Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of ß-Keto Esters.

ß-keto esters are used as precursors for the synthesis of ß-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of ß-keto esters, the first step in the preparation of ß-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening.

Simvastatin protects against cholestasis-induced liver injury.

BACKGROUND: Bile duct obstruction is associated with hepatic accumulation of leukocytes and liver injury. The aim of this study was to evaluate the effect of simvastatin on cholestasis-induced liver inflammation and tissue damage. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with simvastatin (0.02 and 0.2 mg.kg(-1)) and vehicle before and after undergoing bile duct ligation (BDL) for 12 h. Leukocyte recruitment and microvascular perfusion in the liver were analysed using intravital fluorescence microscopy. CXC chemokines in the liver were determined by enzyme-linked immunosorbent assay. Liver damage was monitored by measuring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Hepatic levels of myeloperoxidase (MPO) were also determined. KEY RESULTS: Administration of 0.2 mg.kg(-1) simvastatin decreased ALT and AST by 87% and 83%, respectively, in BDL mice. This dose of simvastatin reduced hepatic formation of CXC chemokines by 37-82% and restored sinusoidal perfusion in cholestatic animals. Moreover, BDL-induced leukocyte adhesion in sinusoids and postsinusoidal venules, as well as MPO levels in the liver, was significantly reduced by simvastatin. Notably, administration of 0.2 mg.kg(-1) simvastatin 2 h after BDL induction also decreased cholestatic liver injury and inflammation. CONCLUSIONS AND IMPLICATIONS: These findings show that simvastatin protects against BDL-induced liver injury. The hepatoprotective effect of simvastatin is mediated, at least in part, by reduced formation of CXC chemokines and leukocyte recruitment. Thus, our novel data suggest that the use of statins may be an effective strategy to protect against the hepatic injury associated with obstructive jaundice.

Nitric oxide reduces organ injury and enhances regeneration of reduced-size livers by increasing hepatic arterial flow.

BACKGROUND: Reduced-size livers suffer from portal hyperperfusion, diminished arterial blood flow and the risk of postoperative liver injury. The aim of this experimental study was to unravel the role of nitric oxide in this setting. METHODS: Rats underwent 85 per cent partial hepatectomy and either substitution of nitric oxide with molsidomine or inhibition of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine methyl ester. Untreated hepatectomized animals served as controls and unresected animals as the sham group. RESULTS: Ultrasonic flowmetry following partial hepatectomy revealed a marked increase in portal venous inflow with a concomitant decrease in hepatic arterial inflow. Nitric oxide substitution counteracted the decline in hepatic arterial inflow and caused a significantly greater increase in cell proliferation after partial hepatectomy compared with control or NOS-inhibited animals. Hepatectomized animals further profited from nitric oxide substitution, as indicated by reduced aminotransferase release and improved liver function. CONCLUSION: Nitric oxide improves the postoperative course of rats with reduced-size livers by modulating hepatic macrohaemodynamics and mediating regeneration and cytoprotection, but not by reducing hepatic hyperperfusion and the accompanying sinusoidal shear stress.

Platelet-dependent accumulation of leukocytes in sinusoids mediates hepatocellular damage in bile duct ligation-induced cholestasis.

BACKGROUND: Although it is well known that extrahepatic cholestasis induces liver damage, the mechanisms are still not completely understood. The aim of the present study was to evaluate the role of platelets and P-selectin in cholestasis-induced liver injury. EXPERIMENTAL APPROACH: C57BL/6 mice underwent bile duct ligation (BDL) and pretreatment with an anti-GP1balpha antibody, which depletes platelets, an anti-P-selectin antibody or a control antibody. Hepatic platelet and leukocyte recruitment as well as microvascular perfusion were determined by intravital fluorescence microscopy. KEY RESULTS: BDL caused significant liver damage and sinusoidal perfusion failure. BDL further induced hepatic platelet accumulation with widespread intravascular platelet aggregates, increased platelet adhesion in postsinusoidal venules and massive platelet accumulation in liver sinusoids. Administration of the anti-GP1balpha antibody reduced systemic platelet count by 90%. Depletion of platelets in BDL mice not only abolished accumulation and adhesion of platelets in sinusoids and venules but also restored sinusoidal perfusion and reduced liver enzymes by more than 83%. Platelet depletion further reduced BDL-associated sinusoidal leukocyte accumulation by 48% although leukocyte-endothelium interactions in venules were not affected. Immunoneutralization of P-selectin also inhibited hepatic microvascular accumulation of platelets and leukocytes, and protected against cholestasis-provoked hepatocellular damage. CONCLUSIONS AND IMPLICATIONS: Platelets play an important role in BDL-induced liver injury by promoting leukocyte recruitment and deteriorating microvascular perfusion. Moreover, our findings demonstrate that cholestasis-induced accumulation of platelets is mediated by P-selectin. Thus, targeting platelet accumulation may be a useful strategy against liver damage associated with obstructive jaundice.

Early antibiotic treatment and later asthma.

The reasons for the asthma epidemic are poorly understood. As the asthma prevalence follows the geographical and temporal trend of antibiotic use into clinical medicine, we examined a possible association in a population-based study of 2,512 children age 5-14 in East Germany. Wheezing was associated with increasing number of antibiotic courses (never versus one time odds ratio 1.9, P = 0.012, 2 to 5 times odds ratio 3.0, P<0.001 and more than 5 times, odds ratio 6.9, P<0.001) which was also seen for asthma diagnosis. The risk increased with earlier administration (never versus second year odds ratio 4.6, month 7-12 odds ratio 5.4 and birth until month 6 odds ratio 7.9, all P<0.001). Also non pulmonary treatment indication was associated with later wheezing (odds ratio 3.9, P<0.001). The most likely possible explanation is reverse causation indicating that frequent upper respiratory infections, an early symptom of asthma, are treated with antibiotics. Antibiotic therapy could also be a proxy of another closely associated genetic or environmental factor. The high dose effect, the time dependency of the administration and the effect by non-pulmonary indications raises the possibility that early antibiotic treatment could itself be related to later asthma.

Genes, factor X, and allergens: what causes allergic diseases?

Genetic disposition and allergen exposure play the main roles in the development of allergic diseases. Another factor that could be involved is the nutritional intake of vitamin D.

Ascaris-specific IgE and allergic sensitization in a cohort of school children in the former East Germany.

BACKGROUND: Helminthic infections induce an IL-4-dependent polyclonal stimulation of IgE synthetization. It is still unclear, however, what role helminths play in allergic sensitization. OBJECTIVE: We sought to determine the relationship between Ascaris-specific IgE and allergic sensitization in a nontropical country. METHODS: In 2 consecutive cross-sectional surveys in 1992-1993 and 1995-1996, data from school entrants (age range, 5 to 7 years), third graders (age range, 8 to 10 years), and sixth graders (age range, 11 to 14 years) were collected. The 2 younger groups were reexamined in the second survey. Data for about 2300 children, including a cohort of 700 subjects, were analyzed. Ascaris IgE and total and specific IgE to inhalant allergens were measured, and skin prick tests were performed. Information about asthma and allergic rhinitis was collected by a questionnaire. RESULTS: Children who were Ascaris-IgE seropositive (>0.35 IU/mL) in both surveys had 10-fold higher levels of total IgE (451 IU/mL vs 45 IU/mL, P < .001) and higher prevalence rates of allergen-specific IgE seropositivity (56.3% vs 26.6%, P < .001). They also had a higher prevalence of allergic rhinitis (12.6% vs 3.7%, P < .001) and asthma (5.7% vs 1.6%, P < .05). In subjects who were Ascaris-seronegative in the first survey but seropositive in the second survey, total and specific IgE increased markedly. Sensitization to Dermatophagoides pteronyssinus increased nearly 3-fold in this group. In contrast, in children who became Ascaris-seronegative, total and specific IgE decreased. CONCLUSIONS: Contact with low doses of helminthic antigen is associated with an increase of total and specific IgE production. Helminthic infections in East German children are not the cause for a low prevalence of allergies in the former East Germany.

Is asthma an endocrine disease?

The prevalence of pediatric asthma has increased in many parts of the world. This increase started more than 30 years ago and is particularly obvious in studies which document the onset of asthma in native populations when they change to a "Western" lifestyle. Besides a genetic influence, numerous environmental factors have been described for the development of asthma. Genetic factors are unlikely to explain the sharp increase within the short time period and also allergen and pollution exposure or any specific infection does not actually seem to be the main cause for this phenomenon. Another factor, however, that fits well into the geographical and temporal background of the asthma epidemic is the mother's oral contraceptive use. We therefore review the epidemiological association with later asthma in the children, give a summary of estrogen effects on immune function and develop a preliminary theory how oral contraception could influence later pregnancy.

Psychosocial characteristics of asthma.

The objective of this study was to compare psychosocial characteristics of children with asthma and children with bronchial hyperreactivity with those of normal children. A population-based study of 2634 children (mean age, 10 years) was carried out. Pulmonary function tests of children were performed in children before and after cold air hyperventilation challenge to determine bronchial hyperreactivity. Parental assessment of children's behavior was evaluated with 15 questions about school/learning habits, level of activity, communication/affection, and sleeping patterns. A factor analysis was performed and the factor loading adjusted for confounders compared in the different groups. Asthmatic children sleep less well than normal and hyperreactive children (p < 0.001). Unexpectedly, however, all other single items did not differ significantly. As a result of the factorial analysis we obtained two factors. On the first factor, measuring school behavior and learning, there was a small difference between asthmatic and normal children, which could not be found on the second factor indicating activity and communication. We conclude that psychosocial differences of asthmatic children are less remarkable than expected. As a result of the examination of the hyperreactive children it is likely that asthmatic children are influenced more by secondary psychosocial factors than by any primary effect of asthmatic disease.

Pertussis infection and allergic sensitization.

BACKGROUND: The immunogenic activity of B. pertussis infection has been described in various laboratory, animal, and clinical studies. There is, however, no information on the impact of pertussis on allergies in the total population. OBJECTIVE: To compare the prevalence of allergic sensitization and allergic rhinitis in children with and without previous pertussis infection. METHODS: A population-based, cross-sectional study was carried out on 13,937 10-year-old children in the western (Munich and Southern Bavaria) and eastern parts of Germany (Leipzig and the region around Halle). A total of 11,969 questionnaires (85.9%) given to the parents were collected. Data from 9,484 German children (questionnaire and skin prick tests with six different allergens) were analyzed. RESULTS: Pertussis was much more common in the western than in the eastern part of Germany. The adjusted odds ratio for any allergic sensitization after pertussis was only slightly increased in western Germany with 1.3 (95% confidence limits 1.2 to 1.5) and in eastern Germany with 1.5, (1.2 to 1.8) but not for allergic rhinitis with 1.0 (0.7 to 1.4) and in Eastern Germany 1.3 (0.8 to 1.9). CONCLUSIONS: Infection with pertussis seems to have only a weak influence on allergic sensitization and does not explain the observed differences in allergic sensitization between western and eastern Germany.

Indoor factors and IgE levels in children.

The objective of the study was to determine indoor characteristics of households in relation to total serum IgE. In a population-based cross-sectional study, 1096 6-12-year-old children were examined in three East German towns (Eisleben, Hettstedt, and Zerbst). Of the questionnaires, 772 (70.4%) were returned by the parents. Serum IgE of 703 children and urinary cotinine in a random subsample of 224 children were analyzed. Linear regression on log(IgE) adjusted for the main covariates was used to assess indoor risk factors such as room size, and the presence of curtains, carpet, and plants in the child's room. Open-heating facilities indoors, passive smoking, and furniture made of chipboard had the most important effect. A higher urinary cotinine/creatinine ratio was associated with higher total IgE level. Total IGE increased also with the number of persons living in the household, independently of indoor smoking. We conclude that indoor air pollution from smoking and open-heating facilities may increase the IgE levels of children. The role of other factors such as chipboard, which could reflect the emission of formaldehyde, or the number of persons per household, which could reflect viral or helminthic infection, remains to be analyzed.

Bronchial hyperreactivity and history of wheezing in children.

UNLABELLED: The objective of this analysis was to determine the relationship between wheezing at different age groups in children and the prevalence of bronchial hyperreactivity at the age of 10. A population-based cross-sectional study was conducted in Leipzig and the region around Halle in Germany. Of 3105 10-year-old children, 2658 questionnaires (85.6%) were returned. In addition 2279 (73.4%) pulmonary function tests were performed before and after cold air challenge. 658 children (24.8%) had recurrent wheezing during their lifetime. In 579 children the individual time course could be evaluated (46 children with and 533 without a physician-confirmed diagnosis of asthma). Wheezing began most frequently in the 1st year of life (44.1% of all wheezing children) with the highest annual prevalence in the 3rd year (71.0% of all wheezing children). Wheezing which started in the first 2 years of life, had disappeared in most of the children by the age of 10. However, if wheezing began later than the 3rd year it was more persisting. Bronchial hyperreactivity measured after cold air challenge was higher in the group with recurrent wheezing (24.1%) than in the group without wheezing (18.8%, P = 0.004). CONCLUSION: Wheezing is a very common symptom in childhood and only partly associated with later bronchial hyperreactivity. On the other hand, asthma is often not diagnosed despite bronchial hyperreactivity and many years of wheezing.

Road traffic and adverse effects on respiratory health in children.

OBJECTIVES: To examine whether road traffic in a big city has a direct effect on pulmonary function and respiratory symptoms in children. DESIGN: Cross sectional study. SETTING: Of all 7445 fourth grade children (aged 9-11 years) in Munich, 6537 were examined. Of the children with German nationality and the same residence during the past five years and known exposure data, 4678 questionnaires and 4320 pulmonary function tests could be analysed. MAIN OUTCOME MEASURES: Variables of pulmonary function by forced expiration and respiratory symptoms reported in a questionnaire; census data on car traffic collected in the school district. RESULTS: Density of car traffic ranged from 7000 to 125,000 cars per 24 hours. Multiple regression analysis of peak expiratory flow showed a significant decrease of 0.71% (95% confidence interval 1.08% to 0.33%) per increase of 25,000 cars daily passing through the school district on the main road. Maximum expiratory flow when 25% vital capacity had been expired was decreased by 0.68% (1.11% to 0.25%). In contrast, response to cold air challenge was not increased. The adjusted odds ratio for the cumulative prevalence of recurrent wheezing with the same exposure was 1.08 (1.01 to 1.16). Cumulative prevalence of recurrent dyspnoea was increased, with an odds ratio of 1.10 (1.00 to 1.20). Lifetime prevalence of asthma (odds ratio 1.04; 0.89 to 1.21) and recurrent bronchitis (1.05; 0.98 to 1.12) were not significantly increased. CONCLUSIONS: High rates of road traffic diminish forced expiratory flow and increase respiratory symptoms in children.

Evaluation of cold air challenge data in a population sample using a model of bronchial hyperreactivity and disposition to bronchial obstruction.

To explore the role of bronchial hyperreactivity and obstruction after cold air challenge, data from a cross-sectional study of more than 7,000 10-year-old children were used. Current knowledge of hyperreactivity is primarily based on pharmacological provocation tests with variable prechallenge flow rates and their decrease relative to baseline. Using forced expiratory volume (FEV) in 1 sec values before and after cold air challenge, however, it is possible to define a subsample of children with predominant hyperreactivity and a subsample with predominant obstruction after challenge. The prevalence of respiratory symptoms and the diagnoses in the two subsamples were compared. The analysis showed that children with bronchial obstruction have nearly the same frequency of respiratory symptoms as those with bronchial hyperreactivity. A combined model of bronchial obstruction and hyperreactivity was, therefore, more predictive of symptoms than a model of hyperreactivity alone.

Month of birth and allergic disease at the age of 10.

The relationship between month of birth and asthma, hay fever and skin sensitization to mixed grass pollen was analysed in a population-based cross-sectional study in Munich and Bavaria 1989-1990 of 6535 10-year-old children. The relative risk of developing atopic disease is calculated by comparing the prevalence in a single month with the prevalence of all other months. A slightly increased risk of developing allergic skin sensitization for grass pollen (n = 1128) was found for February (odds ratio, 1.3, 95% confidence interval 1.0-1.6), May (1.4, 1.1-1.8) and June (1.3, 1.0-1.6). For hay fever (n = 379) an increase was found for May (1.5, 1.0-2.1) and for allergic asthma (n = 277) for August (1.4, 1.0-2.1). A protective effect was observed for certain months of birth; September for allergic sensitization (0.8, 0.6-1.0), October for and November for hay fever (0.6, 0.3-0.9). The occurrence of hay fever and positive prick test is explained by the seasonal variation of atmospheric grass pollen and the peak in August of asthmatic patients by house dust. Date of birth appears therefore to slightly influence the risk of developing an allergic sensitization and allergic diseases.

[Effects of passive smoking on the pediatric respiratory tract]. / Auswirkungen des Passivrauchens auf den kindlichen Respirationstrakt.

OBJECTIVE: To explore the risk of parental smoking to the respiratory health of their children data of a cross sectional study on fourth-grade schoolchildren in Munich and Southern Bavaria were analysed. METHODS: Allergic and asthmatic diseases and symptoms, risk factors like family history, indoor pollution and parental smoking were evaluated by a questionnaire. Pulmonary function tests were performed in 7284 school children aged (9-11 years). Lung function values were adjusted for height, weight, sex and other confounders. RESULTS: The children, whose parents smoke at home, had significantly lower levels of peak flow, MEF75, MEF50 and MEF25 compared to children from non-smoking families, with a dose-response relationship. Smoking of more than 20 cigarettes at home is associated with a mean decrease in MEF75 of 5.7%, in MEF50 of 4.9% and in Peak Flow of 4.9% (p < 0.001). The prevalence of cough and wheezing increased with increasing smoking rates of the parents. CONCLUSIONS: Passive exposure to smoke has direct measurable dose-dependent effects on the respiratory system of children.

[Does breast feeding prevent asthma and allergies? Results of the Munich asthma and allergy study]. / Schützt Stillen vor Asthma und Allergien? Ergebnisse der Münchner Asthma- und Allergiestudie.

The relationship of breast feeding to atopic diseases is studied in a population-based cross sectional study in Munich and Bavaria 1989/1990 in 6,535 german ten year old children. According to the questionnaire answers 1914 (29.3%) children were not breast fed, 2,368 (36.2%) shorter than 2 months, 1,744 (26.7%) 2 to 6 months and 509 (7.8%) more than 6 months. Compared to controls without any allergic disease the relative risk of later asthma, hayfever, atopic dermatitis and of allergic skin sensitization was not diminished by breast feeding. The same result pertained to high risk subgroups defined by positive family history of asthma or hayfever. It is concluded that breast feeding alone has no long term protective effect against atopic diseases.

Genetic risk for asthma, allergic rhinitis, and atopic dermatitis.

In order to explore the genetic risk of a child with a family history of allergies developing asthma, allergic rhinitis, or atopic dermatitis, questionnaires filled in by 6665 families were analysed. The data were collected in a population based cross sectional survey of 9-11 year old schoolchildren living in Munich and southern Bavaria. The relation between asthma, allergic rhinitis, and atopic dermatitis and the number of allergic first degree relatives, and the type of allergic disease was examined. Analyses were done separately for families with single or multiple allergic diseases. In families with one allergic parent the risk of the child developing asthma was increased by asthma in a parent, with an odds ratio (OR) of 2.6 (95% confidence interval 1.7 to 4.0) but not by parental allergic rhinitis with OR 1.0 (0.7 to 1.5) or atopic dermatitis, OR 1.0 (0.6 to 1.6). For allergic rhinitis the highest risk with OR 3.6 (2.9 to 4.6) was observed with allergic rhinitis of one parent, apparently lower for asthma of one parent, OR 2.5 (1.6 to 4.0) or atopic dermatitis, OR 1.7 (1.1 to 2.5). Children with parental atopic dermatitis had a high risk for atopic dermatitis, OR 3.4 (2.6 to 4.4), compared with children with parental asthma, OR 1.5 (1.0 to 2.2), or parental allergic rhinitis, OR 1.4 (1.1 to 1.8). Risk factors in families with combined allergies of two relatives (parents and siblings) were analysed separately for the different combinations. These results support the hypothesis that asthma, allergic rhinitis, and atopic dermatitis are multifactorial diseases brought about by various familial and environmental influences.

[Prevalence of asthma in 6,000 10-year-old children in Munich and Upper Bavaria based on physicians' diagnoses and a symptom score]. / Asthmaprävalenz bei 6000 10jährigen Kindern in München und Oberbayern anhand von Arztdiagnosen und einem Symptomscore.

In the Munich Survey on Asthma and Allergy the parents of 9,349 fourth-class schoolchildren (mean age 9.8 years) in Munich and Southern Bavaria were addressed by a questionnaire to which 8,204 responded (87%). In 7,192 children (76%) a skin prick test was performed and 7,284 (77%) had pulmonary function tests with maximum expiratory flow-volume loops before and after cold air challenge. Of the 6,083 children of German nationality, 160 children (2.6%) had physician-diagnosed asthma, 79 (1.3%) so called asthmoid bronchitis and 373 children (6.1%) spastic bronchitis. Since physician-diagnosed diseases does not reflect all children with respiratory disease, symptoms reported in the questionnaire, results of skin prick and pulmonary function tests were combined to a score of probable asthma (VSA) with 9 items. Of the group with physician-diagnosed asthma, 79.4% of the children (127) have an elevated VSA, of 68.4% (54) of asthmoid bronchitis, 35.9% (134) of spastic bronchitis, 11.4% (192) of simple bronchitis and 3% of (114) never-diagnosed bronchial disease. The cumulative prevalence of asthma in ten-year-old children is therefore estimated at 10.2%. Only half of these children have been diagnosed with asthmatic disease.