SO2 is a pollutant in automobile fumes and may play a role in the exacerbation of airway disease symptoms. We first investigated its effect on mucociliary clearance. MB 10-1 M, increased mucociliary clearance time (MCT) by 254.5 ± 57.3% of control values, (p < 0.001, n = 7). MB 10-4 and 10-2 M did not interfere with mucus clearance time compared to control values. In MB-treated frog palates, MCT did not return to control values after one hour (control, 97.3 ± 6.3% vs. MB, 140.9 ± 46.3%, p < 0.001, n = 7). Scanning EM images of epithelial tissue were morphometrically analyzed and showed a 25 ± 12% loss of ciliated cells in MB palates compared to controls with an intact ciliary blanket. Intact cells or groups of ciliated cells were found in scanning EM micrographs of mucus from MB-treated palates. This was associated with increased matrix metalloproteinase (MMP-9) activity in epithelial tissue and mucus. We suggest that the loss of ciliated cells as a result of MMP-9 activation prevented full recovery of MCT after MB 10-1 M. The mechanism of action may be on epithelial cell-cell or cell-matrix attachments leading to cell loss and a disruption of MCT. Further studies are warranted to determine whether this is an inflammatory mediated response or the result of a direct action on epithelial cells and what role this mechanism may play in the progression to chronic airway diseases with impaired mucociliary clearance.We studied the action of sodium metabisulphite on mucociliary transport in a frog palate epithelial injury model, hypothesizing that it may be useful for the study of mechanisms of airway injury. Sodium metabisulphite (MB) releases SO Particle clearance in the airways is dependant on mucus and cilia . The cil2) on contact with water and has been employed as an aerosol in other airway injury models to study hypersecretion and hyperplasia [2 produced symptoms similar to chronic bronchitis in humans [We have extended the physiological applications of the frog palate model to study the initial events of airway injury. To create an injury model from the fresh frog palate model, a solution of sodium metabisulphite was topically applied to the palate. Sodium metabisulphite has been shown to release sulfur dioxide SO on contaerplasia . In dog n humans .We hypothesize that sodium metabisulphite will interfere with mucociliary clearance on the frog palate by disrupting the action of the ciliated epithelium, vital to the process of mucociliary clearance. The objective of this study was to evaluate the effect of sodium metabisulphite on mucociliary clearance on the frog palate. A further objective was to analyze tissue and mucus samples in ultra-structural and molecular studies to characterize the nature of the injury and to assess the potential involvement of matrix metalloproteinases which have been shown to play a role in airway injury and remodeling ,8 and inRana catesbiana) was excised by cutting in the coronal plane from the lateral border of the mouth on one side of the head to the other. The excised palate was placed horizontally on gauze soaked in frog Ringers (2/3 Ringers + 1/3 distilled water, 207 mosml L-1) in a Petri dish. The palate was placed in an enclosed chamber (20 × 20 × 30 cm) maintained at a constant temperature (22–24°C) and continuously humidified at 100% with aerosolized frog Ringers generated by a Pari Jet® nebulizer at a airflow rate of 8 L/min. The palate was allowed to stabilize for 15–20 min before any procedures were carried out on the palate.A fresh frog palate was prepared as previously described ,2. Brief2 (volume applied = 2 μl), 4.5 (3 μl) 5.5 (4 μl) to 6.5 cm2 (5 μl).Mucociliary clearance time (MCT) was measured by applying a droplet of mucus collected from the inferior (cut) edge of the palate that was placed at the superior edge of the palate near the midline. The action of cilia carries the mucus toward the inferior edge. The effect of various concentrations of sodium metabisulphite on MCT was measured following topical application on the palate. Frog Ringers was used as a control solution and vehicle for sodium metabisulphite. The volume of solution (either frog Ringers or sodium metabisulphite) that was applied to each palate was normalized among different sized palates according to the area of the palates surface. The area of the palate was approximated by measuring across the lateral-most borders of the jaw at the base of the palate, and calculating the area of the equivalent half-circle. The volume of solution applied was normalized to the area of each palate as shown: area = 3.5 cm-4 M was applied to the palate. After two minutes, another five measurements of MCT were recorded followed by a recovery period. At this point in time  was applied followed by the measurement of MCT again. This was followed with a recovery period with the measurement of frog Ringers MCT again, which was shown to be not different from the previous controls.Using bromophenol blue in frog Ringers applied to the palate, it was shown that within two minutes of application, the solution was carried from the superior edge of the palate to the inferior edge by ciliary action. Therefore, when frog Ringers was applied to the palate, two minutes was allowed for the droplet of solution to disperse on the palate. This was followed by the measurement of MCT using a drop of frog mucus collected off the inferior (cut edge) of the palate and marked with carbon particles to enhance its visibility on the palate surface. The movement of the mucus droplet down the palate by ciliary action was observed through a stereomicroscope with a reticulated eyepiece and timed over a set distance of 4 mm, once it reached a steady speed. For each solution tested, five consecutive mucus clearance times were recorded and the average was used as the time point for that particular group of recordings. After a recovery period, sodium metabisulphite 10-1 M were plotted versus time and a line of best fit was determined (data not shown). No change in the significance of the slope of the line  indicated that no significant deterioration of the palate had taken place over time. Within each individual experiment there was, however, some variability in controls. Therefore the control mucociliary clearance times that were used to determine a line of best fit (taken as 100%) were compared to the actual MCT measured at that particular point. Thus for each experiment the actual MCT was expressed as a percentage of the line of best fit of the control time which was extrapolated to the time of application of frog Ringers or sodium metabisulphite to the palate as shown  ×100). Thus, there is variation within and between controls that are shown as a standard deviation for each time point .Frog Ringers following each recovery period also represented a timed control prior to each dose of metabisulphite. However, in order to control for deterioration of palate over the course of the experiment, three sets of frog Ringers controls measured before the application of sodium metabisulphite 10 MCTs for metabisulphite were expressed as a percentage of the line of best fit for frog Ringers controls, extrapolated to the time metabisulphite was applied. An increase in MCT compared to control times, indicated a slowing of the mucociliary clearance time. A minimum of fifteen minutes was allowed after metabisulphite, for MCT to return to the normal range, i.e. within 10% of the frog Ringers MCT, measured prior to metabisulphite. If recovery of MCT had not occurred after twenty minutes to within the range specified, frog Ringers was re-applied and the recovery period was repeated.a priori as indicative of a quantifiable injury to the mucociliary clearance system. Sodium metabisulphite was applied in progressively increasing concentrations from 10-4, 10-2 and 10-1 M. Each test solution was alternated with frog Ringers. A higher concentration of metabisulphite was not applied until the MCT had returned to within 10% of the previously measured frog Ringers control. In several experiments, pH was measured on the surface of the frog palate, using a solid-state micro pH electrode (Lazar Research Laboratories, Los Angeles, CA) connected to an Accumet® pH meter (Model 925, Fisher Scientific, Nepean, ON, Canada) to continuously monitor changes on the palate surface during the application of sodium metabisulphite.A 50% increase in MCT was established Samples of frog palate epithelial tissue and mucus were placed in 2.5% glutaraldehyde solution, immediately after collection and kept in a refrigerator at 4°C until processing. Samples were prepared for the SEM by standard methodology. Briefly samples were post-fixed in 1% osmium tetroxide in Milonig's buffer at room temperature for one hour. They were then washed briefly in distilled water and dehydrated in an increasing series of ethanol (50, 80 and 100%), ten minutes at each concentration, followed by two additional periods of absolute ethanol. The samples were further dehydrated by critical point drying at 31°C for 5–10 minutes, and then mounted on a specimen holder for drying overnight in a desiccator. In the final stage of preparation before viewing, the samples were sputter coated with gold (Edwards, Model S150B Sputter Coater) and examined with a Hitachi 2500S scanning electron microscope. High-resolution digital images were acquired directly to a computer for storage and reproduction.® image analysis software to trace areas of cell loss and determine the areas of loss relative to the field of view. Fifteen fields from 3 samples exposed to sodium metabisulphite 10-1 M were examined as well as samples from control tissue (exposed only to frog Ringers).To quantify the area of cilia loss in fields of view in the electron microscope studies, image files were analyzed using Sigma Scan2, EDTA and Tris-HCl) was added to the samples that were sonicated for 30 seconds and then centrifuged at 14,000 rpm for 15 minutes. The supernatant was collected and an aliquot removed for protein assay (BCA protein assay kit, PIERCE).Samples of frog palate epithelial tissue were removed following mucus clearance studies, snap frozen in liquid nitrogen and stored at -80°C until they were prepared for zymography. At that time the tissue samples were ground with a mortar and pestle to a powder, adding liquid nitrogen to the mort to keep the tissue frozen. Homogenization buffer (KCl, ZnCl2, 0.05% Azide NaN3, 50 mM Tris-Cl, 2 M Tris-HCl). The gel was then stained for 2 hours with 0.05 % Coomassie blue (R-250) in methanol: acetic acid: water (2.5:1:6.5) followed by de-staining in 20% isopropanol in 4% ethanol and 8% acetic acid. The presence of gelatinases (MMP 2 and 9) was shown by clear bands (no staining) corresponding to MMP standards (MMP 2 and 9) run in leftmost lane on the gel. Optical density was measured in a Bio-Rad Scanning densitometer.A 10 μl sample, normalized for protein content, was loaded on a separating gel (acryl amide and gelatin) and run at 120 volts for one hour. After electrophoresis, the gel was washed for one hour in 25% Triton-X100 at room temperature followed by incubation overnight in zymography development buffer  evaluated using planned orthogonal comparisons. For comparisons between two groups (density comparisons between control and MB in zymograms), a Student T-test was used. The level of significance was set at p < 0.05.-4, 10-2 and 10-1 M and 3 consecutive recovery periods following sodium metabisulphite 10-1 M in which frog Ringers was applied to the palate in twenty-minute intervals followed by a measurement of MCT. The average frog Ringers MCT (in 7 frogs) measured initially at 15 minutes following an initial stabilization period was 97.3 ± 6.3 %. No difference in MCT was measured after the application of sodium metabisulphite 10-4 (30 min) and 10-2 M (60 min); whereas 10-1 M sodium metabisulphite (at 100 min) increased MCT by 254.5 ± 57.3% compared to Ringers control MCT (taken as ~100%). Between 10-4 and 10-2 M sodium metabisulphite, there was no significant difference compared to control MCTs. This is illustrated by the dotted line in Figure -1 M, frog Ringers was applied but MCT did not recover to previous frog Ringers control times. Another twenty minutes of recovery was allowed and frog Ringers MCT was still not recovered. After an additional twenty minutes, frog Ringers MCT was measured for the third consecutive time, showing that after one hour of recovery (~170 min in the time course of the experiment), the MCT was still significantly different from the initial frog Ringers MCT (140.9 ± 46.3 vs. 97.3 ± 6.3%, p < 0.001, n = 7).Figure -1 M. To determine if this acute effect was due to pH changes, possibly representing altered ion fluxes in the tissue, a micro pH electrode was placed on the palate to measure pH before and after the application of metabisulphite to the palate surface. This is shown in Figure -4 and 10-2 M. However, after sodium metabisulphite 10-1 M, the pH declined within seconds, reaching a nadir at ~60 seconds. After 300 seconds, there was some recovery toward normal, but the pH was still 0.3–0.5 units below the initial control value. As shown in Figure -1 M and showed continued (but incomplete) recovery after one hour. No corresponding pH measurements were taken at these time points.MCT was significantly increased after sodium metabisulphite 10-1 M sodium metabisulphite was applied. The normal continuous covering of cilia is shown greater detail in Panel C (x3500) and in Panel D after 10-1 M sodium metabisulphite, where a region of exfoliation is shown more clearly at the higher magnification. The absence of cilia and ciliated epithelial cells is visible, with only the extracellular matrix remaining. Morphometry to quantify the area of exfoliation determined in a five different field from three independent experiments, revealed that after sodium metabisulphite 10-1 M, there was a 25 ± 11.8% loss of ciliated epithelial cells from these palates compared to none in control palates.In Figure -4 and 10-2 M, showed no ultra structural changes compared to control palates to which frog Ringers had been applied. Figure -1 M. At lower power (X400) a grouping of ciliated cells are visible in the mucus. At the higher power (x2000), intact ciliated epithelial cells are clearly shown.SEM of the palate surface following sodium metabisulphite 10-1 M treated tissue (two rightmost columns), bands representing MMP 9 activity were seen whereas only faint bands were visible in control tissue. Statistical comparison of densitometry bands showed significant activation (p < 0.05, n = 3). MMP-2 activity (in the bottom row on the zymogram) may have also increased, but since control tissue showed similar activation these results are inconclusive. A similar state of MMP activation in mucus is shown in Figure -4, 10-2 and 10-1 M, and prepared for zymography  while both were greater than MMP-9 activation following application of 10-4 M sodium metabisulphite (*, p < 0.05, n = 3).Figure y Figure . Optical2 on contact with water) has an acute effect on mucus clearance followed by incomplete recovery of mucus clearance time; 3. ultra-structural studies showed that areas of ciliated epithelial cells were lost from the palate surface resulting in an incomplete recovery of mucus clearance. Loss of cilia has been previously reported following exposure to SO2 in dogs [-1 M sodium metabisulphite-treated palates but not from frog Ringers-treated control palates; 4. Gelatinase zymography showed increased activity of MMP-9 after sodium metabisulphite (10-4 to 10-1 M) and this was shown to be a dose-related effect. It is noteworthy that gelatinase zymography showed increased activity of MMP-9 at each concentration of sodium metabisulphite, whereas ultrastructural damage was only found at the highest concentration; 5. The finding that intact ciliated cells were found in the mucus suggests that the action of activated gelatinases was on cell-cell or cell-matrix attachments resulting in the exfoliation of intact ciliated epithelial cells, which may have contributed to a slowing of mucus clearance over the surface of the palate. The important findings of this study are: 1. the development of a model of airway epithelial injury that can be used for study of ultra-structural and molecular events in airway injury that are directly related to the disruption of mucus clearance; 2. that sodium metabisulphite  and assuming a mucus plus periciliary layer of 10 μm, a volume of 5 μl would effectively be diluted by as much as 1–2 orders of magnitude . This calculation would suggest that sodium metabisulphite 10-1 M was effectively and rapidly diluted to 10-2 M or less. It follows that the lower concentrations of metabisulphite would be effectively less than the stock concentrations. Since, the effective concentration was determined experimentally in a dose-response experiment as that dose that produced a 50% or greater increase in the mucus clearance time, and since only the highest concentration of metabisulphite produced this effect, this concentration became physiological relevant to the outcome of these experiments. Lower concentrations (10-4 and 10-2 M) were also used, even though no effect on mucociliary clearance time was observed in the dose-response experiments, to determine if there might be some quantifiable effect at the cellular level, which was not manifested as a decrement in mucociliary clearance.Sodium metabisulphite, when applied to the palate is diluted in the periciliary fluid . The dil-1 M to the palate surface was monitored for 5 minutes. The pH measured on the palate prior to metabisulphite was 6.9 ± 1.4 units. There was a decrease in pH following sodium metabisulphite 10-1 M, reaching a nadir of 6.4 ± 0.25 pH units after 60 seconds. Sodium metabisulphite 10-4 and 10-2 M did not cause any decrease in pH on the palate after application. Although the observed decrease in pH with sodium metabisulphite 10-1 M is relatively minor (<0.5 pH units), it may have been sufficient to influence ion channels, possibly disrupting ciliary beating and causing chemical changes such as the induction of inflammatory mediators [-1 M occurred in a similar time frame to the pH changes. After five minutes, the pH was returning toward normal, and within 20 minutes there was some recovery of mucus clearance time. An in vitro study [In several experiments the continuous pH response following the application of sodium metabisulphite 10ediators . The draro study  examined-1 M sodium metabisulphite (~250% compared to control, ~100%) was followed by a recovery (120 to 170 min in Figure The increase seen in mucus clearance times after 10We have shown from the zymographic studies, taken together with the scanning electron microscope studies, that MMP-9 activation was associated with the loss of ciliated cells from the palate. These results suggest the sustained increase in MCT as measured directly on the frog palate may have been due to the action of sodium metabisulphite to activate MMP-9 leading to a loss of ciliated epithelial cells. How this occurs at the cellular level is a question that remains to be answered. Further studies that clarify a site of action of the MMPs and a source of MMPs in this model will be important to determine the mechanism of action of this effect. How MMPs are activated in the tissue is another important question. An understanding of this injury mechanism may lead to ways to intervene in the early stages of airway diseases with symptomatic signs of impaired of mucociliary clearance.


To determine the correlates of serum lipoprotein (a) (Lp(a)) in children and adolescents in the United States.Cross-sectional study using representative data from a US national sample for persons aged 4–19 years participating in The Third National Health Nutrition and Examination Survey (NHANES-III).P < 0.001). Higher levels of Lp(a) > 30 mg/dl associated with parental history of body mass index and residence in metro compared to nonmetro in Blacks, and high birth weight in Mexican American children in the NHANES-III. In the entire group, total cholesterol (which included Lp(a)) and parental history of premature heart attack/angina before age 50 (P < 0.02) showed consistent, independent, positive association with Lp(a). In subgroup analysis, this association was only evident in white (P = 0.04) and black (P = 0.05) children. However, no such collective consistent associations of Lp(a) were found with age, gender, or birth weight.We observed ethnicity-related differences in levels of Lp(a) > 30 mg/dl, with values being markedly higher in African American (black) than nonhispanic white (white) and Mexican American children in multivariate model (Ethnicity-related differences in mean Lp(a) exist among children and adolescents in the United States and parental history of premature heart attack/angina significantly associated with levels of Lp(a) in children. Further research on the associations of Lp(a) levels in childhood with subsequent risk of atherosclerosis is needed. Levels of serum or plasma Lp(a) above 30 mg/dL are associated with increased risk of coronary artery disease and stroke in adults of European descent -3. GivenFew studies have examined the epidemiology of Lp(a) in representative samples of total populations of children and adolescents,12. HoweData for this analysis was obtained from The Third National Health and Nutrition Examination Survey (NHANES-III) conducted on a nationwide multi-stage probability sample of about 40,000 persons from the civilian, non-institutionalized population aged 2 months and over of the United States excluding reservation lands of American Indians. Of these, 31,311 were examined. Our analysis was restricted to children aged 4–11 years (518 whites, 877 blacks, and 685 Mexican Americans) and adolescents aged 4–19 years (336 Whites, 665 Blacks and 504 Mexican Americans) with valid Lp(a) measurements in Phase II of the survey conducted in 1991–1994. Details of the planning, sampling, operation, informed consent procedures, and measures taken to maintain confidentiality of information have been previously detailed .Demographic, medical history and behavioral information were collected prior to the examination by household interview of the parents or guardians of children and of adolescents aged 12 and over. Parents of children aged 2 months-11 years were asked "How much did the child weigh at birth?". Parents responding "don't know" were asked "Did the child weigh more than 5 1/2 pounds (2500 grams) or less? Responders were then asked, "Did the child weigh more than 9 pounds (4100 grams) or less?" An approximate category of weight at birth was created by combined responses to exact birth weights and the latter two questions. Participants' parent or guardian was also asked, "Has either of the biological parents ever been told by a doctor that he or she had a) high blood pressure or stroke before age 50 b) heart attack or angina before the age of 50? c) high blood cholesterol at any age? d) diabetes at any age? All "Yes" responses were followed by "Which, father, mother, or both?" Other interview variables are described elsewhere .Blood samples were obtained at the examination centers . A subsaSerum total cholesterol were determined at the Centers for Disease Control using a modified ferric chloride technique (GFAA/Perkin-Elmer Model 3030 and 5100) . High-de<0.05. Since substantial proportions of white and Mexican American children had undetectable Lp(a), log or other transformations could not produce an approximately normal distribution of Lp(a) for parametric analyses. Therefore analytic results presented are primarily those using Lp(a) > 30 mg/dL as a categorized variable.Population estimates for many of the variables other than Lp(a) have been published by the National Center for Health Statistics ,17. BecaBlacks had higher median Lp(a) than whites, who had higher levels than Mexican Americans Table . The difP = 0.02) (Table Table 2) Table .P = 0.96).Median Lp(a) did not differ consistently by gender across age or ethnic groups Table . SimilarBirth weight by parental recall was available for children aged 4–11 years (Table P = 0.011), whereas the percentage (Lp(a) > 30 mg/dL) was similar among those with parental history of diabetes and high cholesterol vs. those without . However, no significant differences were seen in other groups.In all the groups combined (age range 4–16 years), the percentage with Lp(a) > 30 mg/dL was significantly higher among those with parental history of heart attack/angina before age 50 years compared to those without (50.0 percent versus 30.2 percent), Chi square 2.72, P = 0.56). Among blacks, a greater percent of metropolitan, compared to non-metropolitan  had Lp(a) > 30 mg/dL. No significant differences were seen in other groups.Among white children, median Lp(a) was lower in the Midwest (8.2 mg/dL) and South (8.2) than in the Northeast (16.4), or West (10.1). No differences were noted for Blacks and too few Mexican Americans lived in the Northeast and Midwest for evaluation. In white children, the percentage with Lp(a) > 30 mg/dL were 15.1 in the Midwest, Northeast 25.1, South 21.2, West 21.4 (P = 0.10). Poverty income ratio was not significantly correlated with Lp(a).Family income < $20,000 was not associated with Lp(a) > 30 mg/dL in whites or Mexicans, but Blacks with low income tended to have a higher percentage of individuals with Lp(a) >30 mg/dL (56.9% versus 50.5%, P = 0.03) and a quadratic association in Mexican Americans (age beta 0.068, SE = 0.013, P < 0.001, age squared beta -0.000, SE beta 0.000, P < 0.001), indicating a lower prevalence of Lp(a) > 30 mg/dL at both ages 4–5 and 16–19 than at 6–15 years. Age and age squared, and sex were entered first in all analyses described below. Controlling for age, sex was not significantly associated with high Lp(a) in any group. Compared to whites, non-hispanic black ethnicity was significantly associated with high Lp(a) after controlling for age and sex (P < 0.001). Mexican American ethnicity was not significantly associated with lower prevalence of high Lp(a) (P = 0.35). Within ethnic groups at ages 4–11 years, low birth weight (<2500 g) was not significantly associated with high Lp(a) after controlling for age and sex. High birth weight (>4100 g) was associated with high Lp(a) (beta 1.87, P = 0.02) only in Mexican Americans.The following additional variables known to influence CVD risk were assessed as correlates of Lp(a) by ethnic group: age, total serum cholesterol, HDL cholesterol, casual triglycerides, hours of fasting, weight, height, body mass index, waist circumference, waist-to-hip ratio, subscapular skinfold thickness, suprailiac skinfold thickness, pulse rate, systolic and diastolic blood pressure, heavy activity frequency or TV hours. Logistic regression analysis with Lp(a) > 30 as dichotomous dependent variable and age in months as independent variable revealed a significant linear association in whites (beta = 0.004, SE = 0.002, P = 0.04) and in blacks (beta -0.80, SE 0.39, P = 0.05). Parental history of heart attack/angina was also significantly associated with high Lp(a) in all children (P = 0.02) after controlling for age, sex and ethnicity. In white children, parental history of high blood cholesterol (P = 0.07) and diabetes mellitus (P = 0.16) were not significantly associated with high Lp(a) after adjustment for age and sex.Parental history of heart attack/angina before age 50 was significantly associated with Lp(a) >30 after controlling for age and sex both in white children (beta -1.14, SE 0.55, P = 0.02). Region, rural/urban code, family income < 20,000 or higher poverty income ratio were not significantly associated with high Lp(a) in white or Mexican American children .Residence in central cities/fringe areas remained significantly associated with high Lp(a) in black children after controlling for age, sex, region, season, and time of the day  after controlling for multiple variables. Total serum cholesterol was significantly associated with Lp(a) after controlling for multiple variables in all three ethnic groups as expected.Body mass index or weight were significant predictors of Lp(a) > 30 mg/dL only among black children after controlling for age, sex, region, rural/urban code, season and poverty income ratio, e.g. weight(kg) The most important findings of this study are that ethnicity significantly associated with Lp(a), and that parental history of heart attack significantly associated with Lp(a) levels >= 30 mg/dl. While non-hispanic black children had significantly higher total Lp(a) level, compared to white and Mexican American children, no consistent associations of age or gender with Lp(a) were found in NHANES-III below age 20. Low birth weight (<2500 g) was not significantly associated with high Lp(a) after controlling for age and sex in the entire group. Higher levels of Lp(a) >30 mg/dl was evident in metropolitan compared to non-metropolitan non-hispanic black children.Lp(a) is a circulating particle that consists of phospholipids, cholesterol, and apolipoprotein B-100 (i.e. a LDL particle), with apolipoprotein(a) attached to the latter at a single point ,24. LikeWhereas levels of Lp(a) above 30 mg/dL was shown to increase risk of coronary heart disease in European samples , no suchA number of studies of adults have compared Lp(a) levels in Whites and Blacks, and have uniformly reported two-fold higher levels in Blacks ,5,11. InNo previous reports of studies comparing Lp(a) in whites and blacks and Mexican American children in the same study were found prior to NHANES-III. In fact, few comparisons of plasma Lp(a) concentrations were found for hispanic children or other children below age 8 years prior to NHANES-III ,31. The Perhaps one of the most noteworthy observation from this study is the significantly higher levels of Lp(a) >30 mg/dl in black, compared to white and Mexican American children Table . Ethnic-The explanation for ethnic-related differences in levels of Lp(a) in the US remains unclear. Other than total cholesterol, no single environmental or biological variable consistently associated with levels of Lp(a) in the NHANES-III sample. While levels of Lp(a) > 30 mg/dl received significant contributions from BMI and residence in metro compared to non-metro in black children, only higher birth weight significantly contributed to levels of Lp(a) in Mexican American children. Contrary to a previous report of association of low birth weight with elevated Lp(a) concentration in black children,  we foundAt the genetic level, heritability estimates were reportedly higher for Whites than for Blacks ,34 despiStudies of adults found an association of higher age and female gender with higher Lp(a) levels . The BogAltogether, our observation from the present NHANES-III study, together with the work of others ,38 foundFew studies of the association of Lp(a) with family history have been reported in children ,39. Our In the NHANES-III study, no association of Lp(a) were seen with family history of stroke, hypertension, or diabetes. Similarly, family history of high cholesterol and diabetes were not significantly associated with levels of Lp(a) >30 mg/dl in the entire sample Figure , except Limitations of the present study include possible bias from survey non-response, missing values for some variables, and confounding by variables not measured. Fortunately, several special studies of earlier NHANES-III data have indicated little bias due to non-response . AlthougIn conclusion, ethnicity significantly associated with levels of Lp(a). Parental history of heart attack/angina before age 50 years associated with levels of Lp(a) > 30 mg/dl in offspring. Collectively, different pathological thresholds may have to be established for elevated serum Lp(a) levels, to be used as a risk marker for coronary heart disease in different populations. Future research should include longitudinal studies of Lp(a) in white, black and hispanic children followed to adulthood. Racial admixture as well as environment and behavioral variables associated with acculturation and urban residence should be studied, especially in Mexican American and black populations. Standardization of methods will facilitate inter-study and longitudinal comparisons.


Cerebral venous sinus thrombosis is an infrequent disease with a variety of causes. Pregnancy, puerperium, contraceptive pills and intracranial infections are the most common causes. The patient may present with headache, focal neurological deficits and seizures.The clinical outcome is highly variable and treatment with heparin is advised.The patient is a 22 year old male who presented with headache, repeated vomiting and papilledema.He was a bodybuilder doing exercise since 5 years ago, who had used nandrolone decaonoate 25 milligrams intramuscularly during the previous 5 months. Brain MRI and MRV showed superior sagital and transverse sinus thrombosis and extensive investigations did not reveal any known cause.We suggested that androgen was the predisposing factor in our patient. Androgens may increase coagulation factors or platelet activity and cause arterial or venous thrombosis.As athletes may hide using androgens it should be considered as a predisposing factor for thrombotic events in such patients. Cerebral venous sinus thrombosis (CVST) is a disease with a wide spectrum of non specific clinical signs and symptoms, including headache, focal neurological deficits, seizures and coma.The clinical outcome is highly variable; patients may recover completely or may develop severe and lasting neurological deficits .There are many causes for this disease but the most common predisposing factors are pregnancy, puerperium, contraceptive pills, coagulopathies and intracranial infections .There are few reports of patients with CVST after androgen therapy. We present a young bodybuilder man who developed CVST with abusing androgens for increasing muscle mass.In May 2004 a 22 year old male was admitted to our department with chief complaints of headache and vomiting.The patient was well till 10 days prior to admission that developed progressive, intense bitemporal headache exacerbated with bending.The patient also had history of malaise, nausea and several episodes of vomiting from 3 days before admission. The only objective finding on physical examination was bilateral papilledema. The patient was a body builder doing exercise from 5 years ago who had used nandrolone decaonoate 25 mg once or twice a week during the last 5 months. He had injected 20 ampoules in this period.th day of treatment headache resolved and warfarin added to heparin.Brain computed tomography without contrast was done for the patient which showed cord sign, emergency MR imaging including T1 – T2, weighted and MRV showed prominent superior sagital and transverse sinus thrombosis. The C.S.F opening pressure was 480 mm/H2o without any other abnormality. Heparin 80 IU/kg started as loading dose then continued 1000 IU/ hr for 10 days. On the 5Laboratory tests including antithrombin III activity, protein C, S factor V leiden, Plasma hemocystein and anticardiolipin were all within normal limits.The patient was discharged in a good condition and was maintained on 6 months warfarin protcol.There are few reports of CVST following androgen therapy , but theThis has led to widespread use of androgenic anabolic steroids by athletes at all levels. Nandrolone decaonoate is a synthetic anabolic steroid. In focus on homeostasis system the most important factors under testosterone regulation are fibrinogen, Plasminogen activator inhibitor-1 (PAI – 1) and platelet aggregability.The current data indicate that testosterone lowers fibrinogen and PAI – 1, however these anticoagulatory and profibrinolytic may be opposed by proaggregatory effects on platelets because high dosages of androgens were found to decrease cycloxygenase activity and thereby increase platelet functions .Proaggregatory effect of testosterone and other synthetic androgens become more reliable theory for CVST, according to recent publication .This case report presents a patient with CVST following exogenous androgen usage with a mechanism which is not completely understood, but it may be related to platelet activation or an increase in coagulation factors. As androgen use may be frequent and hidden in athletes, it may be an underestimated cause of cerebral venous thrombosis in young adults and careful history should be taken in these groups of patients.CVST: cerebral venous sinus thrombosisPAI-1: Plasminogen activator inhibitor-1MRI: magnetic resonance imagingMRV: magnetic resonance venographyThe author(s) declare that they have no competing interests.M.A.S: Admitting and treating the case, preparing the article.M.M : Revising the article.A.R.A : searching previous articles, preparing case presentation part.B.M : searching previous articles, preparing background.The pre-publication history for this paper can be accessed here:


The chest pain unit (CPU) has been developed to provide a rapid and accurate diagnostic assessment for patients attending hospital with acute, undifferentiated chest pain. We aimed to measure the effect of CPU assessment upon psychological symptoms and health-related quality of life.We undertook a single-centre, cluster-randomised controlled trial. Days (N = 442) were randomised in equal numbers to CPU or routine care. Patients with acute chest pain, undiagnosed by clinical assessment, ECG and chest radiograph, were recruited and followed up with self-completed questionnaires (SF-36 and HADS) at two days and one month after hospital attendance.Patients receiving CPU assessment had significantly higher scores on the physical functioning , vitality , and general health  dimensions of the SF-36 at two days, and significantly higher scores on all except the emotional role dimension at one month. They also had significantly lower depression scores on the HADS depression scale at two days  and one month . However, initially lower anxiety scores at two days  were not maintained at one month . CPU assessment was associated with reduced prevalence  and severity  of chest pain at one month, but no significant difference in the proportion of patients taking time off work .CPU assessment is associated with improvements in nearly all dimensions of quality of life and with reduced symptoms of depression. Acute chest pain is a common reason for emergency hospital attendance and admission. Patients with chest pain that remains undiagnosed after clinical assessment, ECG and chest radiograph pose a particular problem. They carry a low, but important risk of an acute coronary syndrome . The potThe chest pain unit (CPU) was developed to provide rapid and accurate diagnosis for patients presenting with acute undifferentiated chest pain . PatientThe ESCAPE  trial was a randomised controlled trial and economic evaluation of CPU versus routine care that showed that CPU care was associated with reduced hospital admission , improveThe Northern General Hospital Emergency Department provides adult emergency care to the 530,000 population of Sheffield, United Kingdom. In 1999 a CPU was established in the emergency department, staffed by three specialist chest pain nurses, and able to accommodate up to six patients with acute undifferentiated pain. Patients were selected using validated clinical predictors and received two to six hours of observation and biochemical cardiac testing, followed by, where appropriate, an exercise treadmill test. Full details of the CPU protocol have been published . Routineth February 2001 to 5th May 2002 the CPU was subject to a cluster randomised controlled trial. Days of the week (N = 442) were randomised to CPU or routine care in equal numbers. All patients attending with acute chest pain were screened for eligibility in the trial. Patients were excluded if they had ECG changes diagnostic for an acute coronary syndrome, clinically obvious unstable angina, co-morbidity or alternative pathology requiring hospital admission (e.g. suspected pulmonary embolus), negligible risk of acute coronary syndrome (e.g. age less than 25 years), or if they were unable to consent to participation. Written, informed consent was requested and patients who agreed to participate were followed up in a review clinic at two days, and by postal questionnaire at one month. The study protocol was approved by the North Sheffield Research Ethics Committee. Full details of the ESCAPE trial have been published [From 5ublished .Health related quality of life was measured using the SF-36 questionnaire . AnxietyA brief additional questionnaire was sent at one month that was designed specifically for the study. This predominantly asked questions about health service use for the economic evaluation, but also asked participants whether they had suffered any further chest pain. If they responded that they had, they were asked to score the severity of the chest pain on a 100 mm visual analogue scale. A further question asked whether the patient had taken time off work since their hospital attendance.The sample size estimate of 988 was based upon the primary outcome measure, the proportion of patients admitted to hospital. Assuming a response rate of 65% to the questionnaires, this sample size would provide 80% power to detect an effect size of 0.25 for these outcomes . Using standard deviations derived from a two-week pilot study, this effect size equates to 1.1 points on the HADS anxiety or depression scores, 11.5 points of the SF-36 physical or emotional role dimensions, and 6 points on the other SF-36 dimensions. Data was analysed using Stata statistical software (version 8.0). Multi-level random effects modelling was used with day of week as a random effect to adjust for clustering by day of week. For the principal analysis no adjustment for confounding was made. For secondary analysis age, gender and past history of coronary heart disease were included as covariates , along with any variable that showed significant (p < 0.05) baseline imbalance between the study groups.During the 442-day study period there were 6957 attendances with chest pain or a related complaint. Of these, 764 11.0%) had ECG changes diagnostic for an acute coronary syndrome, 2402 (34.5%) had clinically obvious unstable angina, 869 (12.5%) had co-morbidity or alternative pathology requiring hospital admission, 1291 (18.6%) had negligible risk of acute coronary syndrome, and 513 patients (7.4%) were unable to participate in the trial or provide consent. The remaining 1118 patients (16.1%) were asked to participate in the trial and 972 agreed (86.9%). Response rates were: 717 (73.8%) to the initial questionnaire and 679 (69.9%) to the one-month questionnaire. The CONSORT diagram and full details of exclusions have been published elsewhere . Baselin1.0% had Table Table Table At one-month follow-up, 143 out of 318 participants (45.0%) receiving CPU care reported having further pain, compared to 168 out of 314 (53.5%) receiving routine care . For those reporting further pain, the mean score on a 100 mm visual analogue pain score was 36.5 mm among those receiving CPU care and 43.0 mm among those receiving routine care . Thus, at one month, CPU care was associated with a reduction in the incidence and severity of subsequent chest pain.One month after hospital attendance, 49 out of 315 participants receiving CPU care (15.6%) reported that they had taken time off work, compared to 58 out of 316 receiving routine care (18.4%). The unadjusted odds ratio for taking time off work after receiving CPU care was 0.82 .Patients with acute, undifferentiated chest pain who received CPU care had improved quality of life and reduced psychological symptoms. All dimensions of quality of life were improved at one month apart from the emotional role dimension. Anxiety was reduced two days after assessment, but there was no significant difference by one month, whereas reduced symptoms of depression at one month were still significant at one month. Patients receiving CPU care reported that subsequent symptoms of chest pain were less frequent and (if present) less severe. However, these reported differences in symptoms and quality of life were not associated with any significant difference in the need to take time off work.Previous studies of CPU care have focussed on cardiac events, process measures and economic measures . One preNevertheless, CPU assessment was associated with reduced anxiety and improved quality of life. This is consistent with a previous study of diagnostic testing by Sox et al  that shoThe main limitation of this study relates to our inability to blind participants to the intervention they received and to fact that they were involved in a trial of CPU care. Participants may have been influenced by this knowledge and improvements in psychological symptoms and quality of life may represent a positive response to receiving a novel form of care, rather than improvements specifically related to CPU care.The use of cluster randomisation has substantial advantages for pragmatic evaluation of changes in organisation, particularly if economic evaluation is undertaken . HoweverAlthough the measures used have been validated, they have not been widely used in the emergency setting. Changes in health status after an episode of chest pain may be very rapid, hence our need to measure outcomes only two days after intervention. Yet the HADS measures anxiety and depression over the previous week, while some SF-36 questions refer to the previous month. A recent episode of chest pain is likely to be an important determinant of reported health, but it may be that, if participants interpreted the questionnaires strictly, the initial questionnaire was recording health status before the intervention. Also, there may be doubts regarding what some of the outcomes are actually measuring. For example, some of the questions in the HADS measure symptoms that are useful markers for depression, such as levels of activity, which may also be changed by other health or social processes. Thus it may be that the reduced scores associated with CPU care measured on the depression scale relate to increased activity in response to the CPU exercise treadmill test, rather than reduced depression.This study suggests that the assessment that patients receive when they present with acute chest pain can have an impact upon their subsequent health, even if this assessment does not, in most cases, provide a definitive diagnosis. It supports the findings of decision analysis modelling  that theYet it is not clear how this effect is achieved. It is possible that early, rigorous testing, particularly the exercise treadmill test, has a valuable effect in reassuring the patient that they are healthy and capable of normal physical functioning. Alternatively, it could be that consistent, reliable advice and attention from specialist chest pain nurses, rather than a variety of different doctors, is the key element. A third possibility, as previously discussed, is that bias plays an important role.Future research needs to determine which of these possibilities is the key factor. This is important for the specific issue of determining whether and how CPU care is effective, and thus what elements of CPU care are essential, and for the more general issue of exploring how diagnostic assessment effects subsequent well being.CPU care for patients attending hospital with acute, undifferentiated chest pain is associated with reduced initial anxiety, reduced depression over the following month, and improvements in most dimensions of quality of life. Further research is required to establish how this effect is achieved.OR: odds ratioCI: confidence intervalECG: electrocardiographCPU: chest pain unitESCAPE: effectiveness and safety of chest pain assessment to prevent emergency admissionHADS: hospital anxiety depression scaleSG conceived and designed the study, analysed the data, drafted the paper, and participated in writing the final paper. JN assisted with study conception and design, supervised data analysis, and participated in writing the final paper.


Drosophila larvae across decaying fruit yields a competitive advantage over mould, by which the larvae achieve a higher survival probability in larger groups compared with smaller ones. Feeding and locomotor behaviour of larger larval groups is assumed to cause disruption of fungal hyphae, leading to suppression of fungal growth, which in turn improves the chances of larval survival to the adult stage. Given the relationship between larval density, mould suppression and larval survival, the present study has tested whether fungal-infected food patches elicit communal foraging behaviour on mould-infected sites by which larvae might hamper mould growth more efficiently.Competition with filamentous fungi has been demonstrated to be an important cause of mortality for the vast group of insects that depend on ephemeral resources (e.g. fruit, dung, carrion). Recent data suggest that the well-known aggregation of Drosophila larvae were offered the choice between fungal-infected and uninfected food patches, larvae significantly aggregated on patches containing young fungal colonies. Grouping behaviour was also visible when larvae were offered only fungal-infected or only uninfected patches; however, larval aggregation was less strong under these conditions than in a heterogeneous environment (infected and uninfected patches).Based on laboratory experiments in which Drosophila larvae to fungal-infected sites leading to suppression of mould growth may reflect an adaptive behavioural response that increases insect larval fitness and can thus be discussed as an anti-competitor behaviour. These observations support the hypothesis that adverse environmental conditions operate in favour of social behaviour. In a search for the underlying mechanisms of communal behaviour in Drosophila, this study highlights the necessity of investigating the role of inter-kingdom competition as a potential driving force in the evolution of spatial behaviour in insects.Because filamentous fungi can be deadly competitors for insect larvae on ephemeral resources, social attraction of  A common idea in animal ecology is that adverse or stressful environmental conditions facilitate the evolution of social behaviour . The forDrosophila as an ecological model system, one benefit that females flies seem to achieve by this spatial aggregation is that larval survival probability to the adult stage is highest at intermediate densities [Drosophila larvae on the same patches have been demonstrated to cause high rates of mortality when larvae feed solitarily or in small groups, whereas larger groups are able to hamper mould growth [In the vast group of insects that depend on ephemeral resources, such as decaying plant tissues, dung and carrion, aggregation in the immature stages across resource patches is the result of the choice of a female to lay batches of eggs and/or to aggregate with conspecifics -8. In stensities ,10, indiensities . Competid growth  d growth ,13,14. Ad growth  seems tod growth ,14.Drosophila can be interpreted as an adaptive behaviour against competing fungi on larval feeding sites in order to enhance offspring survival. These ecological interrelationships might set conditions for facilitating social behaviour in the fly larvae because, at the level of larval behaviour, a more efficient strategy that might control the rapid establishment of noxious fungi would be to exert physical stress directly on fungal colonies. Thus, larvae should display an assortative behaviour on the site on which fungi are growing, rather than moving randomly and independently of each other across a resource patch, by which the fungal tissues might only incidentally be destroyed. In the present study, I have provided groups of Drosophila melanogaster Meigen (Diptera, Drosophilidae) larvae with fungal-infected (2-day-old colonies of Aspergillus niger van Tieghem) and uninfected (control patches) (F-C treatment) food patches and examined whether the distribution of larvae across the patches is driven by fungal infection. In comparison with this naturally occurring heterogeneity in patch quality, I have also studied the distribution of fly larvae when they were offered only infected (F-F treatment) or uninfected (C-C treatment) food patches in order to test for the existence of grouping behaviour in two types of homogenous larval environment. If grouping is irrelevant under the given experimental setting, no deviation from the regular larval distribution across the food patches would be expected, i.e larvae should distribute themselves across patches in order to minimise larval competition for food [Given the relationship between spatial oviposition patterns, Allee effects and the suppression of mould, spatial aggregation in for food .Drosophila is a thoroughly studied model organism in foraging biology [Drosophila communities are characterised by strong intraspecific aggregation across patchily distributed substrates (e.g. decaying plant tissues) [Although  biology ,18, knowtissues) -21. The pl, was used as a measure of the way in which Drosophila larvae distributed themselves between the two types of food patches in the F-C treatment (see method section for details). The number of larvae in both food patches (LARVAE) and the experimental day (DAY) did not influence Δpl (Table pl was significantly different from zero (GLM d.f. = 1, mean square = 4.475, F = 20.13, P < 0.0001, N = 35), the positive value for Δpl  indicating the aggregation of larvae on fungal-infected sites (see method section).The proportion of larvae on fungal-infected patches minus the proportion on uninfected patches, Δpl Table . The estpl|, the absolute value of Δpl, was used as a measure of the general tendency of Drosophila larvae to aggregate with conspecifics in the heterogeneous environment (F-C) and the two types of homogenous environment (F-F or C-C). By using |Δpl|, aggregation in the F-C treatment was quantified independently of whether a food patch was infected with fungi or not. With regard to all three larval environments, the estimated intercepts for |Δpl| were significantly different from zero, and hence indicate larval aggregation (Table pl| (Table d.f. = 2, mean square = 0.0134, F = 3.34, P = 0.0393, N = 105). Significantly fewer larvae were found to be feeding in both patches in the C-C treatment (8.89 ± 1.64 SE) than in the F-C (9.43 ± 0.95 SE) or the F-F treatment (9.46 ± 0.61 SE). However, LARVAE within one type of environment had no effect on larval aggregation  between Drosophila larvae is present in third-instar larvae, a behaviour regulated by a peptide neuromodulator (Drosophila neuropeptide F, dNPF) [Caenorhabditis elegans [C. elegans [C. elegans, the intimate communal digging behaviour in old third-instar Drosophila larvae does not occur in the context of food foraging behaviour but is part of the post-feeding phase prior to pupation [Drosophila larvae, higher levels of dNPF expression in younger larvae seem to suppress strong larval aggregation and communal digging behaviour [Drosophila larvae with respect to social affinity related to foraging for food and attraction to fungal competitors. With regard to the proximate causes of attraction to mould-infected sites, fungal-borne volatiles such as CO2 or ethylene [Group formation in eusocial insects and those living in groups for part or most of their lives is often mediated by pheromones, e.g. cuticular hydrocarbons that induce attraction between individuals ,23. Chemunctions , both chF, dNPF) ; this sh elegans . Moreove elegans , thus pr elegans . In contpupation . Whereasehaviour . Therefoethylene  might beDrosophila larvae  enhance group foraging behaviour. Because the larval-driven reduction in mould growth [Drosophila. Thus, this study highlights the largely unappreciated role of inter-kingdom competition as a potentially important driving force in the evolution of insect behavioural traits. In general, the group formation of Drosophila larvae in response to well-defined ecological conditions might be an interesting model system for the study of proximate and ultimate aspects of social biology.The study presented here demonstrates that fungal-infected food patches (1) attract first-instar d growth  has beend growth ,13,14, sDrosophila larvae using a D. melanogaster strain that originated from wild animals caught in 2003 near Kiel, northern Germany (approx. 54° N, 10° E). Flies had been reared for 18 generations on standard Drosophila medium , and Nipagin) under constant environmental conditions (photoperiod of 16 hours and a temperature of 22°C). In order to obtain first instar larvae, a population of approx. 300 individuals (five to seven days old) were offered a 10 cm Petri dish containing a hard Agar medium (22 gram Agar, 90 cm3 sugar beet syrup and 9.5 cm3 Nipagin (10% in 95% ethanol) per 500 cm3 water), on which they were allowed to oviposit for a period of 16 to 18 hours, including a period of darkness. Subsequently, flies were removed and the eggs were incubated at 22°C over a 16-hour photoperiod. After 24 hours, almost all larvae had hatched from the eggs and were then isolated from the medium by washing them off the Agar plate onto fine-meshed gauze with water. These larvae were used in the experiments.I experimentally analysed the grouping behaviour of 3 hot medium were transferred to each of two small pots (10 mm diameter, 5 mm high) that were glued to the bottom of a Petri dish (45 mm diameter, 13 mm high). Within one Petri dish, the larval food patches were placed at a distance of approx. 10 mm and surrounded by an Agar layer (5 mm high), so that the surfaces of the Agar and the food patches were at the same level. After the food patches had cooled down, one patch was provided with 1 μl of water containing approx. 800 conidiospores of the fungus A. niger (F treatment) and, as a control, the second patch was provided with spore-free water (C treatment). In addition to this F-C treatment, I simultaneously prepared arenas in which both patches were infected with spores (F-F) or both patches remained uninfected (C-C). The arenas were immediately sealed with lids and incubated under the aforementioned conditions. Two days later, the fungal spores had germinated and tiny translucent hyphal colonies were visible. A group of ten Drosophila larvae were transferred, with a fine brush, to each arena at a distance of approx. 10 mm from each of the two food patches. Subsequently, the arenas were sealed with lids and stored at 22°C in an illuminated incubator. In order to avoid any systematic effects on larval distribution behaviour that might be caused by the position of the light tubes, the arenas of all three treatments were randomly arranged in the incubator. After six hours, the number of larvae in each food patch was recorded. Preliminary experiments had shown that this time period was sufficient to obtain final larval distribution patterns across the two patches, which remained nearly constant until the next day. Five to ten replicates for each treatment were simultaneously prepared at four different days.I used the same medium as for fly rearing  to simulate an uninfected or a fungal-infected larval environment as follows: aliquots of 3.5 cmpl. Δpl = 0 would indicate no effect of the presence of fungal-infected and uninfected food sites on larval distribution patterns. Whereas Δpl > 0 would indicate aggregation on fungal-infected sites and thus larval attraction to fungal colonies, Δpl < 0 is expected if larvae avoid fungal colonies and aggregate on uninfected patches. Subsequently, I used the absolute values of Δpl, |Δpl|, that were obtained in all three types of treatment (F-C, F-F and C-C) in order to compare the tendency to aggregate with conspecific larvae in the heterogeneous larval environment (F-C) with larval aggregation in two types of homogeneous environment (F-F or C-C). Note that, because the absolute value of Δpl can only be equal to or larger than zero, |Δpl| measures larval aggregation in the F-C treatment independently of whether a food patch was fungal-infected or not.Based on the number of larvae that were found to be feeding in both patches, I calculated the proportion of larvae in each patch. Larvae that were not found on any of the food patches were ignored. However, I tested for the effect of the number of larvae in both patches (LARVAE) on the degree to which larvae aggregated across the food patches (see below). To obtain a measure of the degree of larval aggregation across the two food patches in the F-C treatment, the proportion of larvae on the fungal-infected patch in each arena was reduced by the proportion of larvae on the uninfected patch, yielding ΔDrosophila larvae aggregated on fungal infected food patches, i.e. if Δpl is significantly larger than 0 (see above). For this only the intercept was tested as an effect in the statistical model [pl (Table pl|) under different environmental conditions , I used the aforementioned GLM procedure with a RANDOM statement to account for possible effects of experimental DAY on larval distribution patterns. In this model, TREATMENT and LARVAE were fixed main effects. Since five to ten replicates for each treatment were prepared at four different days, DAY was considered as a categorical random factor. DAY is nested within TREATMENT and was used as the error term in testing for the effect of TREATMENT [To analyse the effect of TREATMENT (F-C, F-F or C-C) and LARVAE on the general propensity of REATMENT . The res


Plasmodium falciparum and Plasmodium vivax, which infects mosquitoes in Thailand, was (a) in people feeling sufficiently ill with malaria to come to a clinic or (b) in people who had remained in their home villages with some fever symptoms or with none.It was unknown whether the main reservoir of Plasmodium infections and with fever. Malaria patients were also located at a clinic which served these villages. Adults from both sources whose blood slides registered positive for Plasmodium spp. were requested to allow laboratory-bred Anopheles minimus to feed on them. Seven to nine days after the blood feeds the mosquitoes were dissected and checked for presence of oocysts.Mass surveys were carried out in Thai villages to identify people with Plasmodium infection among people in the villages with fever than without fever and much higher rates of infection among clinic patients than among people who had remained in the villages. People with malarial infections identified via the clinic and the village surveys could infect mosquitoes, especially, but not only, if their blood slides showed visible gametocytes. Because only a very small minority of the village populations were visiting the clinic on any one day, assessment indicated that the main reservoir of infection was not primarily among clinic patients but among those in the villages, especially those feeling feverish.There were higher rates of Efficient use of an anti-gametocyte drug to suppress the parasite reservoir in a population requires that it be given, not just to clinic patients, but to infected people located by mass surveys of the villages, especially those feeling feverish. Plasmodium reservoir to mosquitoes is of interest in understanding the epidemiology of malaria and its changes after application of certain types of control measures. Different approaches have been used to investigate this, including direct feeding of mosquitoes through the skin of human subjects [An estimate of the infectiousness of the subjects -5 or feesubjects -13. The This study was approved by the Thai Ethics Committee and the Ethics Committee of London School of Hygiene and Tropical Medicine. Subjects were recruited from mass blood surveys in villages and from a clinic in Muang District, Mae Hong Son Province, which is in north-western Thailand. Anti-malarial drug use is tightly controlled in Thailand and these drugs are generally available on prescription. Self-medication is, therefore, unusual in this area. Mass blood surveys were conducted twice a year. After slides were examined for parasites, the people whose slides were positive and were more than 14 years of age were invited to take part in this study. The Thai ethical committee did not allow direct feeding of mosquitoes on children aged less than 15 years. Adults positive for malaria parasites were informed about the purpose of the study and, if they agreed, a consent form was signed and patients were interviewed.Human subjects for the study were also enrolled from the Vector-borne Disease Control Unit No. 8 (VBDU: described as "Clinic" throughout this paper). In this Clinic, blood smears were taken by the clinic staff and stained with Giemsa to check for malaria. Patients who had presented with clinical malaria and/or parasitaemia and were more than 14 years of age were informed about the purpose of the study and asked to participate in it. If they agreed and signed the consent form, then the mosquito feeding was performed.Anopheles minimus species A (aged 4–6 days), which had been starved for 9–12 hr, were allowed to feed on their arms for 30 minutes. After feeding was completed, anti-malarial drugs were given to all the human subjects by a malaria worker. Two hours after feeding the unengorged mosquitoes were removed from the cups using a sucking tube and destroyed, leaving only fully engorged mosquitoes in the cup. The mosquitoes were brought back to the Chiang Mai insectarium and maintained at 25 to 27 C and 70–80% relative humidity with permanent access to sucrose solution and without any further blood meals. Mosquitoes at 7–9 days post-feed were anaesthetized and the wings and legs were removed. Midguts were dissected on glass slides in a drop of 0.85% NaCl and examined at a 40× magnification. The number of oocysts present on the midgut to each mosquito was counted and recorded individually.Before feeding, patients' arms were cleaned with 70% alcohol. Then, 50 laboratory-bred female Asexual parasitaemia and gametocytaemia were quantified as the number of aseuxal forms/200 white blood cells on a thick film. Throughout the period of the study, 20% of the negative blood smears and all positive slides which had been examined by the microscopist of the Clinic were chosen randomly and re-examined by a team from the Office of Vector-borne Disease Control No.2, Chiang Mai. All cases of positive slides without visible gametocytes and which led to oocyst production were re-examined by an expert team from the London School of Hygiene and Tropical Medicine to ascertain whether a few gametocytes might have been present but were initially missed.The database from mosquito feeding included 1) densities of gametocytes and trophozoites, 2) number of infected mosquitoes, and 3) mean number of oocysts per infected mosquito. The chi-square test or Fisher's exact test was used to examine the significance of differences in the proportion of people in various categories who transmitted malaria to the mosquitoes (i.e. yielded at least 1 oocyst). Regressions on the natural log of gametocyte density were also computed. Stata statistical software (version 6) was used for analysis.Plasmodium falciparum and 10 with Plasmodium vivax. The prevalence of infection of both species from the village surveys was higher in children (15/623 = 2.4%) as compared to adults (37/4604 = 0.8%) (χ2 = 12.8, P < 0.001). The lower part of Table P. falciparum and 47 with P. vivax.The upper part of Table 2 = 0.26, p = 0.6). The probability of infection of mosquitoes from people infected with P. falciparum was significantly less than from people infected with P. vivax (24.7% versus 57.1%, χ2 = 11.6, P < 0.001). 77.1% (44/57) of P. falciparum cases reported fever. Among those, only 27.3% could infect mosquitoes. For P. vivax 84.1% (53/63) reported fever; among those 56.6% could infect mosquitoes. More than half of the infections from whose blood no mosquitoes developed oocysts had parasite density > 3,960/μl. There was no association of probability of mosquito infection with parasite density (χ2 = 1.3, P > 0.05).As shown in Table P. vivax (P= 0.007), but the relationship was only of borderline significance for P. falciparum (Fisher's exact test, P = 0.06). Approximately 50% (7/13) of the P. falciparum cases with observable gametocytes failed to infect mosquitoes and 30% (14/45) of the P. vivax cases with observable gametocytes failed to infect. However, the difference was not significant (Fisher's exact test, P= 0.19). Approximately 21% (13/62) with no observable gametocyte of either species could infect mosquitoes.Considering each species separately, the probability of infectiousness to mosquitoes was significantly related to being observably gametocyte positive in t = 0.87, df= 36, P= 0.39). Similarly, there was not a significant association of mean oocyst load per infected mosquito on the natural log of gametocyte density (t = 0.87, df = 36, P= 0.38).58 of the infections with either species had observable gametocytes (11 cases from the village survey and 47 from the Clinic). Feeds on 37 of these 58 gametocyte carriers led to oocyst production. The regression of percent of mosquitoes infected on the natural log of gametocyte density was not significant  in the blood taken up by mosquitoes than in the blood from finger pricks. In some cases high densities of gametocytes were not infectious and similar results have been reported in most studies assessing human malarial infectivity to mosquitoes ,11,24,25It is clear that, as the Ethical committee only authorized us to request people over 14 years of age to take part in our experiments, this excluded a sector of the reservoir population, the children, from the study. It is also clear that the occurrence of transmission-blocking immunity and prior histories of the subjects taking anti-malarial drugs might have influenced whether they infected mosquitoes. No data on these factors was collected and so no comment can be made on their possible influence on infectivity of people's blood to mosquitoes. However, the fact that availability of anti-malarial drugs is much more tightly controlled in Thailand than in most malarious countries should be emphasized.From the results of the study, an attempt can be made to estimate the number of adults in the catchment area of the Clinic who were reservoirs of infection. On the basis of the number of patients visiting the Clinic per day and the catchment population from which the patients come Figure , it was Aree Pethleart led the data collection team in the field and laboratory, analysed data and drafted the paper;Somsak Prajakwong provided research facilities;Wannapa Suwonkerd organized mosquito rearing and dissection;Boontawee Corthong was in charge of Clinic data and provided facilities for village surveys;Roger Webber was responsible for early planning of the work; andChristopher Curtis supervised data analysis and edited the draft.


Accurate staging of cancers is one of the most important parts of the work up of patients for both prediction of prognosis and determination of the most appropriate treatment. And an essential part of this work up is assessing whether or not there has been lymphatic spread. Current methods include surgical removal of nodes for examination and various types of imaging, ranging from ultrasound to newer technologies such as magnetic resonance imaging (MRI). All these methods have problems; some are very invasive, others are very time consuming, and none are completely reliable.However in one of the more exciting crossovers from chemistry into medicine, researchers have developed nanoparticles to improve the diagnostic accuracy of MRI. The nanoparticles contain a central superparamagnetic iron oxide core and are covered by dextran, imparting long circulation times and biocompatibility. When injected intravenously, the nanoparticles localize to lymphoid tissue, and are internalized into macrophages. There is then a decrease in signal intensity on T2- and T2*-weighted images, and when metastases are present there is a recognizably abnormal pattern on MRI scans.New England Journal of Medicine, Ralph Weissleder and colleagues described using these nanoparticles to assess lymphoid spread in patients with prostate cancer. Now, in a paper published in this month's PLoS Medicine, they have gone further by extending the analysis to patients with different types of cancer, and producing an algorithm that allows semiautomation of the procedure.In a previous paper published in the The authors developed the algorithm in a training group of 36 patients and then validated it in a group of 34 patients. The results are encouraging: the analysis showed a sensitivity of 98%  and a specificity of 92% . The advantages of automating this procedure are substantial, not least because it can remove the problem of different observers assessing data differently.And what is more, once the data have been collected and assessed it is possible to reconstruct a virtual picture of the patient's lymph nodes, thus potentially allowing accurate surgical removal of the nodes.


To function properly, cells must keep constant tabs on the environmental conditions around them, such as the presence of growth hormones in the blood or the proximity of neighboring cells. These external cues are relayed into the cell through a cascade of chemical and physical reactions referred to as signal transduction. Signal transduction pathways inform and regulate almost all activity within the cell, from protein production to cell division. Understanding these processes is fundamental to biology, but the sheer number of molecules and interactions in some pathways makes thorough documentation difficult.Taking a holistic approach that combines both computational models and experimental manipulations, scientists have described the web of interactions involved in the aryl hydrocarbon receptor (AHR) signal transduction pathway. AHR belongs to the Per–Arnt–Sim (PAS) superfamily of sensor molecules that regulate functions like development, the sleep-wake cycle, and cellular reaction to oxygen deprivation. Unlike many receptors that are embedded in the cell membrane, AHR floats freely in the main body of the cell, called the cytosol. There it waits for a stimulus or ligand, such as a dioxin molecule, to enter the cell and bind to it. Once bound, AHR undergoes a host of changes, glomming on to additional molecules before it enters the cell nucleus and acts as a transcription factor, initiating the production of enzymes to digest foreign, or xenobiotic, compounds. The AHR pathway is a curiosity; though found in all vertebrates, the natural, or endogenous, ligand remains unknown. Without this knowledge, researchers are limited in the kind of experiments they can perform to evaluate the pathway.Christopher Bradfield and colleagues used yeast as a model system to elucidate the steps involved in this pathway, which regulates vertebrate cell response to pollutants like dioxins. To first assess the molecules involved in the AHR pathway, the team used 4,507 yeast “deletion” strains, each strain missing one gene from its genome. They then inserted the AHR gene into the strains using small rings of movable DNA called plasmids. Though yeast does not naturally possess AHR, it is an ideal genetic model for studying signaling pathways due to its quick generation time, small, well-characterized genome, and similarity to vertebrate systems.Bradfield's team exposed each strain to a receptor stimulus or agonist and screened them for AHR response. If a deletion strain showed significantly reduced activity, they concluded that the missing gene was a key component to the signal pathway. The researchers identified 54 genes that had a significant influence on AHR response. Only two of these genes, termed modifiers, had been previously identified.Signaling pathways usually boil down to a series of discrete steps. To identify steps of the AHR pathway, the researchers constructed a spider web-like map called a "protein interaction network," or PIN, based on previously known interactions between the proteins encoded by the 54 modifier genes. The resulting map revealed groups of highly connected, related modifiers, which the authors proposed to be steps in the pathway. Though other studies have used the newly developed PIN strategy to investigate cellular processes, Bradfield's team also annotated their PIN through a series of experiments both to support the identity of and to better understand the protein groups, referred to as functional modules.With tests based on discrete receptor signaling events, known active structural regions, reaction to different types and concentrations of agonists, and functional location within the cell, Bradfield's team organized the functional modules into five steps. One group of modifiers is involved in AHR folding, the conformational change that occurs when the receptor binds to a toxin. With the help of other modifiers, the new AHR complex is then translocated into the cell nucleus. Once in the nucleus, a series of modifiers assist the AHR in its role as a transcription factor. The researchers also identified a step in the pathway that controls production of AHR itself and another unknown "step" that takes place inside the nucleus.As AHR is thought to be a prototype PAS receptor, understanding the steps in this pathway will likely guide future research on the entire family, allowing scientists to study in detail individual steps in these complex pathways. The highly integrated method reported here could also be used to study most other mammalian signaling pathways, giving scientists a new tool as they attempt to understand how cells respond to their changing environment.


Chronic smoking is the main risk factor for chronic obstructive pulmonary disease. Knowledge on the response to the initial smoke exposures might enhance the understanding of changes due to chronic smoking, since repetitive acute smoke effects may cumulate and lead to irreversible lung damage.We investigated acute effects of smoking on inflammation in 16 healthy intermittent smokers in an open randomised cross-over study. We compared effects of smoking of two cigarettes on inflammatory markers in exhaled air, induced sputum, blood and urine at 0, 1, 3, 6, 12, 24, 48, 96 and 192 hours and outcomes without smoking. All sputum and blood parameters were log transformed and analysed using a linear mixed effect model.Significant findings were: Smoking increased exhaled carbon monoxide between 0 and 1 hour, and induced a greater decrease in blood eosinophils and sputum lymphocytes between 0 and 3 hours compared to non-smoking. Compared to non-smoking, smoking induced a greater interleukin-8 release from stimulated blood cells between 0 and 3 hours, and a greater increase in sputum lymphocytes and neutrophils between 3 and 12 hours.We conclude that besides an increase in inflammation, as known from chronic smoking, there is also a suppressive effect of smoking two cigarettes on particular inflammatory parameters. Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality world-wide, and its prevalence is still rising . In ordeUntil now, only a few studies have investigated acute effects of smoking in humans . UnfortuIn the present study we investigated acute effects of smoking of two cigarettes by healthy intermittent smokers who refrained from smoking nine days before the study period. In this way, temporary effects on the airways due to chronic smoking will probably not affect the acute response to smoke. We assessed the time effects of cigarette smoking on both induction and resolution of the inflammatory response in exhaled air, induced sputum, blood and urine. We hypothesised that smoking of two cigarettes would induce an increase in inflammatory cells and markers within a limited time interval.1) were performed immediately before (baseline) and 1, 3, 6, 12, 24, 48, 96 and 192 hours after smoking and at the same time points in the no smoking period. Sputum was induced at 3, 6, 12, 24, 48, 96, 192 hours after smoking and no smoking. All subjects smoked two cigarettes from the same brand within 30 minutes and were encouraged to inhale deeply . Adequacy of smoke inhalation was verified by the investigator. The working groups sputum induction from the ERS stated recently that sputum inductions should not be repeated within 48 hours to avoid carry over effects [carry over effect. We have analysed the results of the control arm as a separate study in order to investigate the induction and resolution of the inflammatory response generated by repeated sputum inductions [We performed a randomised, two-period cross-over, pilot study. Subjects were randomised into smoking two cigarettes or no smoking. Subjects refrained from smoking during nine days before each study period, verified by exhaled carbon monoxide (CO < 6 ppm) and urinary cotinine (< 25 ng/ml). The time interval between the two study periods varied between 9 to 20 days. Measurements of exhaled CO, exhaled Nitric Oxide (NO), blood sampling and Forced Expiratory Volume in 1 second . Excluded were subjects with: 1) a history of asthma, allergic rhinitis, or allergic eczema; 2) atopy, confirmed by a positive skin prick test; 3) any current respiratory disease, symptoms of cough or sputum production; 4) a respiratory tract infection within the preceding 8 weeks or a nasal infection within the preceding 4 weeks; 5) treatment with glucocorticosteroids within the preceding 8 weeks; 6) use of aspirin, NSAIDs, paracetamol or antihistamines within the preceding 4 weeks. Subjects were asked to avoid places with high environmental tobacco smoke exposure during the study periods. The study was approved by the medical ethics committee of the University Medical Center Groningen, the Netherlands. Written informed consent was obtained from all subjects.Sixteen healthy intermittent smokers were recruited by advertisements in the local newspaper. Intermittent smoking was defined as smoking more than one cigarette a month, but not daily, during the last 6 months. We chose to investigate intermittent smokers because they are able to refrain from smoking for a certain time period (in contrast to most current smokers) and they are used to inhale smoke (in contrast to non-smokers). Included were subjects older than 40 years, with normal lung function . Exhaled CO levels were measured using an infrared CO analyser (UNOR 6N, Maihak AG, Hamburg, Germany) [FEV Society , using a Society , exhalinBlood differential cell counts were analysed automatically with a haematology flow cytometer (Coulter-STKS, Beckman Coulter, Miami, USA). Flow cytometric analysis was performed on blood cells using peridinin chlorophyll protein (PerCP) labelled anti-human leukocyte antigen (HLA)-DR, phycoerythrin (PE) labelled anti-CD11b, allophycocyanin (APC) labelled CD14 and fluorescein-isothiocyanate (FITC) labelled CD63 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ USA). HLA-DR, CD63 and CD11b are activation markers for respectively monocytes and granulocytes. CD14 is used to discern between monocytes and granulocytes. Functional assays were performed on unstimulated and lipopolysacharide  stimulated blood cells, measuring tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8 and IL-10 by ELISA (Sanquin, Amsterdam, the Netherlands).Sputum was induced according to a modified standard technique , using 4Flow cytometric analysis was performed on sputum cells using PerCP labelled anti-HLA-Dr, PE labelled anti-CD11b, APC labelled CD14 and FITC labelled CD63 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ USA). Immunocytology was performed to quantify the percentage of inducible NO synthase (iNOS) positive macrophages. Cytospins were double stained with a monoclonal antibody against CD68 (IgG1 isotype, Dako, Glostrup, Danmark) as a marker for macrophages and rabbit polyclonal antiserum against iNOS (Transduction Laboratories, Lexington, KY, USA).2-/NO3- was measured using the Griess reaction, eosinophilic cationic protein (ECP) using the fluorenzyme immunoassay UniCAP ECP . IL-8 and Leukotriene B4 (LTB4) were measured by a commercial ELISA (IL-8: Sanquin, Amsterdam, the Netherlands, LTB4: Amersham Biosciences, UK). Matrix metalloproteinase-9 (MMP-9) was measured by gelatine zymography [The following soluble mediators were measured in sputum supernatant. NOmography .Before inhalation of smoke (or control), a urine portion was collected to measure urine cotinine. Cotinine was measured by gaschromatography-mass-spectrometry (Pharmacy Department, Groningen, the Netherlands). Furthermore, urine was collected over 24 hours in five consecutive fractions: 0–1 hour, 1–3 hours, 3–6 hours, 6–12 hours and 12–24 hours from all subjects to assess leukotriene E4 levels (ELISA, Amersham Biosciences, UK).Since the start and duration of the acute effects of smoking of two cigarettes on our parameters were unknown, time series of all variables were plotted. Based on visual inspection of these plots the time intervals to be analysed were selected. The slopes of parameters were estimated using linear mixed effect models  by incluClinical characteristics of the 16 subjects are listed in table 1 .In order to better understand the effects of chronic smoking, it is important to study the initial (acute) response to cigarette smoke, since repetitive acute smoke effects may cumulate and ultimately lead to irreversible damage. We therefore investigated the acute effects of cigarette smoking on both induction and resolution of the inflammatory response in healthy intermittent smokers. This study shows that smoking of two cigarettes acutely suppresses blood eosinophils. Furthermore, smoking induces a biphasic response in sputum lymphocytes, after an initial smoke-related suppression, the cells increase more with smoking than without. Finally, smoking increases sputum neutrophils and the release of IL-8 from whole blood cells.A remarkable finding in our study is that smoking of two cigarettes decreases eosinophils in blood. Three other studies have reported similar results: eosinophils decreased in blood from healthy female smokers within two hours after smoking 12 cigarettes , and in This study is the first to report that sputum can be used to study acute smoke effects. The number of sputum neutrophils increased between 3 and 12 hours after smoking. In line with this, we demonstrated a higher release of IL-8 by LPS stimulated blood cells after smoking, which may have contributed to increased neutrophil chemotaxis. The rise in neutrophils is in line with two studies on acute effects of smoking in humans, showing increased neutrophils in bronchoalveolar lavage fluid 1 hour after smoking  and incrSmoking also shortly suppressed the number of lymphocytes in sputum. Thereafter sputum lymphocytes increased more with smoking than without. The initial decrease might result from increased adherence of lymphocytes in the lung tissue due to the fast upregulation of adhesion molecules after smoking  or may aSmoking did not affect all inflammatory markers we investigated. A few factors may contribute to this lack of response. First, the number of subjects and the number of cigarettes (n = 2) might have been too low. Second, we may have included a heterogeneous group of subjects regarding their response to cigarette smoke. We know that approximately 80% of all smokers never develop COPD. Therefore it is conceivable that a part of our healthy smokers does not respond to cigarette smoking. Third, we included subjects with a broad range in current and past smoking. Fourth, sputum may reflect only a part of the acute inflammatory changes of the airway wall [Studying the acute effects of smoking in intermittent healthy smokers has both advantages and disadvantages. We choose the presented model for a number of reasons. First, intermittent smokers can refrain from smoking for three weeks in contrast to most current smokers. Second, intermittent smokers have a normal lung function (in contrast to COPD), and likely (nearly) no structural airway changes, which may affect a normal response to cigarette smoke. Third, we assumed that detecting an acute inflammatory response to cigarette smoking after an abstinence period of 9 days would be easier than detecting a response on top of chronic smoke exposure. Finally, intermittent smokers are used to inhale cigarette smoke (in contrast to non-smokers). We realise that our model has the disadvantage that the results of our study cannot easily be extrapolated to the chronic effects of smoking or COPD development. Nevertheless, when comparing the airway inflammation of our subjects with that of smoking COPD patients, both show increased levels of neutrophils, lymphocytes and IL-8 in sputum. However, in COPD patients after quitting smoking lymphocytes and neutrophils do not normalise , in contWe conclude that besides an increase in inflammation, as known from chronic smoking, there is also a suppressive effect of smoking of two cigarettes on particular inflammatory parameters. Although this seems beneficial, it may disturb physiologic responses, like repair processes, in which inflammatory cells play a role.The author(s) declare that they have no competing interests.HV: Participated in the design of the study, performed the study and drafted the manuscript.DP: Conceived the study, participated in the design and co-ordination of the study and helped draft the manuscript.WT: Participated in the design of the study and helped draft the manuscript.MH: Participated in the design of the study, co-ordinated the FACS analyses and helped draft the manuscript.BW: Performed some of the laboratory analyses, participated in the design of the study and helped draft the manuscript.HB: Performed statistical analyses and helped draft the manuscript.JV: Performed statistical analyses and helped draft the manuscript.DR: Performed and co-ordinated most laboratory analyses and helped draft the manuscript.HK: Co-ordinated laboratory analyses, participated in the design of the study and helped draft the manuscript.NH: Conceived the study, participated in the design and co-ordination of the study and helped draft the manuscript.9/L) after smoking and no smoking. Table Table Click here for file