical course of the case along with a review of the relevant literature.

Cochineal dye-induced immediate allergy: Review of Japanese cases and proposed new diagnostic chart.

BACKGROUND: Cochineal dye is used worldwide as a red coloring in foods, drinks, cosmetics, quasi-drugs, and drugs. The main component of the red color is carminic acid (CA). Carmine is an aluminum- or calcium-chelated product of CA. CA and carmine usually contain contaminating proteins, including a 38-kDa protein thought to be the primary allergen. Severe allergic reactions manifest as anaphylaxis. The aim of this study was to review all Japanese reported cases and propose useful diagnostic chart. METHODS: All reported Japanese cases of cochineal dye-induced immediate allergy were reviewed, and newly registered cases were examined by skin prick test (SPT) with cochineal extract (CE) and measurement of CE and carmine-specific serum IgE test. Two-dimensional (2D) western blotting using patient serum was conducted to identify the antigen. RESULTS: Twenty-two Japanese cases have been reported. SPT and the level of specific IgE test indicated that six cases should be newly registered as cochineal dye allergy. All cases were adult females, and all cases except three involved anaphylaxis; 13 cases involved past history of local symptoms associated with cosmetics use. Japanese strawberry juice and fish-meat sausage, and European processed foods (especially macarons made in France) and drinks were recent major sources of allergen. 2D western blotting showed that patient IgE reacted to the 38-kDa protein and other proteins. Serum from healthy controls also weakly reacted with these proteins. CONCLUSIONS: SPT with CE and determination of the level of CE and carmine-specific IgE test are useful methods for the diagnosis of cochineal dye allergy.

Evaluation of [¹8F]MK-0911, a positron emission tomography (PET) tracer for opioid receptor-like 1 (ORL1), in rhesus monkey and human.

Antagonism of the central opioid receptor like-1 receptor (ORL1) has been implicated in cognition, and has been a focus of drug discovery efforts to ameliorate the cognitive deficits that remain during the stable treatment of schizophrenia with current antipsychotics. In order to facilitate dose selection for phase II clinical testing an ORL1-specific PET tracer was developed to determine drug plasma concentration versus occupancy relationships in order to ensure that the doses selected and the degree of target engagement were sufficient to ensure adequate proof of concept testing. MK-0911 is a selective, high affinity antagonist for the ORL1 receptor radiolabeled with high specific activity (18)F for positron emission tomography (PET) studies. Evaluation of [(18)F]MK-0911 in rhesus monkey PET studies showed a pattern of brain uptake which was consistent with the known distribution of ORL1. In vitro autoradiography with [(18)F]MK-0911 in rhesus monkey and human brain tissue slices showed a regional distribution that was consistent with in vivo imaging results in monkey. Pre-treatment of rhesus monkeys with high doses of structurally diverse ORL1 antagonists MK-0584, MK-0337, or MK-5757 achieved blockade of [(18)F]MK-0911 in all gray matter regions. Baseline PET studies with [(18)F]MK-0911 in healthy human subjects showed tracer distribution and kinetics similar to that observed in rhesus monkey. Quantification of [(18)F]MK-0911 uptake in repeat human baseline PET studies showed a test-retest variability in volume of distribution (V(T)) averaging 3% across brain regions. Humans dosed orally with MK-5757 showed reduced [(18)F]MK-0911 tracer concentration in brain proportional with MK-5757 dose and plasma level. [(18)F]MK-0911 was useful for determining MK-5757-induced receptor occupancy of ORL1 to guide MK-5757 dose-selection for clinical proof-of-concept studies. Additionally, [(18)F]MK-0911 may be a useful tool for studying the pharmacology of ORL1 in various human populations and disease states.

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion.

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5ß1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.

Transient expression of mouse pro-α3(V) collagen gene (Col5a3) in wound healing.

The α3(V) chain is poorly characterized among type V collagen chains. Pro-α3(V) collagen is expressed in newly synthesized bone as well as in the superficial fascia of developing muscle. Present study examined the expression in a mouse model of wound healing. Real-time reverse transcriptase polymerase chain reaction and in situ hybridization revealed transient expression of pro-α3(V) chain at a lower level than other fibrillar collagen genes after injury. Immunohistochemistry showed a similar expression pattern in the injured skin. In addition, electron microscopy showed that pro-α3(V) chain was localized in the amorphous nonfibrillar region, but not in fine or dense fibrils. The pro-α3(V) chain co-localized with heparan sulfate, which appeared in the skin after injury and might bind via an acidic segment of the pro-α3(V) chain. The matrix containing the pro-α3(V) chain may therefore be needed for the initiation of wound healing.

Scald Burns While Bathing Among Elderly People in Japan.

Night baths are an essential and beloved tradition in Japanese households. The main purpose of taking a bath at the end of the day, besides hygiene, is relaxation. The aging population is rapidly growing in Japan, as one in three people are approaching the age of 65 years or older. Furthermore, with the progress of nuclear families, the number of households with only elderly people and the need for elderly care is increasing. In recent years, elderly people experienced burns caused by hot water during bathing. We report two cases of water bath burns experienced by elderly people. The first is a case of a 68-year-old woman who presented with a history of type 2 diabetes and osteoarthritis in both knees. She did not notice that the bath stopper was unplugged while she was taking a bath, and she added hot water at around 44°C. She was exposed to hot fluid on the right foot and suffered deep dermal burns. However, due to knee osteoarthritis, it became difficult for her to move. Two hours after taking a bath, she was removed from the bathtub. The second is a case of a 71-year-old woman who presented with a history of type 2 diabetes and osteoarthritis in both knees. Because the temperature of the bath was approximately 44°C, she tried to cool down the water, but it was difficult for her to move because of knee osteoarthritis. She called out for help from her family living in the neighborhood, but she could not get out. She was sitting for about 2 hours before being noticed by her family. As a result, she suffered second-degree burns on both the buttocks and soles of her feet. Prolonged exposure to thermal liquids and burns such as low-temperature burns are caused by individual factors, such as decreased perception, orthopedic disease, and difficulty in moving due to fainting, and social factors such as delay in discovery in elderly people living alone. These factors lead to an increased depth of the burn.

A clinical and statistical analysis of abscess in cellulitis.

The features of abscess that forms in cellulitis patients were investigated. Among 449 cellulitis cases, about 15% developed abscess, and about 70% of teenaged patients were abscess cases. Furthermore, abscess in the younger age group occurred almost exclusively among male patients. The lesion was predominantly concentric at all ages in the abscess group and in teenagers in the non-abscess group. In contrast, it was diffuse in patients ≥20 years old in the non-abscess group. The clinical symptoms and laboratory values in the abscess group were generally more severe than those in the non-abscess group, especially in the older age group. The abscess sizes increased with the initial area of the lesions. Most abscess cases received incision, and post-incision ulcers were mostly conservatively treated. The duration required for treatment termination was >30 days longer in the abscess group than in the non-abscess group. When post-incision ulcers were conservatively treated, the duration until healing increased with abscess size. However, the durations did not significantly differ between the conservatively and surgically treated groups. When surgically treated, many ulcers healed within 19 days after operation and the outpatient follow-up period tended to be relatively short. Most teenaged patients were student athletes, and thus needed an early return to competition. The main cause in these patients was bruising during sports, with rugby football the most common causative sport. Ulcer closure by operation was suggested to be preferable for shortening the outpatient follow-up period, especially for student athlete patients.

Dermatopontin promotes epidermal keratinocyte adhesion via alpha3beta1 integrin and a proteoglycan receptor.

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by alpha3beta1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through alpha3beta1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

Prolonged ischemia increases severity of rejection in skin flap allotransplantation in rats.

To investigate the relationship between ischemic time and rejection against allotransplants, vascularized cutaneous flaps from the groin of Brown Norway rats were transplanted to Lewis rats. The ischemic time was set at 1 hour and 6 hours for comparison. Cycrosporine A was used as the immunosuppressant. The results showed more severe rejection in the 6 hours ischemic time group in vivo, and in vitro examination using mixed lymphocyte reaction assay also demonstrated a greater antidonor response in 6 hours-ischemic group than that in 1 hour-group. Immunohistochemical study demonstrated more MHC class II antigen expression in 6 hours-ischemic group than in 1 hour-group. These results suggest that longer ischemic time induces more severe rejection against allo-transplanted tissue compared with the shorter one through an upregulation of MHC class II antigen. It is expected that these findings contribute to the studies for investigating the mechanism of rejection against the allo-transplants.

Solitary subungual neurofibroma with glomus tumor-like appearance: a case report.

We present a case of subungual neurofibroma which clinically presented a glomus tumor-like appearance. Six months after surgical resection, no clinical recurrence or pain was reported. We herein report the difficulty of its preoperative diagnosis, including literature review.

Statistical analysis of anti-mamushi venom serum injection time and clinical course.

AIM: Early injection of anti-mamushi venom serum (antiserum) is believed to be effective for the treatment of patients with mamushi bites. However, there is no firm information that indicates the time range constituting "early" injection. We tried to quantify the cut-off time of antiserum injection that brings favorable clinical courses by clarifying the relationship between the injection time and clinical outcome. METHODS: We retrospectively analyzed the relationships between the time after bite, injection time of the antiserum, swelling grades, and laboratory values. RESULTS: The injection time of the antiserum in severe cases was significantly delayed as compared with non-severe cases. The best cut-off time of the antiserum injection that could distinguish non-severe and severe cases was 14 h. In the group that received the antiserum within 14 h, the antiserum injection may have successfully arrested the grade progression in a substantial number of cases. In the other group receiving the antiserum beyond 14 h, the grades in many cases possibly may have peaked by the time of antiserum injection. CONCLUSION: The cut-off time of early injection for favorable clinical course was determined to be 14 h. A statistical basis concerning the appropriate antiserum injection time was made to help prevent a severe clinical course due to delayed injection.

Synthesis and biological evaluation of imidazole derivatives as novel NOP/ORL1 receptor antagonists: exploration and optimization of alternative pyrazole structure.

Nonpeptidic small-molecule NOP/ORL1 receptor antagonists with an imidazole scaffold were designed and synthesized to investigate alternatives to the pyrazole analog. Systematic modification of the original pyrazole lead [Kobayashi et al., Bioorg. Med. Chem. Lett.2009, 19, 3627; Kobayashi et al., Bioorg. Med. Chem. Lett., in press] to change the heterocyclic core, substituted side chain, and pendant functional group demonstrated that examining the structure-activity relationship for novel templates allowed the identification of potent, fully substituted 4-aminomethyl-1H-imidazole and 2-aminomethyl-1H-imidazole. These compounds exhibited excellent potency for ORL1 receptor with minimal P-gp efflux and/or reduced hERG affinity.

Discovery of novel arylpyrazole series as potent and selective opioid receptor-like 1 (ORL1) antagonists.

The synthesis and biological evaluation of new potent opioid receptor-like 1 antagonists are presented. A structure-activity relationship (SAR) study of arylpyrazole lead compound 1 obtained from library screening identified compound 31, (1S,3R)-N-{[1-(3-chloropyridin-2-yl)-5-(5-fluoro-6-methylpyridin-3-yl)-4-methyl-1H-pyrazol-3-yl]methyl}-3-fluorocyclopentanamine, which exhibits high intrinsic potency and selectivity against other opioid receptors and hERG potassium channel.

Optimization of benzimidazole series as opioid receptor-like 1 (ORL1) antagonists: SAR study directed toward improvement of selectivity over hERG activity.

A structure-activity relationship (SAR) study on the benzimidazole series of opioid receptor-like 1 (ORL1) antagonists related to 1 is described. Optimization of 1 by introduction of a hydrophilic substituent into the thioether part resulted in identification of potent ORL1 antagonists with high selectivity over binding affinity for hERG and other opioid receptors.

Identification of MK-1925: a selective, orally active and brain-penetrable opioid receptor-like 1 (ORL1) antagonist.

Structure-activity relationship studies directed toward improving the metabolic stability of compound 1 resulted in the identification of 3-[5-(3,5-difluorophenyl)-3-({[(1S,3R)-3-fluorocyclopentyl]amino}methyl)-4-methyl-1H-pyrazol-1-yl]propanenitrile 39 (MK-1925) as a selective, orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonist. The compound also showed in vivo efficacy after oral dosing. Therefore, compound 39 was selected to undergo further studies as a clinical candidate.

Discovery of substituted 2,4,4-triarylimidazoline derivatives as potent and selective neuropeptide Y Y5 receptor antagonists.

Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline (1), which had an IC(50) of 54nM. Subsequent optimization led to the identification of several potent derivatives.

Aryl urea derivatives of spiropiperidines as NPY Y5 receptor antagonists.

Continuing medicinal chemistry studies to identify spiropiperidine-derived NPY Y5 receptor antagonists are described. Aryl urea derivatives of a variety of spiropiperidines were tested for their NPY Y5 receptor binding affinities. Of the spiropiperidines so far examined, spiro[3-oxoisobenzofurane-1(3H),4'-piperidine] was a useful scaffold for producing orally active NPY Y5 receptor antagonists. Oral administration of 5c significantly inhibited the Y5 agonist-induced food intake in rats with a minimum effective dose of 3mg/kg. In addition, this compound was efficacious in decreasing body weight in diet-induced obese mice.

Identification of novel and orally active spiroindoline NPY Y5 receptor antagonists.

A series of spiroindoline-3,4'-piperidine derivatives were synthesized and evaluated for their binding affinities and antagonistic activities at Y5 receptors. Potent Y5 antagonists were tested for their oral bioavailabilities and brain penetration in rats. Some of the antagonists showed good oral bioavailability and/or good brain penetration. In particular, compound 6e was orally bioavailable and brain penetrant, and oral administration of 6e inhibited bPP-induced food intake in rats with a minimum effective dose of 10mg/kg.

2-Cyclohexylcarbonylbenzimidazoles as potent, orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonists.

The synthesis and biological evaluation of new potent opioid receptor-like 1 (ORL1) antagonists are presented. Conversion of the thioether linkage of the prototype [It is reported prior to this communication as a consecutive series.: Kobayashi, K.; Kato, T.; Yamamoto, I.; Shimizu, A.; Mizutani, S.; Asai, M.; Kawamoto, H.; Ito, S.; Yoshizumi, T.; Hirayama, M.; Ozaki, S.; Ohta, H.; Okamoto, O. Bioorg. Med. Chem. Lett., in press] to the carbonyl linker effectively reduces susceptibility to P-glycoprotein (P-gp) efflux. This finding led to the identification of 2-cyclohexylcarbonylbenzimizole analogue 7c, which exhibited potent ORL1 activity, excellent selectivity over other receptors and ion channels, and poor susceptibility to P-gp. Compound 7c also showed satisfactory pharmacokinetic profiles and brain penetrability in laboratory animals. Furthermore, 7c showed good in vivo antagonism. Hence, 7c was selected as a clinical candidate for a brain-penetrable ORL1 antagonist.