Ursolic acid and oleanolic acid, members of pentacyclic triterpenoid acids, suppress TNF-α-induced E-selectin expression by cultured umbilical vein endothelial cells.

E-selectin is an early response adhesion molecule expressed on the surface of endothelial cells during inflammatory responses. We examined the effects of two pentacyclic triterpenoid acids, ursolic acid (UA) and oleanolic acid (OA), on the expression of E-selectin by cultured human umbilical vein endothelial cells (HUVECs). Treatment of the cells with UA or OA alone did not influence expression of E-selectin. Expression of E-selectin mRNA and surface antigen by HUVECs was induced by treatment with tumor necrosis factor-α (TNF-α) in a dose- and time-dependent manner. TNF-α-induced up-regulation of E-selectin was abrogated by pre-treatment of the cells with UA or OA which decreased expression of E-selectin mRNA. The repression of E-selectin mRNA expression caused by the pentacyclic triterpenoid acids paralleled the inhibition of NF-κB activation and nuclear translocation, as evaluated by electrophoretic mobility shift assays, although the degree of repression by UA was approximately two times more effective than that of OA. The results suggest that UA and OA suppress the inflammatory cytokine-induced expression of E-selectin in endothelial cells by decreasing E-selectin transcription via inhibition of NF-κB activation. Thus, UA and OA function as anti-inflammatory agents. The differences in the inhibitory efficacy between UA and OA may be due to conformational differences in ring-E of the two pentacyclic triterpenoid acids.

Screening of Nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition.

South-East Asian population is daily exposed to strong sunlight. As a result, the majority of population will have darker, ethnic skin. Moreover, many people suffer from dark spots, hyperpigmentation, which is considered to be a skin disorder and causes psychological disturbance. To treat dark spots, most of the population will still rely on traditionally used crude drugs, knowledge about which is transferred from generation to generation. Fifty-two crude drugs were selected based on the survey performed among local healers and beauticians of different ethnic origin. These crude drugs were screened for mushroom tyrosinase inhibitory activity, as tyrosinase inhibitors are becoming increasingly important as cosmetic and medicinal products, primarily to control hyperpigmentation. Among the tested crude drugs, methanolic extracts of Glycyrrhiza glabra, Morus alba, Syzygium aromaticum, Citrus aurantifolia, Cypreae moneta, Punica granatum and Citrus aurantium, at the final concentration of 50 microg mL(-1), showed mushroom tyrosinase inhibitory activity of 78.9%, 71.0%, 69.4%, 59.0%, 56.0%, 53.4 and 51.9%, respectively, with 91.4% inhibitory activity of kojic acid taken as positive control. To our knowledge, this is the first report that extracts of Cypreae moneta shell and Syzygium aromaticum flowering bud have tyrosinase inhibitory activity. These potent extracts were further evaluated at different concentration. The final concentration of the extracts in reaction mixtures was 50, 25 and 5 microg mL(-1) for the initial concentration of 1000, 500 and 100 microg mL(-1), respectively. They showed concentration-dependent inhibition of mushroom tyrosinase. Those extracts expressing relatively weak tyrosinase inhibitory activity may act through different inhibition pathway which is not based on tyrosinase activity. Further evaluation of the most potent tyrosinase inhibitors in in vivo conditions would be recommended.

Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent allergy in Japan. Recently, the Japanese cedar pollen allergen Cry j 3 was cloned as a homologue of Jun a 3, which is a major allergen from mountain cedar (Juniperus ashei) pollen. However, native Cry j 3 has not been isolated and there are no reports on its allergenic activity. The aims of this study were to isolate native Cry j 3 and assess its immunoglobulin E (IgE)-binding capacity in patients with Japanese cedar pollinosis. METHODS: Native Cry j 3 was purified from Japanese cedar pollen by multidimensional chromatography. We assessed the IgE-binding capacity using sera from patients allergic to Japanese cedar pollen by immunoblot analysis and ELISA. Moreover, we assayed the capacity of Cry j 3 to induce histamine release from the patients' leukocytes. We cloned cDNA corresponding to purified Cry j 3 from a cDNA library of Japanese cedar pollen. RESULTS: We isolated native Cry j 3 as a 27-kDa protein. The IgE-binding frequency of Cry j 3 from the sera of patients allergic to Japanese cedar pollen was estimated as 27% (27/100) by ELISA. Cry j 3 induced the release of histamine from leukocytes. We cloned the cDNA and named it Cry j 3.8. Cry j 3.8 cDNA encoded 225 amino acids and had significant homology with thaumatin-like proteins. CONCLUSIONS: Cry j 3 is a causative allergen in Japanese cedar pollinosis and may play crucial roles in the cross-reactivity with oral allergy syndrome.

Osteogenic protein-1 promotes the formation of tissue-engineered cartilage using the alginate-recovered-chondrocyte method.

OBJECTIVE: This study examined the effects of a growth factor, recombinant human osteogenic protein-1 (rhOP-1), on the formation of tissue-engineered cartilaginous tissue by adult bovine articular chondrocytes using the alginate-recovered-chondrocyte (ARC) method. DESIGN: To ascertain if rhOP-1 enhances the formation of the cell-associated matrix (CM) and the characteristics of CM formation, bovine articular chondrocytes were first cultured for up to 14 days in alginate beads in medium supplemented with serum, with or without rhOP-1. Then, the recovered chondrocytes and their associated CM were resuspended in medium, with or without OP-1, seeded onto culture inserts, and incubated for an additional 14 days. The fabricated ARC tissues were subjected to biochemical and histological analyses. RESULTS: The addition of rhOP-1 to the medium in the alginate bead culture step resulted in an increased accumulation of both proteoglycan (PG) and collagen, with a ratio of PG to collagen that was higher than that found in native adult cartilage. The addition of rhOP-1 in the second step had a similar stimulatory effect during 14 days of culture. Histological examination of the tissue formed under all conditions revealed a cartilage-like matrix, stained strongly by toluidine blue. The thickness of the tissues obtained from culture conditions that included the addition of rhOP-1 was four times greater than that of the tissues cultured without rhOP-1. CONCLUSIONS: Using the ARC method, rhOP-1 enhanced the formation of matrix and generated a voluminous tissue-engineered cartilaginous construct. These characteristics may be beneficial in generating constructs that can cover large defects.

Identification of peptides containing T-cell epitopes of Japanese cedar (Cryptomeria japonica) pollen allergen (Cry j 1) in dogs.

Japanese cedar (Cryptomeria japonica, CJ) pollen has been known to cause atopic dermatitis in dogs in Japan. However, since the mechanism of the CJ antigen recognition is not well understood in dogs, it is difficult to develop effective immunotherapy for atopic dermatitis caused by sensitization to CJ pollen. In order to aim at development of a peptide immunotherapy, we tried to identify T-cell epitopes of a major allergen of CJ pollen, Cry j 1, in dogs sensitive to CJ pollen allergen. Peripheral blood mononuclear cells (PBMCs) obtained from 22 dogs experimentally sensitized to CJ pollen allergen and 5 atopic dogs sensitive to CJ pollen allergen were used for mapping of T-cell epitopes of Cry j 1 using 35 kinds of synthesized overlapping peptides of Cry j 1. Reactive peptides were identified based on the results of blastogenic responses of PBMCs against the peptides when the stimulation indices were beyond 2.0. Three reactive peptides were identical in a relatively high population of experimental dogs, which were Nos. 8 (p71-90) (41%), 10 (p91-110) (50%), and 11 (p101-120) (41%). It was considered that these synthesized peptides should contain T-cell epitopes of Cry j 1 in the dogs. However, there were no reactive peptides identical among the five atopic dogs spontaneously sensitive to CJ pollen. The population of dogs experimentally sensitized to CJ pollen antigen will be used in order to investigate effects of a peptide immunotherapy using the reactive peptides. The results in atopic dogs sensitive to CJ pollen antigen will also provide useful information on necessity to develop a tailor-made immunotherapy using reactive peptides in each dog.

Increase of CC chemokine receptor 4-positive cells in the peripheral CD4 cells in dogs with atopic dermatitis or experimentally sensitized to Japanese cedar pollen.

BACKGROUND: Since dogs frequently develop allergic diseases, similar to those in humans, dogs represent a possible animal model for allergy in humans. In human atopic dermatitis (AD), CC chemokine receptor 4 (CCR4) has been shown to play an important role in the development of allergic inflammation of AD; however, the association between allergic reaction and CCR4 is not well understood in dogs. OBJECTIVE: To examine CCR4 expression in peripheral blood CD4+ cells in dogs that had AD and were experimentally sensitized with Japanese cedar pollen. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 17 dogs with AD. The proportion of CCR4+ cells in peripheral blood CD4+ cells (CCR4/CD4) was evaluated by flow cytometry and compared with that in 10 healthy dogs. Similarly, in dogs that were experimentally sensitized to Japanese cedar pollen antigen, the proportion of CCR4/CD4 was examined pre- and post-sensitization. RESULTS: The proportion of CCR4/CD4 in dogs with AD was 40.3+/-3.3%, which was significantly higher than that in normal dogs (23.6+/-4.3%) (P<0.01). In the experimentally sensitized dogs, the proportion of CCR4/CD4 was 25.4+/-2.6% at pre-sensitization and it was significantly increased (29.8+/-2.9%) at post-sensitization (P<0.01). CONCLUSION: The proportion of CCR4+ cells in peripheral blood CD4+ cells was measured in dogs with allergic conditions. The present findings indicate that CCR4+ cells may be involved in the pathogenesis of allergy in dogs as in humans.

Comparative effects of dietary fat types on hepatic enzyme activities related to the synthesis and oxidation of fatty acid and to lipogenesis in rats.

The effects of different types of dietary fat on the activities of hepatic enzymes related to fatty acid synthesis [glucose-6-phosphate dehydrogenase (G6PDH) and acetyl-CoA carboxylase (ACC)], oxidation [acyl-CoA synthetase (AST), carnitine palmitoyl transferase (CPT), and peroxisomal beta-oxidation (PbetaOX)], and lipogenesis [phosphatidate phosphohydrolase (PAP), diacylglycerol acyltransferase (DGAT), and phosphocholine diacylglycerol transferase (PCDGT)], and plasma and liver lipid levels were investigated in male Wistar rats. The animals were 6 weeks old and about 120 g of body weight, and were fed on test diets containing 20% of a mixture of tripalmitin, tristearin and corn oil (SFA), olive oil (OLI), sunflower oil (SUN), linseed oil (LIS), and sardine oil (SAR) for 2 weeks. The concentrations of plasma total cholesterol (T-CHOL), high-density lipoprotein-cholesterol (HDL-CHOL), triacylglycerol (TG) and phospholipid (PL) were generally higher in the rats fed on SFA and OLI than in those given SUN, LIS and SAR. The rats fed on OLI had a higher level of liver T-CHOL than those fed on the other fats. The liver TG content was nearly higher from the intake of SFA and OLI than from SUN, LIS and SAR, although the liver PL level was not affected by the type of dietary fat. The SFA and OLI groups had the highest activities of hepatic G6PDH and ACC, and the SAR group, the lowest activities. The activities of AST and CPT, and peroxisomal PbetaOX in the liver were higher in the rats fed on the LIS and SAR diets than in those given the other diets. The hepatic PAP activity was higher from the intake of OLI and SUN, and tended to be higher from SFA than from LIS and SAR. The activity of liver DGAT was higher from SFA and inclined to be higher from OLI, SUN, and LIS than from SAR, while the PCDGT activity in the liver was not effected by the type of dietary fat. The concentrations of plasma and liver TG were generally positively correlated with the activities of liver enzymes related to the synthesis of fatty acids and lipids, and negatively with those involved in fatty acid oxidation. Based on these results, it is suggested that the levels of plasma and liver TG were controlled by different types of dietary fat through changes in the hepatic enzyme activities related to fatty acid synthesis, lipogenesis, and fatty acid oxidation.

IgE reactivity and cross-reactivity to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens in dogs with atopic dermatitis.

The natural occurrence of Japanese cedar (Cryptomeria japonica) pollinosis has been reported in dogs with atopic dermatitis. However, the reactivity to Japanese cypress (Chamaecyparis obtusa) pollen allergens in these dogs has not been reported. The present study was designed to investigate the reactivity to Japanese cypress pollen allergens in dogs sensitized to Japanese cedar pollen allergens. In 19 dogs with specific IgE to C. japonica pollen allergen, we measured the specific IgE to C. obtusa pollen allergen and examined the reactivity to the allergen by intradermal test. Of the 19 dogs, 18 had specific IgE to crude and purified major allergens (Cha o 1) of C. obtusa pollen. Most of the dogs showed a positive reaction to C. obtusa pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in C. japonica pollen) was observed by the ELISA inhibition method. Dogs sensitized to Japanese cedar pollen allergens demonstrate reactivity to Japanese cypress pollen allergens.

Low power diode laser treatment using indocyanine green for eradication of esophageal varices.

BACKGROUND AND STUDY AIMS: Endoscopic variceal ligation (EVL) is an alternative to sclerotherapy for the treatment of esophageal varices, but is associated with higher rates of recurrence and subsequent bleeding than sclerotherapy. To prevent recurrence of varices after EVL, we have developed a low-dose diode laser therapy combined with the injection of indocyanine green, which allows enhanced tissue absorption of the laser beam selectively around varices. In this study we investigated the efficacy and safety of this technique. PATIENTS AND METHODS: Eight patients with F2 or F3 esophageal varices were enrolled. At 1 week after EVL, indocyanine green solution (1 mg/ml) was injected submucosally around the remaining varices. A diode laser (power 10 watts) was applied to the surface from the esophagogastric junction to 5 cm above it. The spot size was kept to 5 mm in diameter. RESULTS: Laser irradiation was performed safely, without bleeding from the varices, or perforation. There were no major complications. Endoscopy 1 month later showed F0 forms in seven patients, F1 in one patient, and no red color sign in any patient. No recurrence of varices has been observed in any of the patients during the follow-up period of at least 12 months. CONCLUSION: This technique may provide a simple, safe and effective procedure, as an additional treatment to EVL, for the prevention of recurrence of esophageal varices.

Comparison of the effects in vitro of tea tree oil and plaunotol on methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

The effects in vitro of tea tree oil (TTO) and plaunotol were examined by monitoring the growth of a standard strain of Staphylococcus aureus FDA 209P and of fourteen methicillin-susceptible strains of S. aureus (MSSA), together with twenty methicillin-resistant strains (MRSA). The minimum inhibitory concentrations (MIC) and the doses for 50% inhibition of growth (ID50) were determined by the micro-broth dilution (MD) method, and the broth dilution with shaking (BDS) method, respectively. The MIC of plaunotol for 50 and 90% of the MSSA and MRSA were assessed by the MD method, as 16 microg/ml and > or = 1,024 microg/ml, respectively. No antibacterial effects of TTO on MSSA and MRSA were detected by the MD method. The growth-inhibitory effects of TTO on S. aureus by the BDS method were examined, and it appeared that TTO was effective over a lower range of concentrations than previously reported. It seems that TTO is very effective in vitro against MSSA and MRSA at high concentrations but less effective below 40 microg/ml of TTO.

A new secoiridoid glucoside, amaronitidin, from the Peruvian folk medicine "Hercampuri" (Gentianella nitida).

A new secoiridoid glucoside designated amaronitidin (1) was isolated from the Peruvian folk medicine "Hercampuri" (Gentianella nitida) along with three known secoiridoid glucosides. Their structures were determined by extensive spectroscopic investigation.

Seasonal rhinitis in a cat sensitized to Japanese cedar (Cryptomeria japonica) pollen.

A cat showing seasonal allergic symptoms of rhinitis was examined for reactivities to Japanese cedar (Cryptomeria japonica, CJ) pollen allergen by intradermal skin test (IDST), Prausnitz-Kustner (P-K) test, and lymphocyte blastogenic response. In IDST for 26 common allergens. the cat showed a positive reaction to CJ pollen allergen. P-K test using CJ pollen allergen also showed a positive reaction, indicating the presence of serum IgE specific to CJ pollen. In the lymphocyte blastogenic response, the stimulation index in the presence of CJ pollen allergen was 2.4. These data suggested that the seasonal rhinitis observed in the cat was caused by the sensitization to CJ pollen allergen.

Analysis of the canine IgE-binding epitope on the major allergen (Cry j 1) of Japanese cedar pollen with anti-Cry j 1 monoclonal antibodies.

In our previous study [Immunology 91 (1997) 161] using monoclonal antibodies (mAbs) specific to Cry j 1, a major allergen in Japanese cedar (Cryptomeria japonica) pollen, we identified five independent epitopes (EP-1-EP-5) on the molecule and found that EP-1 and EP-5 are the predominant allergic epitopes for humans and monkeys, respectively. In this study, we analyzed the epitopes recognized by IgE in the sera of 10 dogs sensitive to C. japonica pollen allergen using an IgE-ELISA inhibition method with these mAbs. The IgE reaction patterns varied among dogs. In eight of the 10 dogs, IgE recognized EP-5 which is a predominant allergic epitope for monkeys with the pollenosis. In four dogs, IgE recognized EP-1 which is a predominant allergic epitope for human patients with the pollenosis. In three dogs, IgE recognized EP-4 which is a heat-stable epitope. EP-5 is a predominant allergic epitope for dogs and some, but not all, dogs have IgE reaction patterns to the epitopes similar to those of humans.

Experimental sensitization with Japanese cedar pollen in dogs.

Japanese cedar pollinosis is a type I allergic disease mediated by immunoglobulin E (IgE) antibodies to Japanese cedar (Cryptomeria japonica) pollen antigen (CPAg). By using 22 dogs consisting of 20 dogs aged 3 months and 2 dogs aged 3 years, immunization was performed by subcutaneous injections of CPAg with aluminum hydroxide gel. Variable levels of CPAg-specific IgE antibody response were detected in 21 of the 22 immunized dogs two weeks after the second immunization. This study provided an experimental sensitization system with CPAg in dogs, which will be useful for further immunological studies on Japanese cedar pollinosis.

In vivo and in vitro tests showing sensitization to Japanese cedar (Cryptomeria japonica) pollen allergen in atopic dogs.

Using both in vivo and in vitro tests, dogs with atopic dermatitis were examined for sensitization with Japanese cedar (Cryptomeria japonica, CJ) pollen allergen. Ten dogs with clinical manifestation of atopic dermatitis were shown to be sensitized to CJ pollen based on the results of intradermal skin test and serum antigen-specific IgE test. In vitro lymphocyte stimulation test showed blastogenic response after stimulation with crude antigen of CJ pollen in all of the 5 cases examined. The peripheral leukocytes showed increased histamine release after stimulation with crude antigen of CJ pollen in 2 cases examined. These data indicate that a proportion of dogs with atopic dermatitis is sensitized to CJ pollen in a cell-mediated manner and show immediate phase reaction of type I hypersensitivity.

Abnormal air-righting reflex in striatal rats.

To understand dynamic postural control of the higher brain, we compared air-righting reflexes in various decerebrate rats. Post-operative 3-5 d thalamic and mesencephalic rats displayed almost similar righting movements as intact. However, in striatal animals, coordination of righting movements was disrupted. The higher brain without cortical control could interfere with the brainstem center of the air-righting reflex.

IgE-reactivity to major Japanese cedar (Cryptomeria japonica) pollen allergens (Cry j 1 and Cry j 2) by ELISA in dogs with atopic dermatitis.

The present study investigated IgE-reactivity to two major Japanese cedar (Cryptomeria japonica, C. japonica) pollen allergens (Cry j 1 and Cry j 2) in dogs with atopic dermatitis by use of a fluorometric ELISA. The serum samples from 27 dogs that showed IgE-sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to the two major allergens. All 27 dogs had anti-Cry j 1 IgE, and 10 (37%) had anti-Cry j 2 IgE. Inhibition of binding of dog specific IgE to crude C. japonica pollen allergen was carried out by addition of Cry j 1. When serum samples containing anti-Cry j 1 IgE but no anti-Cry j 2 IgE were incubated with Cry j 1, specific IgE binding to crude C. japonica pollen allergen was almost abolished. These findings suggest that Cry j 1 is a major allergen in dogs.

Isolation of 10-hydroxycoronaridine from Tabernaemontana penduliflora and its estrogen-like activity.

The methanol extract of Tabernaemontana penduliflora was found to appreciably inhibit [3H]-estradiol binding to estrogen receptors. Activity-guided fractionation led to the isolation of two known alkaloids, 10-hydroxycoronaridine (1) and its 10-O-methyl ether, voacangine (2). These alkaloids together with other related alkaloids were tested for their estrogenic activities. Among these molecules, 1 was found to be the most potent estrogen agonist and is distinctly more active than genistein.

Positive reactions to common allergens in 42 atopic dogs in Japan.

Clinically important allergens for the diagnosis and treatment of atopic dermatitis vary geographically. In order to identify the most prevalent allergens in atopic dogs in Japan, 42 dogs with a clinical diagnosis of atopy were tested using both in vivo (intradermal skin test (IDST)) and in vitro (antigen-specific IgE assay) allergy tests. Allergens used for IDST included 26 allergen extracts from eight allergen groups: trees, weeds, grasses, house dust mites (HDM), molds, foods, epithelia, and arthropods. Immunodot assay was used to measure antigen-specific IgE against 24 allergens from these eight groups and against fish such as cod and sole. In the 42 dogs, the most common positive allergen reaction was to HDM on both IDST (29/42 dogs or 69%) and in vitro testing (23/42 or 54.8%). The second most frequent positive allergen reaction was to Japanese cedar pollen (21/42 or 50.0% for IDST and 7/42 or 16.7% for in vitro testing). In both tests, less than 20% of dogs had positive reactions to molds or foods. Positive reactions to cat epithelia were frequently found on IDST, but rarely found on in vitro testing. Agreement between the two tests was found in 26 instances: HDM (21 dogs), Japanese cedar pollen (five dogs) and wheat (one dog). In this study, the two most common allergens involved in atopic dermatitis in dogs in Japan were HDM and Japanese cedar pollen.