Reevaluation of the earlobe types in Koreans.

The shape of the earlobes has a variety of genetic significance. This study analyzed the frequencies of the earlobe shapes in the Korean population. Data were collected on randomly selected 500 males and 500 females in Daegu Metropolitan City, with all participant ages being in their twenties. Obtuse angled earlobes accounted for 41.2% of the earlobes observed, while acute angled earlobes prevalence was calculated at 38.8% and right angled earlobe was 20.0% of the total (sexes combined). In men, the acute angled earlobe was the most frequent type (43.0%), while the obtuse angled earlobe was the most frequent type in females (45.2%). These differences were statistically significant (p = 0.015). Overall, attached type earlobe (61.2%) was more frequent than free type earlobe. The attached type earlobe was more common in both sex groups (57.0% in male and 65.4% in female), and the proportion was significantly higher for females (p = 0.006). In conclusion, the findings in this study suggest that the attached earlobe type is the most common among Koreans, and the proportion of earlobe types among males and females is significantly different. Further studies are needed to understand the genetic background of earlobe types among Koreans.

Increased sputum levels of thymus and activation-regulated chemokine in children with asthma not eosinophilic bronchitis.

BACKGROUND: Thymus and activation-regulated chemokine (TARC), a member of the CC chemokine family, plays a crucial role in Th2-specific inflammation. We aimed to determine the concentration of sputum TARC in children with asthma and eosinophilic bronchitis (EB) and its relation with eosinophilic inflammation, pulmonary function, and bronchial hyper-responsiveness. METHODS: In total, 90 children with asthma, 38 with EB, and 45 control subjects were enrolled. TARC levels were measured in sputum supernatants using an ELISA. We performed pulmonary function tests and measured exhaled fractional nitric oxide, eosinophil counts in blood, and sputum and serum levels of total IgE in all children. RESULTS: Sputum TARC levels were significantly higher in children with asthma than in either children with EB (p=0.004) or the control subjects (p=0.014). Among patients with asthma, sputum TARC concentration was higher in children with sputum eosinophilia than in those without sputum eosinophilia (p=0.035). Sputum TARC levels positively correlated with eosinophil counts in sputum, serum total IgE levels, exhaled fractional nitric, and the bronchodilator response. Negative significant correlations were found between sputum TARC and FEV1/FVC (the ratio of forced expiratory volume in one second and forced expiratory vital capacity) or PC20 (the provocative concentration of methacholine causing a 20% decrease in the FEV1). CONCLUSION: Elevated TARC levels in sputum were detected in children with asthma but not in children with EB. Sputum TARC could be a supportive marker for discrimination of asthma from EB in children showing characteristics of eosinophilic airway inflammation.

A multicenter study on anaphylaxis caused by peanut, tree nuts, and seeds in children and adolescents.

Peanut (PN) and tree nuts (TNs) are common causes of anaphylaxis in Western countries, but no information is available in Korea. To feature clinical characteristics of anaphylaxis caused by PN, TNs, and seeds, a retrospective medical record review was performed in 14 university hospitals in Korea (2009-2013). One hundred and twenty-six cases were identified, with the mean age of 4.9 years. PN, walnut (WN), and pine nut accounted for 32.5%, 41.3%, and 7.1%, respectively. The median values of specific IgE (sIgE) to PN, WN, and pine nut were 10.50, 8.74, and 4.61 kUA /l, respectively. Among 50 cases managed in the emergency department, 52.0% were treated with epinephrine, 66.0% with steroid, 94.0% with antihistamines, 36.0% with oxygen, and 48.0% with bronchodilator. In conclusion, WN, PN, and pine nut were the three most common triggers of anaphylaxis in Korean children, and anaphylaxis could occur at remarkably low levels of sIgE.

IgE cross-reactivity of peanut with walnut and soybean in children with food allergy.

BACKGROUND: Peanut allergies are common and can be life-threating for sensitised individuals. Peanut allergens share significant amino acid homology with those of other legumes and tree nuts, but their cross-reactivity still remains unclear. OBJECTIVE: We sought to determine the clinical significance of the cross-reactivity of peanut allergens with those of walnut and soybean. METHODS: Pooled sera from eight subjects with both peanut and walnut specific IgE were investigated in an inhibition test. After the sera were incubated with either peanut or walnut protein extracts, the quantity of IgE antibodies against the peanut and walnut was measured using an immunoCAP test. Likewise, pooled sera from 18 subjects with both peanut and soybean specific IgE antibodies were incubated with either peanut or soybean protein extracts and evaluated with a peanut and soybean immunoCAP test. SDS-PAGE and immunoblotting were also performed with peanut, walnut and soybean protein extracts and relevant sera. RESULTS: Peanut specific IgE was inhibited up to 20% and 26% by walnut and soybean protein extracts, respectively. In reverse, walnut and soybean specific IgE were inhibited up to 21% and 23% by peanut protein extracts, respectively. In the immunoblot analysis, pooled serum from the subjects with peanut specific IgE antibodies reacted with walnut protein extracts significantly. CONCLUSION: Although the clinical significance of the cross-reactivity of peanut specific IgE with walnut and soybean protein extracts has not been established, we believe that individuals who are allergic to peanuts need to be cautious about consuming walnuts and soybeans.

Relationship between sputum clusterin levels and childhood asthma.

BACKGROUND: Clusterin is a sensitive cellular biosensor of oxidative stress and has been studied as a biomarker for inflammation-associated diseases. Clusterin levels in childhood asthma have not been evaluated. OBJECTIVES: (1) To evaluate sputum clusterin levels in children with asthma compared to a control group. (2) To assess the relationships between sputum clusterin levels and airway inflammation, pulmonary function, and bronchial hyperresponsiveness. METHODS: This study included 170 children aged 5-18 years with stable asthma (n = 91), asthma exacerbation (n = 29), or no asthma (healthy controls; n = 50). Induced sputum, pulmonary function, and methacholine challenge tests were performed. Stable asthma was classified into two groups according to the severity. Clusterin levels in sputum were measured using an enzyme-linked immunosorbent assay. RESULTS: Children with stable asthma had a higher clusterin level than healthy controls [4540 (3872-5651) pg/mL vs. 3857 (1054-4369) pg/mL, P < 0.001]. The clusterin level was also more elevated in eosinophil-dominant sputum than in non-eosinophilic sputum in stable asthma [5094 (4243-6257) pg/mL vs. 4110 (1871-4839) pg/mL, P = 0.0017]. Clusterin levels were associated with asthma severity. Paradoxically, clusterin levels were lower during asthma exacerbation than in stable asthma [1838 (350-4790] pg/mL vs. 4540 (3872-5651) pg/mL, P < 0.001]. Clusterin levels were strongly correlated with the methacholine concentration that caused a 20% decrease in the forced expiratory volume in 1 s (r = -0.617, P < 0.001); there was no significant correlation between clusterin levels and other pulmonary function parameters. CONCLUSIONS AND CLINICAL RELEVANCE: Clusterin levels were altered in children with stable asthma and asthma exacerbation because of its antioxidant and anti-inflammatory effects. Clusterin may be a marker that reflects airway inflammation and severity of symptoms, and it can be used in the assessment and management of childhood asthma.

The role of interleukin-17 in mouse models of atopic dermatitis and contact dermatitis.

BACKGROUND: Atopic dermatitis (AD) and contact dermatitis (CD) are both T cell-mediated eczematous disorders. Interleukin (IL)-17, expressed by T helper (Th)17 cells, is involved in recruitment of inflammatory cells into AD and CD skin. AIM: In this study, we investigated whether IL-17 regulates immune dysregulation and affects skin barrier in oxazolone (OXA)-induced AD-like and CD-like disease models in mice, by comparing IL-17 null mutant (IL-17(-/-) ) vs. wild-type (WT) mouse strains in the models. METHODS: IL-17(-/-) and WT Balb/c mice were used for OXA induction of AD-like and CD-like skin diseases. Ear swelling was measured by a micrometer. Skin biopsies were obtained for RNA isolation and histology. Real-time quantitative PCR analysis was performed to quantify mRNA expression of Th2 cytokines. Skin permeability was measured by a vapometer, and structural changes in the skin were evaluated by electron and confocal microscopy. RESULTS: Both OXA-induced AD and CD responses were alleviated in IL-17(-/-) mice relative to WT, as demonstrated by reductions in ear swelling, inflammatory cell infiltration and levels of Th2 cytokines. These endpoints were used to characterize inflammatory dysregulation in both AD and CD models. Skin-barrier dysfunction, measured by increases in transepidermal water loss and dysfunction of lamellar bodies, and reductions in lipid distribution, were seen in both AD and CD in WT mice. In IL-17(-/-) mice, however, these responses were significantly diminished. CONCLUSIONS: The results indicate that the IL-17 gene may play a role in modulating immune dysregulation and affecting skin barrier in OXA-induced AD-like and CD-like skin disease models in the Balb/c mouse.

Chitinase induce the release of IL-8 in human airway epithelial cells, via Ca2+-dependent PKC and ERK pathways.

Chitinases are produced in significant quantities by hosts defending against infections with chitin-containing organisms. However, little is known about the immune response of exogenous chitinase in human epithelial cells. IL-8 has been suggested to have a role in the pathogenesis of the allergenic inflammation of bronchial asthma. We examined whether Streptomyces griseus (S. griseus) chitinase-induced IL-8 on airway epithelium and identified the involvement of intracellular signalling pathways. H292 cells were treated with S. griseus chitinase with different concentrations and times. The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction. Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to S. griseus chitinase. Cells exposed to S. griseus chitinase showed higher level of IL-8 protein production and mRNA expression. Cells stimulated by S. griseus chitinase resulted in the activation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-kB) pathways. Inhibitors of Ca(2+)-dependent PKC (Ro-31-8220, calphostin C and Go6976) significantly abolished chitinase-induced expression of IL-8. However, Ca(2+)-independent PKC inhibitor (rottlerin) did not inhibit IL-8 expression. Through ERK inhibitor (U0126) and NF-kB inhibitor (caffeine acid phenethyl ester) treatment, it was proven that ERK and NF-kB regulated chitinase-induced IL-8 expression. We concluded that S. griseus chitinase-induced IL-8 expression was regulated by the activation of Ca(2+/-)-dependent PKC, ERK and NF-kB in human airway epithelial cells.

Increased serum B cell-activating factor level in children with atopic dermatitis.

BACKGROUND: B cell-activating factor (BAFF) is a tumour necrosis factor superfamily member best known for its role in the survival and maturation of B cells. BAFF activity is seen in naïve and effector/memory T cells. AIM: To investigate the level and role of BAFF in serum of patients with atopic dermatitis (AD). METHODS: Levels of serum BAFF, a proliferation-inducing ligand (APRIL) and total serum IgE level, and total eosinophil count were measured in 245 children. RESULTS: Patients were characterized as having atopic eczema (AE) (n = 90) or non-AE (n = 77); the remainder were healthy control subjects (n = 78). Serum BAFF level in children with AE (1625.04 +/- 708.32 pg/mL) was significantly higher than in non-AE children (1194.69 +/- 448.44 pg/mL, P < 0.0001) or healthy controls (1062.89 +/- 444.74 pg/mL, P < 0.0001). Serum APRIL level was not different between the three groups. Serum BAFF level significantly correlated with total serum IgE level (gamma = 0.42, P < 0.0001) and total eosinophil count. It was also positively correlated with serum BAFF and egg-specific IgE level (gamma = 0.252, P = 0.045) in AE. CONCLUSIONS: Serum BAFF level is high in AE and might be a useful marker for AE.

Comparison of laboratory detection methods of aspirin resistance in coronary artery disease patients.

Aspirin reduces the prevalence of nonfatal myocardial infarction, stroke, and death by 25.0% in high risk group of patients with cardiovascular disease. Previous studies have estimated that about 5.5-56.8% of the population are aspirin resistant. The mechanisms of aspirin resistance (AR) have not been fully understood. We compared the detection methods for AR using traditional platelet aggregometry and VerifyNow system. One hundred and seventy-two coronary artery disease patients who had taken aspirin only or combinations with aspirin and clopidogrel for over 7 days were included. Of the 55 patients with aspirin only, aggregometer detected six AR (10.9%) and VerifyNow identified 10 AR (18.2%) cases. Among 117 patients with combined therapy, none (0.0%) and 10 (8.5%) of AR were detected by aggregometer and VerifyNow, respectively. There were six (3.4%) patients of AR defined by both methods and they all received aspirin monotherapy. Although the correlation between the aggregometry and VerifyNow was low, with defined criteria both methods gave 91.9% agreement to find AR. VerifyNow showed a higher sensitivity to detect AR. Further studies are required to biologically define AR and to alter therapy based on platelet function tests.

Caspase-3 activation as a key factor for HBx-transformed cell death.

OBJECTIVES: Nuclear factor-kappa B (NF-kappaB) activation has been associated with the tumorigenic growth of hepatitis B virus X protein (HBx)-transformed cells. This study was aimed to find a key target for treatment of HBx-mediated cancers. MATERIALS AND METHODS: NF-kappaB activation, endoplasmic reticulum-stress (ER-stress), caspase-3 activation, and cell proliferation were evaluated after Chang/HBx cells permanently expressing HBx viral protein were treated with inhibitors of NF-kappaB, proteasome and DNA topoisomerase. RESULTS: Inhibition of NF-kappaB transcriptional activity by transient transfection with mutant plasmids encoding Akt1 and glycogen synthase kinase-3beta (GSK-3beta), or by treatment with chemical inhibitors, wortmannin and LY294002, showed little effect on the survival of Chang/HBx cells. Furthermore, IkappaBalpha (S32/36A) mutant plasmid or other NF-kappaB inhibitors, 1-pyrrolidinecarbonidithioic acid and sulphasalazine, were also shown to have little effect on the cell proliferation. By contrast, proteasome inhibitor-1 (Pro1) and MG132 enhanced the HBx-induced ER-stress response and the subsequent activation of caspase-12, -9 and -3 and reduced cell proliferation. Camptothecin (CPT), however, triggered activation of caspase-3 without induction of caspase-12, and reduced cell proliferation. In addition, CPT-induced cell death was reversed by pre-treatment with z-DEVD, a caspase-3-specific inhibitor. CONCLUSIONS: Detailed exploitation of the regulators of caspase-3 activation could open the gate for finding an efficient target for development of anticancer therapeutics against HBx-transformed hepatocellular carcinoma.

Combined effect of tumour necrosis factor-alpha and interleukin-13 polymorphisms on bronchial hyperresponsiveness in Korean children with asthma.

BACKGROUND: TNF-alpha and IL-13, two pivotal pro-inflammatory cytokines, are increased in asthmatic airways and may be linked to asthma susceptibility and/or bronchial hyperresponsiveness (BHR). OBJECTIVE: We investigated the association between the TNF-alpha-308G/A polymorphism and asthma susceptibility or asthma-related phenotypes in Korean children with asthma, and tested for a combined effect with IL-13 polymorphisms. METHODS: Asthmatic children (n=719) and non-atopic healthy control children (n=243) were evaluated for asthma phenotypes including total serum IgE and BHR to methacholine. Genotypes were determined by PCR-restriction fragment length polymorphism analysis. RESULTS: The allele frequency of TNF-alpha-308A in asthmatics (14.1%) was higher than that in control children [8.7%, odds ratio (OR) 1.72, 95% confidence interval (CI) 1.05-2.82]. Significantly lower PC(20) values were found in asthmatic children carrying one or two copies of the TNF-alpha risk allele (-308A) vs. those homozygous for the common allele (P=0.026). Combined analysis revealed that atopic asthmatic children co-inherited the risk alleles of TNF-alpha-308G/A and IL-13 +2044G/A more frequently than control children (aOR 1.91, 95% CI 1.00-3.65), and asthmatic children co-inheriting both risk alleles had significantly lower PC(20) values vs. asthmatic children homozygous for the common alleles (P=0.024). CONCLUSION: The TNF-alpha promoter polymorphism (-308G/A) may be associated with asthma susceptibility and BHR in Korean children with asthma. In addition, there appears to be a synergistic effect between the TNF-alpha promoter polymorphism and an IL-13 coding region polymorphism in terms of asthma susceptibility and BHR in this population.

Regulation of German cockroach extract-induced IL-8 expression in human airway epithelial cells.

BACKGROUND: Cockroaches have been known as a cause of respiratory allergies such as asthma. IL-8 plays an integral role in the coordination and persistence of the inflammatory process in the chronic inflammation of the airways in asthma. OBJECTIVE: We investigated the mechanism by which German cockroach extract (GCE) triggers IL-8 release from human airway epithelial cells. METHODS: Chemical inhibitors were pretreated before addition of GCE for promoter activity and protein synthesis of IL-8. The Transcriptional activity of IL-8 promoter was analysed by mutational, deletional anaylsis and electrophoretic mobility shift assay (EMSA). RESULTS: Stimulation of H292 cells with GCE resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. IL-8 promoter deletion analysis indicated that position -132 to +41 was essential for GCE-induced IL-8 transcription, and mutants with substitutions in activator protein (AP)-1, nuclear factor (NF)-IL6 and NF-kappaB-binding sites revealed a requirement for NF-kappaB and NF-IL6, but not AP-1, in GCE-induced activation of the IL-8 promoter. The DNA-binding activities of NF-kappaB and NF-IL6 were induced by GCE, as determined by EMSA. The chemical inhibition of extracellular signal-regulated kinase (ERK) attenuated GCE-induced transcriptional activity and protein synthesis. In addition, through aprotinin treatment and PAR2 small interfering RNA transfection, it was proven that protease of GCE is consistent with the regulation of GCE-induced IL-8. CONCLUSION: We conclude that GCE with protease activity-induced IL-8 expression is regulated by transcriptional activation of NF-kappaB and NF-IL6 coordinating with the ERK pathway in human airway epithelial cells.

Basement membrane thickening and clinical features of children with asthma.

BACKGROUND: Asthma is a chronic inflammatory disease, characterized by airway inflammation, bronchial hyper-responsiveness, and airway obstruction. Although asthma induces partially reversible airway obstruction, obstruction can sometimes become irreversible. This may be a consequence of airway remodeling, which includes a number of structural changes, such as epithelial detachment, basement membrane (BM) thickening, smooth muscle hypertrophy, and new vessel formation. This study evaluated children with asthma for the presence of BM thickening. METHODS: Eighteen children with asthma and 24 control subjects underwent flexible bronchoscopy with endobronchial biopsy. Light microscopy was used to measure BM thickness in paraffin-embedded biopsy sections. The association between BM thickening and age, sex, duration of asthma, asthma severity, FEV(1), FEV(1)/FVC, FEF(25-75%), methacholine PC(20), eosinophil count, and presence of atopy was examined. RESULTS: Basement membrane thickness was greater in subjects with asthma (8.3 +/- 1.4 microM) than in control subjects (6.8 +/- 1.3 microM, P = 0.0008). Multiple regression analysis revealed that sex, FEV(1)/FVC, total IgE, and atopy (IgE for Dermatophagoides pteronyssinus >0.34 kUA/l) were significant predictive factors for BM thickness. There was no significant association between BM thickness and age, duration of asthma, FEV(1), FEF(25-75%), methacholine PC(20), eosinophil count, or asthma severity. CONCLUSIONS: Basement membrane thickening has been known to be present in children with asthma. In addition, we report an association between BM thickness and sex, FEV(1)/FVC, total IgE, and the presence of IgE specific to D. pteronyssinus.

Interleukin-18 is a critical factor for vascular endothelial growth factor-enhanced migration in human gastric cancer cell lines.

Cell migration and angiogenesis are key steps in tumor metastasis. However, the mechanism of migration regulated by vascular endothelial growth factor (VEGF), a potent regulator of angiogenesis, is not completely understood. This study examined the relationship between VEGF and migration, along with the mechanism involved in the VEGF-regulated migration of human gastric cancer cells. The level of cell migration was increased by recombinant human (rh)VEGF-165 in the VEGF receptor-2-expressing SNU-601 cells. Interleukin (IL)-18 is associated with the malignant progression of tumors. Accordingly, this study examined the effect of IL-18 on the migration of cancer cells in order to identify the factors involved in VEGF-enhanced migration. Inhibiting IL-18 markedly reduced the level of VEGF-enhanced migration, and IL-18 increased cell migration directly through filamentous-actin polymerization and tensin downregulation. It was confirmed that rhVEGF-165 increased IL-18 production significantly. An antioxidant and an extracellular signal-regulated kinase (ERK)1/2-specific inhibitor blocked rhVEGF-165-enhanced IL-18 production. Accordingly, rhVEGF-165 increased the generation of region of interest (ROI) and activated the ERK1/2 pathway. These results suggest that rhVEGF-165 enhances IL-18 production via the generation of ROI and ERK1/2 phosphorylation, which results in the increased migration of gastric cancer cells.

Increased serum thymus and activation-regulated chemokine and cutaneous T cell-attracting chemokine levels in children with atopic dermatitis.

BACKGROUND: Thymus and activation-regulated chemokine (TARC) and cutaneous T cell-attracting chemokine (CTACK) are responsible for the trafficking of T helper type 2 lymphocytes into sites of allergic inflammation. OBJECTIVE: We tested whether these cytokines are useful markers for childhood atopic dermatitis (AD), and evaluated age-related differences in the levels of these chemokines. METHODS: Serum TARC and CTACK levels, total serum IgE levels, total eosinophil counts, and specific IgE levels were measured in 401 children. The patients were characterized as having atopic eczema (n=157), non-atopic eczema (n=107), or as healthy control subjects (n=137). RESULTS: Both TARC and CTACK levels in children with AD were significantly higher than those in healthy control subjects. Serum TARC and CTACK levels significantly correlated with disease severity both in children with atopic eczema and in children with non-atopic eczema. Serum TARC levels in children with AD significantly correlated with their serum CTACK levels. Serum TARC and CTACK levels decreased in accordance with their ages. CONCLUSION: Serum TARC and CTACK levels might be useful markers for disease severity both in children with atopic eczema and with non-atopic eczema. Serum TARC and CTACK levels decreased in accordance with their ages.

Prediction of buckwheat allergy using specific IgE concentrations in children.

INTRODUCTION: Buckwheat (BW) is considered to be one of the most important food allergens, and positive skin tests are found in about 5% of Koreans. We investigated the positive and negative predictive values of BW-specific IgE in subjects with a BW allergy in order to reduce the need for buckwheat challenge, which can be more riskier than other causes of food allergies. METHODS: Twenty-eight BW allergic subjects with symptoms after BW Open food challenge and 16 asymptomatic control subjects with positive skin test to BW were recruited. Serum samples from all patients were analyzed for BW-specific IgE antibodies using the Pharmacia CAP System. RESULTS: According to the receiver operator characteristic (ROC) analysis, the optimal cutoff level of BW-specific IgE, as the definitions of serum BW-specific IgE positive, was 1.26 kUA/l. With this selected cutoff level, the sensitivity, specificity, positive and negative predictive values were 93.10, 93.33, 79.75 and 97.96%, respectively. CONCLUSION: The use of the optimal cutoff level, 1.26 kUA/l, that simultaneously maximizes sensitivity and specificity, would be helpful for avoiding unnecessary risky challenge in children with a strong clinical history and skin test responses.

Staphylococcal enterotoxin B upregulates fas-mediated apoptosis of peripheral blood mononuclear cells in childhood atopic dermatitis.

Staphylococcal infection-producing superantigens, such as staphylococcal enterotoxin B (SEB), are presumed to play an important role of inflammatory processes in atopic dermatitis (AD). The aim of this study was to elucidate the apoptotic response of peripheral blood mononuclear cells (PBMCs) from children with AD. PBMCs from AD children were sampled and cultured with SEB stimulation. Levels of apoptosis and Fas expression were measured using flow cytometry; the soluble Fas ligand (sFasL) was also measured using the enzyme-linked immunosorbent assay method, and the expression of FasL in PBMCs was observed using reverse transcriptase-polymerase chain reaction. There was no difference in the initial levels of apoptosis and Fas expression in precultured PBMCs of AD patients and healthy donors. After culturing for 48 h under SEB stimulation, the apoptosis level and Fas expression were significantly upregulated in the PBMCs from AD children compared with that from the normal controls. In patients, the sFasL was significantly increased, and the expression of FasL was observed in messenger RNA of peripheral monocytes. These results suggest that the Fas/FasL system is involved in the apoptosis induced by SEB in AD, with simultaneous increases in sFasL and expression of FasL.

Expression of recombinant human granulocyte macrophage-colony stimulating factor (hGM-CSF) in mouse urine.

We have generated transgenic mice expressing human granulocyte macrophage-colony stimulating factor (hGM-CSF) in urine. In particular, the expression plasmid DNA containing mouse uroplakin II promoter was used to direct uroepithelium-specific transcription of transgene. In this study, hGM-CSF transcript was detected only in bladder uroepithelium as determined by northern blot analysis. Furthermore, hGM-CSF protein was detected in the suprabasal layer of the uroepithelium and ureter by immunohistochemistry. The hGM-CSF was secreted into urine at high level (up to 180 ng/ml), and enhanced proliferation of hGM-CSF-dependent human acute monocyte leukemic cells, suggesting that transgenic urine-derived hGM-CSF was bioactive. This is the first case of demonstrating biological activity of a cytokine produced in the urine of a transgenic animal. Our results demonstrate that bladder can be used as a bioreactor to produce biologically important substances. In addition, it suggests a potential application of bladder expression system to livestock for high-yield production of pharmaceuticals.

Pharmacokinetics of a new antitumor 3-arylisoquinoline derivative, CWJ-a-5.

1-(4-Methylpiperazinyl)-3-phenylisoquinoline hydrochloride (CWJ-a-5) is a newly developed from benzo[c]phenanthridine alkaloids and derivative and has exhibited potent antitumor activities, in vitro and in vivo. The pharmacokinetics of this novel antitumor 3-arylisoquinoline derivative was studied after intravenous (i.v.), oral (p.o.) and hepatoportal (p.v.) administration in rats. A simple high performance liquid chromatographic method was developed to determine the concentrations of CWJ-a-5 in plasma, bile and urine. Plasma concentration profiles of CWJ-a-5 were best fitted by the two-compartment model after i.v. administration and showed a linear pharmacokinetic behavior up to 20 mg/kg doses. The half-life of CWJ-a-5 in the post-distributive phase (t1/2beta), total-body plasma clearance (CLt), and volume of distribution at steady-state (Vdss) were 86.9 min, 5.72 l/h per kilogram and 9.79 l/kg, respectively, after i.v. administration of 10 mg/kg. Biliary and urinary excretion of CWJ-a-5 was < 1% after i.v. injection of 10 mg/kg. The bioavailability of CWJ-a-5 after p.o. and p.v. administration (50 and 10 mg/kg, respectively) was 52.9 and 72.2%, respectively. Gastrointestinal bioavailability was calculated to be 73.3%. The apparent partition coefficient (log P) of CWJ-a-5 between n-octanol and water was 2.64. Plasma protein binding of CWJ-a-5 measured by the ultrafiltration method was > 95%.

Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency.

Mice lacking the Gadd45a gene are susceptible to ionizing radiation-induced tumors. Increased levels of Gadd45a transcript and protein are seen after treatment of cells with ionizing radiation as well as many other agents and treatments that damage DNA. Because cells deficient in Gadd45a were shown to have a partial defect in the global genomic repair component of the nucleotide excision repair pathway of UV-induced photoproducts, dimethylbenzanthracene (DMBA) carcinogenesis was investigated because this agent produces bulky adducts in DNA that are also repaired by nucleotide excision repair. Wild-type mice and mice deficient for Gadd45a were injected with a single i.p. dose of DMBA at 10-14 days of age. The latency for spontaneous deaths was slightly decreased for Gadd45a-null mice compared with wild-type mice. At 17 months, all surviving animals were killed, and similar percentages of each genotype were found to have tumors. However, nearly twice as many Gadd45a-null than wild-type mice had multiple tumors, and three times as many had multiple malignant tumors. The predominant tumor types in wild-type mice were lymphoma and tumors of the intestines and liver. In Gadd45a-null mice, there was a dramatic increase in female ovarian tumors, male hepatocellular tumors, and in vascular tumors in both sexes. In wild-type mice, this dose of DMBA induced a >5-fold increase in Gadd45a transcript in the spleen and ovary, whereas the increase in liver was >20-fold. Nucleotide excision repair, which repairs both UV- and DMBA-induced DNA lesions, was substantially reduced in Gadd45a-null lymphoblasts. Mutation frequency after DMBA treatment was threefold higher in Gadd45a-null liver compared with wild-type liver. Therefore, lack of basal and DMBA-induced Gadd45a may result in enhanced tumorigenesis because of decreased DNA repair and increased mutation frequency. Genomic instability, decreased cell cycle checkpoints, and partial loss of normal growth control in cells from Gadd45a-null mice may also contribute to this process.