Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. The aqueous humor (AH) is a clear fluid that fills the anterior segment of the eye and bathes the lens, iris, and corneal endothelium . It is sPrevious AH proteomic studies were limited by a number of factors including interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. In 1998 Rohde et al.  used a pThe study subjects were patients who were scheduled to undergo standard cataract surgery at a tertiary referral center, Price Vision Group . Exclusion criteria were as follows: previous intraocular surgery, history of conjunctivitis or any ocular infection within the previous 3 months, intraocular inflammation, or any eye disease. An independent Institutional review board (IRB) approved the study and all subjects signed a written Informed Consent document. Before undergoing cataract surgery, the patient's eye was anesthetized topically with proparacaine. A stab incision was made in the peripheral cornea, and 0.1 to 0.2 ml of anterior chamber fluid was aspirated using a 30-gauge needle. Aqueous humor samples were stored frozen in liquid nitrogen until analysis. A single surgeon (F.W.P.) collected all the samples. Any sample suspected of being contaminated with blood or iris pigment was discarded. Samples from 12 subjects were analyzed; 50% were from females, mean age was 65±6.3 years, 11 were Caucasian, and 1 was Asian .The following sample preparation and mass spectrometric analyses were carried out at Monarch LifeSciences . All reagents and chemicals were purchased from Sigma-Aldrich  unless indicated otherwise. AH samples were pooled and fractionated into bound and flow-through (depleted) fractions using Sigma-Aldrich ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit. The fractionation was performed via manufacturer’s procedures. Protein concentration for each fraction was determined by the BCA protein concentration assay . The albumin-depleted fraction contained 116 µg, and the albumin-bound fraction contained 221 µg of protein. Each fraction was denatured with 8 M urea, reduced with 10 mM dithiothreitol (DTT), and alkylated with 55 mM iodoacetamide. Urea concentration was lowered to 1.6 M with 10 mM Ammonium bicarbonate (pH 8). Trypsin  was prepared by adding 100 µl of ice cold 0.1 M ammonium bicarbonate to the trypsin vial to make a 1µg/µl solution, then added in a 1:20 (trypsin:protein) ratio and incubated overnight at 37 °C. Peptides (100 µl) were precipitated with 800 µl of diethylether and 400 µl of methanol to remove salt. Then, each sample was dissolved in 0.1% formic acid, and 40 µg of protein digest was loaded onto SCX column  at 3 µl/min with an Agilent 1100 high pressure liquid chromatography (HPLC) system . Peptides were eluted with a 2.5 mM to 250 mM NaCl gradient for 2 hr collecting fractions every 8 min, thus yielding 16 fractions by strong cation exchange chromatography. Each of these 16 fractions for both the albumin-bound and albumin-depleted fractions was dried and resuspended in 40 µl of 0.1% formic acid. Columns were equilibrated with 3% acetonitrile (ACN) and fractions were loaded onto the trapping column at 5 µl/min. Salt was washed away for 15 min with 3% ACN before peptides were injected onto the C18-reversed-phase column. Zorbax C18  resin was used for both the trapping column  and the analytical column  and were self-packed in house. Peptides were eluted with a linear gradient from 5 to 45% ACN over 120 min at room temperature, at a flow rate of 500 nl/min, and the effluent was electro-sprayed into the LTQ mass spectrometer  with a nanospray source. The source was operated in positive ion mode with 3.0 kV electrospray potential, a sheath gas flow of 15 arbitrary units, and a capillary temperature of 200 °C. The source lenses were set by maximizing the ion current for the 2+ charge state of angiotensin. Data were collected in the triple play mode. Maximum ion time was 200 msec for the full scan and 500 msec for the zoom scan and the MS/MS scan.X!Tandem algorithms and the human protein database from IPI (version 3.48). Parameters were set as follows: a mass tolerance of 2 Da for precursors and 0.7 Da for fragment ions, two missed cleavage sites allowed for trypsin, carbamidomethyl cysteine as fixed modification, and oxidized methionine as optional modification. The q-value represents peptide false identification rate and was calculated by a recently published method [GO) tools available at. The Swiss-Prot accession numbers listed in The acquired data were filtered by a proprietary algorithm that was developed by Higgs, et al.  and descd method  which ind method  with priAppendix 1. The Swiss-Prot protein accession number, protein common name, and molecular weight are provided in A statistical summary provided in A representative tandem mass spectrum corresponding to a peptide (FDEFFSEGCAPGSK) of the protein transferrin is illustrated in Rather than focus on differentially expressed proteins in the AH of patients with a particular ocular pathology, we chose to concentrate our efforts on a thorough investigation of the human AH proteome isolated from patients who did not have any significant health problems other than cataracts, which are just part of the normal aging process. We utilized albumin depletion to remove the known interfering high abundant protein and associated proteins and therefore increase lower abundant protein identification. We also incorporated 2D-LC-MS/MS to further fractionate samples and facilitate identification of lower abundant proteins. By doing so, we were able to overcome obstacles of previous AH proteome analyses and provide the highest confidence information to date on the protein composition of healthy human aqueous humor in a population of aged adult subjects.In 1998 Rohde et al.  attempteIn 2005, Funding et al.  used twoO. cuniculus) proteins with the rest being identified as other various mammalian proteins. This limited the analysis for two reasons: 1) the rabbit is not always useful as a model of human ocular pathology; for example the rabbit corneal endothelium regenerates unlike that in humans [In 2007, Stastna et al.  employedn humans , and 2) In 2008 Duan et al.  used 2-DGO Term Finder” tool for function and component GO terms revealed several interesting findings. The component analysis revealed that almost half of the proteins identified were predicted to be extracellular space proteins and that proteins associated with several different lipoprotein particles were present in the AH, the relevance of which is discussed below. Regarding function, many AH proteins were found to be involved in lipid transport and binding and in endopeptidase inhibition. A manual search of the literature detailed below has revealed a potential role for several of these proteins in a variety of physiological functions of AH, many of which were identified for the first time here.Gene ontological analyses performed using the “Generic Lipoproteins in the AH are thought to provide a significant source of lipids for avascular tissues such as the lens in which de novo cholesterol synthesis is considered insufficient to meet even 50% of its total cholesterol need ,24. We iThe roles of several other proteins identified here have already been purported in the AH. For example, though the exact role of Glutathione Peroxidase in the AH is unclear, it is known to protect cells and other proteins from oxidative damage and most likely serves a similar function in the AH . ProstagAnti-angiogenic proteins are key molecules in the AH as there are several tissues that must remain avascular to maintain their function such as the cornea which serves as a “clear window” to the eye. Indeed, it is known that the AH suppresses angiogenesis , but theIn this study, we employed multiple prefractionation techniques and tandem mass spectrometry to identify lower abundant proteins and extensively characterize the human AH proteome. These multi-peptide, high-confidence, priority 1 proteins, widely regarded as definitive identifications, provide a reliable foundation and should enable the generation of new hypotheses regarding pathologies of the eye as well as normal AH function. Though these IDs should be considered definitive, it is still possible that there were AH proteins not identified with high confidence in this study. Proteins identified at priority levels 2-4 could be considered candidates for future investigation in this regard. The exact number of human AH proteins is unknown, and it is possible that tens if not hundreds of lower abundance proteins exist in the AH and many of these could fall below current detection limits. Nevertheless, based on previous investigations of AH and other body fluids, the AH would not be expected to contain as many proteins as a typical cell lysate. Other areas for future study include determining differences in AH protein levels between patients in different age groups with different ocular pathologies, as well as differences between men and women; aspects that could not be addressed in the current study.


The study was designed to establish relationship between the plasma concentration and QTc interval prolonging effect of fexofenadine and demonstrate the phenomenon of anticlockwise hysteresis.Six subjects were given fexofenadine 60 mg tablet orally under stable conditions, and their drug concentrations were measured at regular intervals. At predetermined time, their ECGs were recorded. Data were analyzed and plotted graphically.Randomized parallel design, single group study conducted at clinical research organization of Ahmadabad.max) was around 3 h and the value of average maximum plasma concentration was 460.63 ng/mL, the effect of fexofenadine on the heart  was maximum (Emax) after 6 h and average QTc interval was 469.75 ms. Thus, the time to maximum concentration of fexofenadine did not match with the maximum effect on the heart as measured by QTc interval.In all subjects time taken for maximum plasma concentration of fexofenadine (TThe relationship between the drug concentration and drug effect on the heart are at two different time scales. It can be understood by two-compartment model of pharmacokinetics, and this retardation or lagging of an effect behind the concentration is known as hysteresis. The increase of QTc was not beyond 500 ms and not sustained, demonstrating overall cardiac safety of fexofenadine. Significant cardiac toxicity has been associated with some older antihistamines  when their plasma concentrations are increased, due to which some of them were withdrawn from the market. There is24Results of clinical safety and efficacy trials with fexofenadine HCl doses up to 240 mg twice daily in patients with seasonal rhinitis have further demonstrated its safety, indicating a large therapeutic window for this drug. The drug7This study was undertaken to assess the relationship of plasma concentration of fexofenadine with the QT interval prolongation.The QT interval represents the duration of ventricular depolarization and subsequent repolarization, beginning at the initiation of the QRS complex and ending where the T-wave returns to isoelectric baseline. While thThis study was conducted in the Pharmacological Department of Synchron Research, a clinical research organization of Ahmedabad. Ethics Committee approval was obtained for this study by an Institutional Ethics Committee. During the course of a routine bioequivalence study, there were six subjects, who consented to the additional measurement of QT intervals by periodic ECG.These subjects were between the ages of 21–40 years with weight of >50 kg [but within the range of the LIC (Life Insurance Corporation of India) height–weight chart], heart rate 60–90 beats/min; normal body temperature, respiratory rate (RR) 15–25 breaths/min, systolic blood pressure (SBP) 100–130 mmHg, and diastolic blood pressure (DBP) 50–90 mmHg.Informed consent was obtained as per ICH–GCP guidelines. The subjects tested negative for drugs of abuse and had routine laboratory investigation values within normal range. The subjects were housed in the department on the previous day of the study, till end of study on the next day. They were free from any renal, hepatic, or cardiac diseases.The subjects were relaxed and provided standard food, but were asked to abstain from caffeine, tobacco, and alcohol at least 24 h before the vital parameters were recorded . This was followed by ECG recording. Consistent operator technique was maintained by ensuring skin preparation, correct leads placement, subject position, and data acquisition practice.ECG was recorded with validated electronic device, “NASAN-ECG1997” with paper speed 25 mm/s, calibration 10 mm/mv.The ECGs were recorded by an inbuilt software programme, which also calculates QTc by Bazett’s formula (QT/square root of R–R) where QTc is the rate corrected in milliseconds and R–R interval in seconds.Blood (9 mL) was sampled from antecubital or cubital veins and collected into sodium citrate-containing tubes at 0, 1, 2, 3, 4, 6, 8, and 10 h after the administration of each fexofenadine tablet formulation (180 mg). The blood samples were centrifuged at 2500g for 10 min at 4°C, and the separated plasma was collected and stored at −20°C until drug analysis.All plasma samples were analyzed for fexofenadine concentration according to a sensitive, selective, and accurate high-performance liquid chromatography (HLPC) method, which was developed and validated before the study. The chromatographic separations and quantitative determination were performed using a high-performance liquid chromatograph from E. Merck, equipped with a degassing unit, quaternary pump, autoinjector, UV detector, and controlled by Merck software.2id.; 5 μm particle size) HPLC column. The mobile phase consisted of acetonitrile and potassium dihydrogen phosphate . The mobile phase was eluted at a flow rate of 1.4 mL/min at 52°C. UV detection of fexofenadine and the internal standard (cetirizine) was performed at 195 nm. Each analysis required a maximum of 13 min. The calibration curves were linear over a range of 10–1000 ng/mL using 1.0 mL plasma samples. All samples from each volunteer were measured on the same day in order to avoid inter-assay variation.Chromatographic separation was performed using a C18  in humans.3This lack of propensity to cause QTc irregularities has been confirmed in two long-term studies in volunteers. Fexofenadine 60 mg twice daily for 6 months or 240 mg once daily for 12 months had no significant effect on QTc relative to placebo.12Cmax) reaches in 3 h after oral administration of fexofenadine tablets, and the maximum effect on heart (Emax) as QTc interval prolongation takes 6 h after oral administration of the drug, so the cause of this time lag can be explained by a two-compartment model.[The maximum concentration of fexofenadine nt model.14Cmax react-test, comparing mean of drug concentration in plasma and mean of QTc interval; P value was found to be 0.028 (<0.05). Thus, a statistically significant relationship existed between these two parameters.When this data were analyzed by paired Vd(5.4–5.8 L/kg).[Fexofenadine has high volume of distribution; .8 L/kg). In the c.8 L/kg).Although fexofenadine is relatively safer, the clinical significance of the hysteresis phenomenon seen in the case of fexofenadine is that the monitoring of drug concentration level in plasma will not be helpful in deciding its effects and ECG monitoring for such drugs affecting QTc interval should be done ideally between 4 and 8 h after dosing in individuals at risk.Hysteresis may be defined as “the retardation or lagging of an effect behind the cause of the effect.” The two main reasons for the lag phase are limited access to the site of drug action or slow receptor kinetics. Both these characteristics produce an anticlockwise hysteresis, in which time moves anticlockwise in the change in the relationship between plasma concentration and observed effect with time. A clockwEmax(6 h) is in agreement with related studies in literature.[The values of terature. The resu


KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (<50 years) and old patients (>70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients.The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in  The incidence of colorectal cancer  has increased in the western world including Norway during the last 50 years KRAS (HGNC:6407), BRAF (HGNC:1097), and PIK3CA (HGNC:8975) and the tumor suppressor gene PTEN (HGNC:9588) are all affected in response to cytokines, growth factors and hormones signaling through RTK.Receptor tyrosine kinase (RTK) signaling is essential for maintaining the metabolism, proliferation, survival and motility of a cell KRAS and BRAF have been shown to have activating mutations in ∼70% of CRCs PTEN and PIK3CA, mutations result in constitutive activation of the PI3K pathway by accumulation of phosphatidylinositol  triphosphate (PIP3), which then catalyzes the phosphorylation of AKT. The activated serine-threonine kinase AKT regulates a broad range of target proteins involved in a variety of downstream signaling pathways TP53 (HGNC:11998), a transcription factor that integrates information from many different types of cellular stress, and execute downstream responses appropriate for the given input TP53 is found mutated in about half of all colorectal carcinomas Both KRAS and TP53 status has been associated with clinical end points, the former only with weak association in larger series and the latter only if subgroups of mutations are considered PIK3CA mutations may carry prognostic information in tumor stages I–III PTEN mutations.MSI status in the primary tumor has prognostic impact in CRC patients KRAS and BRAF are not mutated KRAS and BRAF not all respond to this therapy PIK3CA and PTEN, i.e. only quadruple mutation negative tumors will respond to treatment The metastatic disease treatment targeting EGFR is found to be efficient only if KRAS, BRAF, PIK3CA, PTEN, and TP53 in tumor samples from patients younger than 50 years at diagnosis and without known hereditary syndromes, and stratified according to MSI status and elderly CRC patients.In the present study we have compared the DNA sequence mutation status of the genes Clinicopathological data for the whole series (n = 181), divided into three age groups, are presented in The MSI- and mutation-frequencies of the analyzed genes are presented in KRAS, affecting codons 12, 13 and 61. Twenty-eight tumors had the BRAF V600E change. Eight PIK3CA mutations were identified in exons nine and twenty. Fourteen and fifteen tumors had PTEN mutations and deletions, respectively. Ninety-three tumors had TP53 mutations. Details of all mutations observed, can be found in In total, nine different mutations were observed in BRAF mutations were statistically significantly correlated (P<0.001). No such correlation was found in the <50 age group. The MSI tumors within the <50 age group, the 51–70 age group and the >70 age group had 17%, 54% and 79% BRAF mutations, respectively (P = 0.04). Furthermore, among the six MSI tumors in the <50 group only half of the samples carried mutations in the genes analyzed, whereas among MSI tumors in the other two age groups, all samples displayed one or several mutations.The relationships between the clinical and mutational parameters within each age group are given in KRAS mutations were found in 57 (32%) tumors in the total series. The mutation frequencies were slightly different between the age groups, KRAS and BRAF mutations were mutually exclusive in the total series. The frequency of tumor mutations in either KRAS or BRAF increased with patient age, both when comparing age groups and with age as a continuous variable.PIK3CA mutations, whereas five (7%) and three (4%) were found in the 51–70 group and >70 group, respectively.Surprisingly, none of the tumors in the <50 age group had PTEN mutations were identified in seven of nine exons (no mutations were observed in exon 4 and 9). The mutations were observed in all age groups, as was the case for PTEN deletions. Deletions detected by MLPA were found in eight percent of the samples in total, and coexistence of deletion and alterations detected by sequencing were found in three tumors. Overall, 14% of the tumors had indication of impaired PTEN function. The frequency of PTEN aberrations showed no statistically significant difference between the age groups; 18%, 10% and 16% in <50 group, 51–70 group and >70 group, respectively.Eighteen TP53 mutated tumors differed among the three age groups (P = 0.02). This difference was also significant when analyzing MSS tumors only, (P = 0.03). Mutations were most prominent in the DNA binding domain, encoded by exons 5–8, and only about five percent of the mutations were observed in exon 4 and exon 11. Moreover, in the <50 age group, TP53 mutations and MSI were mutually exclusive, and inversely correlated in the 51–70 and >70 groups, respectively.The number of For a subset of the samples (n = 41) a high resolution dataset of copy number changes obtained by array comparative genome hybridization data (aCGH) was available  P = 0.08), and the younger patients tended to have a more advanced tumor stage than elderly patients. The prevalence of stage III–IV tumors was 54%, 46%, and 36% in the <50, 51–70 and >70 groups, respectively.No difference in survival was found comparing the age groups, even when adjusting for tumor stage at diagnosis. There was a trend that the proportion of rectal tumors decreased with increasing age (P<0.001). In univariate analysis, patients with TP53 mutated tumors had poorer survival rates than patients with wild-type TP53, 938±31 days vs. 1016±23 days (P = 0.04), respectively. However this difference was not significant when correcting for tumor stage. TP53 mutations were of higher prognostic significance in right-sided tumors, 1051±26 days vs. 883± 65 days for wild type and mutated TP53, respectively (P = 0.005).In the total sample set, tumor stage was the most powerful prognostic variable with regard to three-year overall survival, , respectively. None of the other mutational parameters showed any statistically significant difference with regard to survival, neither within nor between age groups.Among patients in the youngest age group, those with We found that the mutation status of a known cancer gene set varies in CRC patients depending on age at onset of disease, and provide further evidence of differences in the somatic development of tumors in young and elderly patients.In all age groups, MSI was statistically significantly associated with right-sided tumors, as expected BRAF mutations were not statistically significantly associated with MSI in the <50 years group, contrary to what was expected BRAFV600E is observed to induce down-regulation of essential DNA repair genes BRAF mutations are not observed in tumors from patients with Lynch syndrome BRAF wild-type samples in the present series are from clinically undetected Lynch syndrome patients.In patients younger than 50 years only 50% of the MSI samples were mutated in one or more of the genes analyzed, whereas in patients older than 50 all MSI samples displayed gene alterations. Additionally, KRAS, BRAF, PIK3CA, PTEN, and TP53 were found to be within previously reported range supporting the representativeness of the series Mutations frequencies in BRAF and KRAS mutations is presumed to be incompatible with proliferation, hence a negative selection for concurrent existence of both mutations KRAS or BRAF mutation was observed with increasing age, in agreement with a recent report on young patients with colorectal cancer The coexistence of BRAFV600E were typically TP53 wild type and PTEN mutated  which might indicate a time difference in tumor development.i.e. a higher degree of genomic complexity. Contrary, as mentioned above, the tumors from elderly patients had the highest gene mutation index, even though this group included fewer advanced stages. Taken together, one may speculate that among the young patients there are carriers with genetic predisposition affecting genes encoding proteins that ensure correct chromosome segregation during mitosis, rather than specific pathways.From aCGH data on a subset of 41 samples, we observed that patients younger than 50 years had significantly more aberrations in their tumors than patients >70 years did, et al.KRAS-BRAF-PIK3CA-PTEN gene set have the highest probability of response to anti-EGFR therapies. However, currently only KRAS testing is recommended prior to decision with regard to such treatment, and BRAF testing is optional Bardelli PTEN and/or mutated PIK3CA exclusively, the youngest age group displayed 16% (n = 7) mutations, compared to 1.4% (n = 1) in the oldest age group. This implies that for older patients, KRAS and BRAF mutation status alone will reveal 98% of the patients unsuitable for anti-EGFR therapy, as compared to 71% in patients <50 years. The effect of mutations in PIK3CA and PTEN are debated with regard to anti-EGFR therapy In the present series of primary cancers, mutation frequencies in these four genes were equally distributed between all tumor stages. Therefore, we have in the following hypothesized the number of potential responders based on the whole dataset . When coThe consecutive sample series of colorectal carcinomas from a single hospital showed overall distribution of clinicopathological data as expected for a Norwegian cohort TP53 is still controversial TP53 mutations had significantly shorter survival than wild type patients. Furthermore, for patients with right-sided or MSI tumors TP53 mutations showed even greater prognostic value, in line with results reported by the TP53-CRC collaborative study group The prognostic and predictive value of PIK3CA mutations were found to serve as surrogate markers for poor survival in colorectal cancer patients PIK3CA mutations were observed in our sample set, we could not confirm these results. However, mutations were found only in samples from older patients, and were not present in patients with distant metastases, and only few in stage III patients. Thus, our data suggest that PIK3CA mutations are important for tumorigenesis among elderly patients, but do not serve as a marker for the ability to metastasize.In a recent study, We have found distinct differences in the genetic make-up of carcinomas from young and elderly patients. The tumors from young patients whom are not carriers of known hereditary CRC syndromes have less gene mutations, but more copy number aberrations across the genome. These data suggest that some young patients may have a potential increased risk for cancer caused by alterations in gene(s) involved in maintaining correct chromosome segregation.Written informed consent was obtained from all subjects included. The research biobanks are registered according to national legislation and the research studies are approved by the Regional Committee for Medical Research Ethics .i.e. a consecutive series of 132 patients enriched with a series of 49 patients with disease at young age (<50 years). Each series are described in the following paragraphs. Clinical data were collected prospectively. Death data were retrieved from either the hospital records or from the Norwegian Population Registry. The clinical and pathological data of these two series are shown in A total of 181 patients were included in the study, Patients undergoing elective resection of colorectal adenocarcinoma in 2005–2007 at Oslo University Hospital, Aker, Oslo, were included. Patients with FAP- or Lynch syndrome were excluded based on clinical criteria. Tumor tissue was sampled immediately after resection of the specimen and instantly frozen in liquid nitrogen. All tissue specimens were evaluated by a pathologist to establish differentiation grade of the carcinoma and tumor cell content.Samples from a biobank from a multi-hospital study , including Oslo University Hospital, Aker, Oslo and five other hospitals in the same geographical region of Norway enrolled from 2003–2008, were included in the present study. The inclusion criteria in the INFAC-study were age <55 years, excluding those who fulfilled criteria of all known CRC syndromes. Tumor tissue and corresponding normal mucosa samples were taken from the resected specimen in the operation theatre and promptly incubated in RNAlater. The tissue was transferred to new tubes after 36–72 hours, and stored at minus 80°C until use. DNA from normal and cancerous tissues was extracted, and used for analysis.For a subset of the patients (n = 41) data from aCGH analyses were available. This subset included 24 patients younger than 50 years, and 17 older than 70 years at primary diagnosis. Clinical and mutational data for these patients are presented in Patients from the early onset series were included to enrich the number of early onset patients in the total sample set. Patients from the early onset series were selected to match patients younger than 55 years in the consecutive series with regard to gender, median age, tumor stage and localization, et al.For all patients, tumor and corresponding normal tissue or blood was analyzed using the Bethesda markers BRAF and KRAS, activating mutations at positions known to frequently carry alterations are reported PIK3CA gene mutations in colorectal cancers cluster in exons 9 and 20 TP53 and PTEN mutations are reported throughout the whole coding sequence For TP53 and PTEN, the total coding sequence was amplified in multiplex PCR reactions, using multiplex PCR kit as recommended by the vendor  PIK3CA, KRAS and BRAF amplicons PIK3CA exons 9 and 20 were amplified, in KRAS, exons 2 and 3, including the frequently mutated codons 12, 13 and 61, and in BRAF exon 15, including the mutation in codon 600, were amplified. Primers, running conditions and fragment details are described in For PCR products were purified using Sephadex columns  prior to incorporation of dye labeled ddNTP's and sequencing on the 3730 DNA Analyzer  PTEN gene, a minimum of two probes for each of the nine exons. Also, 10 probes located elsewhere on chromosome 10, and additional 12 reference probes located on other chromosomes serves as controls. Amplified fragments were analyzed on a 3730 DNA Analyzer (Applied Biosystems). Raw data were analyzed with Coffalyzer® (MRC Holland) with default settings. Cutoffs of 1.2 and 0.8 were used for scoring of gains and losses, respectively, in addition to individual evaluation of the plot from each sample. This scoring is determined according to the following calculation: one copy gain or loss in 60% of the cells in a triploid tumor equals 1.2 and 0.8, respectively. At least two neighboring probes, located in the same exon, with concomitant gain or loss, were confined for assigning aberrant copy number in a region.Salsa MLPA kit P225-B2 PTEN  were used according to instructions from distributor. In total, 25 probes cover the P<0.05 was considered significant.Clinical variables were tested for associations with mutational results for all genes analyzed, and all genes were tested for inter-relational associations. All statistical analyses were performed using the software package PASW 17.0 . Fisher's exact test was used for cross-tabulated variables. Student T-test was performed when calculating the relationship between a continuous variable and a categorical variable. Survival analyses were performed using the Kaplan–Meier life table analyses with three years overall survival, and log rank test used to compare the survival curves. For all statistical tests, a two-tailed Table S1Relation of mutational and clinical data on tumors from included patients. NS = not significant, NA = not applicable. All P-values <0.20 are shown in the table, and P<0.05 is high-lighted in bold.(0.03 MB XLS)Click here for additional data file.Table S2KRAS, BRAF, PIK3CA, PTEN and TP53. * F = female, M = male. # patients included in Details of clinical and mutational data from (0.05 MB XLS)Click here for additional data file.Table S3Clinical characteristics in different age groups. Distribution of clinical data in the total consecutive series, young in consecutive series (patients under age 55 years), early onset series (<55 years), and in the total series of patients.(0.03 MB XLS)Click here for additional data file.Table S4TGTAAAACGACGGCCAGT). #M13 reverse primer (CAGGAAACAGCTATGACC).PCR and primer details for genes analyzed. *M13 forward primer ((0.03 MB XLS)Click here for additional data file.


The hypogonadal (hpg) mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland.Initially, Western blot and immunohistochemistry were used to analyse tissues from hpg mice to identify potential sites of action of estradiol. In the main study, hpg mice were treated for 50 days with either an estradiol implant or daily injections of recombinant human FSH, or a combination of both, to determine whether estradiol would have an additive or synergistic effect with FSH on testis development, as assessed by histological analysis and stereological quantification of Leydig, Sertoli and germ cell proliferation.Western blot analysis revealed ERα immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERα in nuclei of anterior pituitary cells and Leydig and peritubular cells in hpg mice. Histological and morphometric analyses revealed that estradiol treatment alone was as effective as FSH in promoting Sertoli cell production and proliferation of the seminiferous epithelium, resulting in the production of elongating spermatids. Combined estradiol and FSH treatment did not produce a greater effect than either treatment alone, though an increased dose of FSH significantly increased seminiferous tubule volume and testis weight and increase Sertoli cell numbers further within the same time frame. In contrast, estradiol caused substantial increases in the wet weight of the seminal vesicles, whereas FSH was without effect on this tissue, and did not augment the actions of estradiol.hpg mice is predominantly via the stimulation of pituitary FSH release.As ERalpha receptor is abundantly expressed in the pituitary gland of hpg mice, and estradiol did not exert effects on testis development over and above those of FSH, we conclude that the action of estradiol on testis development in  Traditionally, the regulation of mammalian spermatogenesis has been viewed as being dependent upon the interactions of gonadotrophins and androgens, however, recent observations in experimental rodents and man have indicated additional roles for estrogen in this process ,2 for re for re2 hpg) mouse [Hpg mice are infertile because they do not produce gonadotropin and, in consequence, testis development does not proceed beyond the neonatal stage [hpg mice with LH can stimulate steroidogenesis [hpg mice [hpg mice or whether the effects of estradiol in inducing spermatogenesis are more indirect, occurring via the rise in FSH production.We and other research groups have exploited the hypogonadal (g) mouse , which cg) mouse ,10. Hpg al stage . It is wogenesis  and treaogenesis ,14. Howehpg mice ,15,16, thpg mice, in order to identify putative sites of estrogenic action in promoting spermatogenesis in this mutant strain. The main objective was to measure the qualitative and quantitative effects of estradiol on testis development and spermatogenesis in hpg mice in the presence and absence of rhFSH. If combined estradiol and rhFSH treatment proved to be more effective than either of these treatments alone (i.e. there was a synergistic effect), we would conclude that these hormones exert separate actions. If, however, combined estradiol and rhFSH treatment were to be only as efficacious as would be expected from a higher rhFSH dose (i.e. an additive effect) then we would conclude that estradiol most likely acts via stimulation of FSH secretion.This aim of the study was to investigate the relative contribution of direct (testis) and indirect (pituitary) actions of estradiol in stimulating spermatogenesis. The initial objective of the study was to use immunohistochemistry and Western blotting to localise ERα in the pituitary gland and testis of hpg male mice were identified by their micropenis, reduced anogenital distance and small scrotal sac.All animal procedures were approved by the University of Nottingham Local Ethical Review Committee and carried out in accordance with the Animals Scientific Procedures Act (UK) 1986 (project licence PPL 40/2372). Laboratory Animal Science Association (LASA) guidelines were followed for administration of substances . Adult hhpg and age-matched littermates mice (approximately 135 days old) were placed in phosphate-buffered saline at pH7.4 (PBS) containing a protease inhibitor. Tissues were sonicated on ice for 3 × 10 sec, centrifuged at 1300 RPM for 5 min and the supernatant extracted and stored at -20°C until used. Testis and pituitary protein were incubated at 100°C for 5 mins in the presence of 5% mercaptoethanol. Samples were then immediately cooled on ice and loaded onto the gel. Protein samples were separated on a 10% SDS gel and transferred to a nitrocellulose membrane (Amersham). Membranes were blocked with 5% milk (Marvel) in PBS at 25°C for 1 hour before incubation overnight at 4°C with a polyclonal antisera raised in rabbit recognising ERα . After overnight incubation membranes were washed in PBS, then PBS/Tween 20, and subsequently incubated with 1:2000 anti-rabbit IgG peroxidase conjugate . Visualization of the protein was achieved using the supersignal system . The molecular weight of the protein was estimated by comparing its mobility with standard marker proteins of known molecular weights. The relative amounts of the protein indicated by the intensity of the bands was evaluated by analysing scans of the film and performing densitometry using the NIH Image computer package.Pituitary glands and testes from Testis and pituitary tissue was fixed by immersion in 4% paraformaldehyde, then embedded in paraffin wax so that 5 um sections could be cut on a microtome. Mounted sections were then placed in xylene to remove the paraffin wax, and placed in decreasing concentrations of ethanol and then tap water to rehydrate the tissue. The pituitary and testis sections were subject to antigen retrieval in 0.01 M citrate buffer pH6 by microwaving at full power (650 W) for 2 × 5 mins. For the studies on estrogen receptor distribution, sections were washed in phosphate buffered saline (PBS) buffer and blocked for endogenous peroxidase with 3% hydrogen peroxide. Following rinsing in PBS, samples were blocked with normal goat serum  then incubated overnight at 4°C with the previously validated rabbit polyclonal ERα antiserum  at a dilution of 1:50. Sections were washed in PBS and incubated with a biotinylated anti-rabbit IgG at 25°C for 1 hour followed by an avidin biotin horseradish peroxidase complex . Immunoreactivity was revealed by incubation with diaminobenzidine (DAB) and the reaction stopped by immersing sections in tap water. Sections were counterstained with eosin. After dehydration through increasing concentration of ethanol, sections were mounted using DePeX mounting medium .For the studies on where the expression of the steroidogenic enzymes 3β hydroxysteroid dehydrogenase (3βHSD) was used to identify Leydig cells, a similar protocol was followed but omitting the pre-treatment with 3% hydrogen peroxide. Sections were incubated overnight in a polyclonal rabbit anti-3βHSD antiserum (dilution of 1:1000) donated by Prof Ian Mason, University of Edinburgh, and previously validated for use in rodents ,19. Secthpg mice ranging in age from 3–7 months were implanted with either a subcutaneous capsule containing 2% estradiol (n = 11) or cholesterol (n = 15) as previously described [hpg mice bearing estradiol implants were then treated daily with 1 IU rhFSH (n = 6) or vehicle (n = 5) for 50 days. Correspondingly, subgroups of hpg mice bearing cholesterol implants were treated daily with 1 IU rhFSH (n = 5) or 5 IU rhFSH (n = 6) or vehicle (n = 5) for 50 days. The doses of rhFSH were based on those used in a previous dose-response study in hpg mice and were designed to produce a submaximal (1 IU/day) and a maximal (5 IU/day) effect [Under general anesthesia, male escribed . Subgrou) effect . On expeThe primary morphometric aims were to estimate volumes of seminiferous tubules and seminiferous epithelium within testes. To this end, stereological estimates ,21 were v). To this end, we obtained estimates of section thickness  and the mean diameters  of nuclei for each cell type. To estimate the mean nuclear diameter, the diameters of 10 randomly selected nuclei were measured. On the explicit assumption that each cell contains a single nucleus, the total number of specific cell types per testis (N) was then calculated fromQuantitative assessment of Leydig and Sertoli cell numbers was made in order to obtain relative measures of net cell proliferation. Estimates were obtained by randomly superimposing a forbidden line unbiased counting frame  on enlartestis represents testis volume (cm3) was calculated by dividing testis mass (g) by tissue density. The latter was taken to be 1.05 g/cm3 [where V05 g/cm3 .Serum FSH concentrations were measured using both a commercially available human FSH ELISA kit (IDS Ltd UK) to determine the levels of recombinant hFSH circulating, and a rat FSH immunoradiometric kit (IDS Ltd UK) in order to estimate the total circulating FSH concentrations (i.e. endogenous and exogenous). FSH concentrations in extracts of mouse pituitary gland, or in untreated wild-type mouse serum, did not dilute in parallel with the rat standards, and there was substantial cross reactivity of rhFSH in the rat IRMA (1 mIU rhFSH reading as approximately 1.5 ng/ml of the rat FSH standard) so all experimental samples were compared at the same dilution in a single assay and must be considered as relative concentrations rather than absolute values. The minimum detection limit in the rat FSH IRMA was 0.2 ng/ml and the intra-assay CV was 2.0%. The minimum detection limit in the human FSH ELISA was 1 mIU/ml and the intra-assay CV was 7.5%.Data for organ weights were analysed using one-factor ANOVA followed by Tukey's multiple comparison test . FSH values were analysed using Kruskal-Wallis non-parametric tests  because when values were below the limit of detection of the assay they were assigned that value for the purposes of statistical analysis.hpg mice at a molecular weight of approximately 64 KDa  and wild type (n = 4) mice. ERα-immunoreactive bands were also observed at approximately 64 KDa in extracts of testis from both hpg and wild-type mice, plus a slightly lower molecular weight band which is likely to be a degradation artefact  increased testicular weight in ols Fig. . The comols Fig. . The higols Fig. , top. Thols Fig. , whereasols Fig. . Treatmeols Fig. . Likewisols Fig. , whereasols Fig. .hpg mice receiving estradiol only, there was a significant increase (p < 0.05) in circulating FSH . Previous studies have provided rather conflicting evidence for ERβ expression in the mouse testis, some studies demonstrating ERβ expression in the Leydig cells and elongating spermatids of adult mice [The first objective of these studies was to confirm the expression of ERα receptors in the pituitary gland and testis of he mouse . Intereshe mouse . Our obsult mice , others ult mice . As ERβ ult mice , our stuhpg mice, consistent with our previous studies [hpg mice [hpg mice has increased Sertoli cell production [hpg mice treated with the high dose of FSH. Stimulatory actions of FSH on Leydig cell activity and morphology have been reported previously in hpg mice and are likely to reflect indirect actions mediated by paracrine output from Sertoli cells [hpg mice it seems likely that the FSH treatments would have increased testicular androgen production [We again observed robust stimulatory effects of estradiol treatment on the testis in  studies ,15,16. Thpg mice , though hpg mice  reportedhpg mice  did not hpg mice , and witoduction . We alsoli cells . Unfortuli cells ,15 or inoduction .hpg mice at an "adult" age. We have previously observed that there is a degree of spontaneous testicular development in hpg mice probably reflecting a low level of GnRH-independent production of gonadotropins [Though model-based, our estimates of cell numbers are consistent with those obtained by ourselves and others using more robust, design-based stereological procedures ,27,30,31otropins ,15 this not produce a greater degree of stimulation of the seminiferous epithelium that treatment with either hormone alone. This is contrary to our initial prediction, and to studies in the neonatal rat where combined treatment with estradiol benzoate and purified human FSH produce effects on development of the seminiferous epithelium not seen after treatment with either hormone alone, including increased tubule diameters, induction of a lumen and increased numbers of pachytene spermatocytes [hpg mouse experimental model. Although this stimulatory effect of estradiol may seem like a paradoxical response in a male mammal, it was demonstrably the case that circulating FSH levels were increased in this and previous studies in the hpg mouse. This paradoxical effect in the male is not due to incomplete masculinization of the hpg mouse [hpg mouse is arrested at a neonatal stage [are highly likely to be direct actions. In the current study we observed that estradiol treatment produced a substantial increase in the wet weight of the seminal vesicles, whereas FSH had no effect on this tissue nor did it augment the actions of estradiol. This is consistent with a previous study in the hpg mouse [The key outcome of the current studies is that the combined estradiol and rhFSH did atocytes . The obspg mouse , rather al stage ,11 when pg mouse , which apg mouse .hpg mouse represent a physiological role in co-ordinating early development. Whilst estradiol production acts within the testis to retard early androgen production, it also acts in the pituitary gland to stimulate FSH production and thereby promote early Sertoli cell proliferation. Thus, when pubertal increases in LH production eventually occur and thereby stimulate androgen production, the somatic cell complement within the tubules has already been established and will support the androgen-driven spermatogenesis.Recent evidence points to an autocrine or paracrine role for estradiol in the testis during fetal and early neonatal development whereby it inhibits production of androgens by Leydig cells . PerhapsThe author(s) declare that they have no competing interests.HB carried out the tissue processing and stereological analyses on the seminiferous tubules, MOW carried out the in vivo studies and the Western Blot and ER immunohistochemistry, GRH and RAW carried out the immunostaining for 3β-HSD and the quantitative assessment of Leydig and Sertoli cell numbers, TMM developed the stereological procedures and oversaw their application, FJPE managed the project and wrote and revised the manuscript.


Entomopathogenic fungi infection on malaria vectors increases daily mortality rates and thus represents a control measure that could be used in integrated programmes alongside insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS). Before entomopathogenic fungi can be integrated into control programmes, an effective delivery system must be developed.Metarhizium anisopliae ICIPE-30 and Beauveria bassiana I93-825 (IMI 391510) (2 × 1010 conidia m-2) applied on mud panels , black cotton cloth and polyester netting was evaluated against adult Anopheles gambiae sensu stricto. Mosquitoes were exposed to the treated surfaces 2, 14 and 28 d after conidia were applied. Survival of mosquitoes was monitored daily.The efficacy of M. anisopliae conidia on mud panels had a greater daily risk of dying compared to those exposed to conidia on either netting or cotton cloth (p < 0.001). Mosquitoes exposed to B. bassiana conidia on mud panels or cotton cloth had similar daily risk of death (p = 0.14), and a higher risk than those exposed to treated polyester netting (p < 0.001). Residual activity of fungi declined over time; however, conidia remained pathogenic at 28 d post application, and were able to infect and kill 73 - 82% of mosquitoes within 14 d.All fungal treatments caused a significantly increased mortality in the exposed mosquitoes, descending with time since fungal application. Mosquitoes exposed to Both fungal isolates reduced mosquito survival on immediate exposure and up to 28 d after application. Conidia were more effective when applied on mud panels and cotton cloth compared with polyester netting. Cotton cloth and mud, therefore, represent potential substrates for delivering fungi to mosquitoes in the field. To eliminate malaria, vector control programmes will need to incorporate novel tools to complement the use of insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS). Both ITNs and IRS are highly effective against anthropophagic and endophilic species, but their efficacy is threatened by emergence of resistance to synthetic insecticides ,2. ThereBefore entomopathogenic fungi can be integrated into control programmes, additional information regarding isolate selection, optimisation of production and formulation is required. While many successful laboratory evaluations of the efficacy of entomopathogenic fungi have been conducted ,13, moreThe aim of the current study was to test different surface substrates that can be used to infect mosquitoes with fungal conidia and to examine whether the surface affected the availability, efficacy and persistence of the conidia.Anopheles gambiae sensu stricto were reared at the Ifakara Health Institute (IHI) insectary . This colony is being supplemented with newly collected field mosquitoes to maintain its vigor. Larval and adult stages of the mosquitoes were raised using methods described by Huho et al [ho et al . All bioMetarhizium anisopliae var. anisopliae Sorokin isolate ICIPE-30 and Beauveria bassiana Vuillemin isolate I93-825 (IMI 391510) were used in all bioassays. Metarhizium anisopliae was isolated from the maize stalk borer, Busseola fusca , in 1989 in Western Kenya and imported as dry conidia from Wageningen University, The Netherlands . Beauveria bassiana was isolated from a chrysomelid beetle (Coleoptera) in the USA and imported as dry conidia from the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia and Penn State University, USA . Dry conidia of M. anisopliae were produced at IHI, after passaging and re-isolation from infected mosquito cadavers. Conidia were harvested from 15 day old cultures grown on rice grains. The viability of conidia (>85% germination) was confirmed before each bioassay by inoculating 0.2 ml of suspended 107 conidia/ml onto SDA. Three agar plates were used per each fungal isolate. After 16 - 18 h incubation at 26°C and 80% Relative humidity (RH) the germination of 300 conidia in total for each of the three agar plates was quantified from different fields of the inoculum. The conidia viability was then calculated as an average of the percentage germination in the three agar plates.Two fungal isolates, ®, BP South Africa LTD) or Shellsol  oils. The Enerpar and Shellsol oils have no effect on conidia germination or infectivity . To homogenize the mixture it was shaken vigorously, vortexed for 25 sec and then sonicated for 3 min in an ultrasonic bath, 40 kHz frequency . Dilutions of 1:10, 1:100 and 1:1000 were then prepared in the same oil mixture and the concentration of conidia calculated using a Neubauer haemocytometer  and compound microscope  at 400 × magnification. The solution was adjusted to 2 × 1010 conidia m-2 by further dilution in oil.Conidia were formulated in oil before application. Fungal stock solution was prepared by suspending 1-2 g of conidia in 20 ml of a 1:1 mixture of highly refined Enerpar (Enerpar M002The delivery surfaces examined were:1) polyester netting (intended to target host-seeking mosquitoes) was obtained from Safi Net™, A to Z Textile Mills Ltd, Arusha, Tanzania. White netting was used.2) mud panels similar to the walls of adobe houses (intended to target resting mosquitoes). About 20 mm layer of mud, collected from natural soils near Ifakara, was lined onto the inner surface of plywood panels. The panels were kept to dry under room temperature for about three weeks. Panels had screws, thus the individual sections could be assembled to obtain mud cages for the bioassays Figure .3) black cotton cloth (intended to target resting mosquitoes). The cloth was obtained from the local shops, Ifakara, Tanzania.®, SATA, Germany) held 50 cm away and at a right angle to the surface. About 23 ml of fungal formulation (2 × 1010 conidia m-2) was applied onto each of the surfaces. Treated materials were left to dry for 48 h. Sections of netting and black cotton cloth were then joined using Velcro strips, to fit over 20 cm3 wire frame cages. Mud-lined plywood panels were assembled into 20 × 20 × 20 cm cages. Mosquitoes were then exposed to the treated surfaces  and maintained at 26 ± 1°C and 90 ± 5% relative humidity (RH) with access to 9% glucose/water (w/v) solution ad libitum. Mosquito survival was monitored daily for a maximum of 28 d. Dead mosquitoes were collected and put onto moist filter paper in petri dishes, sealed with parafilm, and kept at 26 ± 1°C and 90 ± 5% RH for 3-4 d, after which they were examined for evidence of fungal sporulation. After the initial evaluation, all treated surfaces were stored at 26 ± 1°C and 90 ± 5% RH and separate mosquito cohorts were exposed 14 and 28 d later to determine the persistence of conidia on different delivery surfaces.To test the efficacy of conidia applied to different delivery substrates, 40-55 adult p > 0.05. Bonferroni method was employed to compensate for multiple comparisons. SPSS version 16  was applied.Mosquito survival data were analysed using Cox regression to determine the relative risk of dying (hazard ratios) for the specific treatment group compared with the control group and with each other. The explanatory factors included in the analysis were treatment, delivery surface and time of exposure post-application. The Kaplan-Meier method was applied to obtain median survival times for treated and untreated groups of mosquitoes. Survival curves were considered not statistically different at M. anisopliae  or B. bassiana  was more than two-fold higher than that of control mosquitoes  or cotton cloth  regardless of the time at which mosquitoes were exposed post conidia application  regardless of the time at which mosquitoes were exposed post conidia application. The risk of death for mosquitoes exposed to conidia on the mud panels and cotton cloth was higher than that for mosquitoes exposed to conidia on the polyester netting  of fungal-exposed mosquitoes compared to the controls regardless of delivery surface or time of exposure post-application Table . The daiM. anisopliae 2 d and 14 d post-application, had a similar risk of death . The residual efficacy of conidia of M. anisopliae had significantly declined by 28 d post-application, with the relative risk of death for mosquitoes exposed at 2 d post-application being higher  and 28 d  post application. Mosquitoes that were exposed to B. bassiana at 14 d and 28 d post-application had similar risk of death  varied among the candidate delivery materials. The efficacy of M. anisopliae conidia was highest when applied on mud panels compared with cotton cloth or polyester netting. For B. bassiana, the efficacy of conidia was highest when applied to either mud panels or cotton cloth compared with polyester netting. The lowest efficacy of either fungal isolate was consistently recorded for the polyester netting. Rapid decline in conidial efficacy over time after application on the netting has also been reported in other studies [This study was intended to provide fundamental information for developing delivery formats in advance of field evaluations of entomopathogenic fungi against malaria vectors. Consistent with published literature ,12,13, t studies . Variati studies . In the Bacillus thuringiensis [Even though the residual activity of fungal isolates declined over time, conidia remained pathogenic up to 28 d post application (and possibly longer) and were still able to infect and kill 73 - 82% of mosquitoes within 14 d. When conidia were applied on the netting, the residual activity declined much more rapidly compared with mud panels and cotton cloth. Decline in the residual activity of conidia has also been reported elsewhere ,7. The rngiensis ,22. The ngiensis , which ingiensis .2). As such the netting had a smaller surface area for both conidial attachment and exposure of mosquitoes. The higher efficacy of fungus observed when applied to mud and cotton cloth could therefore have simply been due to availability of more conidia per unit area, maximizing mosquito exposure and probability of picking up more conidia.It is important to note that the treatable surface area of polyester netting was much smaller than mud and cotton cloth due to the holes in this material , many conidia could have been readily available on the surface of each substrate. With time, however, conidial viability and virulence could have decreased thus the conidia became less efficacious to mosquitoes. This may have accounted for a quick drop in efficacy of fungi applied on cotton cloth and netting between the initial and subsequent exposures. A drop in efficacy could also be explained by reduced virulence of the conidia. For the mud panel, the decline was fairly constant. Others have reported a decline in residual activity resulting from fluctuating environmental conditions , but thiThe aim was to develop a delivery tool(s) that facilitates rapid dissemination of fungal conidia to mosquitoes and remains effective for a prolonged amount of time. The high mortality (82% within 14 d) since exposure of mosquitoes to conidia on mud and cotton cloth 28 d post application emphasizes the potential of these two surfaces as target tools. The delivery surfaces examined in the current study were selected because they could be easily adapted for practical dissemination of conidia under realistic field conditions. Cotton cloth could be placed to partially cover eave openings, on ceilings , and on Mosquitoes exposed to entomopathogenic fungi expressed a reduced survival from conidia used between two and 28 days after application. Conidia were more effective when applied on mud panels and cotton cloth compared with polyester netting. Cotton cloth and mud, therefore, present useful and practical tools for applying fungi against resting mosquitoes in the field. These tools should be used such that mosquito contact to conidia is maximized in order to correctly predict the efficacy and residual activity of fungi.The authors declare that they have no competing interests.Conceived and designed the experiments: LLM TLR BJK WT. Performed the experiments: LLM DWL MWM. Analyzed the data: LLM TLR MJK. Wrote the paper: LLM TLR MJK. Reviewed the paper: BJK WT. All authors read and approved the final manuscript.


During development, neurons modify their axon growth mode switching from an elongating phase, in which the main axon stem reaches the target territory through growth cone-driven extension, to an arborising phase, when the terminal arbour is formed to establish synaptic connections. To investigate the relative contribution of cell-autonomous factors and environmental signals in the control of these distinct axon growth patterns, we examined the neuritogenesis of Purkinje neurons in cerebellar cultures prepared at elongating (embryonic day 17) or arborising  stages of Purkinje axon maturation.in vitro, Purkinje cells of both ages undergo an initial phase of neurite elongation followed by the development of terminal ramifications. Nevertheless, elongation of the main axon stem prevails in embryonic Purkinje axons, and many of these neurons are totally unable to form terminal branches. On the contrary, all postnatal neurites switch to arbour growth within a few days in culture and spread extensive terminal trees. Regardless of their elongating or arborising pattern, defined growth features (e.g. growth rate and tree extension) of embryonic Purkinje axons remain distinct from those of postnatal neurites. Thus, Purkinje neurons of different ages are endowed with intrinsic stage-specific competence for neuritic growth. Such competence, however, can be modified by environmental cues. Indeed, while exposure to the postnatal environment stimulates the growth of embryonic axons without modifying their phenotype, contact-mediated signals derived from granule cells specifically induce arborising growth and modulate the dynamics of neuritic elongation.When placed Cultured Purkinje cells recapitulate an intrinsically coded neuritogenic program, involving initial navigation of the axon towards the target field and subsequent expansion of the terminal arborisation. The execution of this program is regulated by environmental signals that modify the growth competence of Purkinje cells, so to adapt their endogenous properties to the different phases of neuritic morphogenesis. The wiring of neural circuits is accomplished during embryonic and postnatal development through a complex multistep process. Neurons first elongate their main axon stem across long distances to reach the appropriate target territory. Here, they develop a highly branched terminal arborization to establish synaptic connections. Elongation of the main axon stem and formation of the terminal arbour represent distinct growth modes The cellular and molecular mechanisms that mediate the transition from elongating to arborising growth are not well understood. Several lines of evidence indicate that cell-autonomous mechanisms determine the mode of axon growth in vivo. Thus, to ask whether such properties can be modified by extrinsic cues, we co-cultured embryonic PCs with different cellular components of the postnatal cerebellar milieu.To investigate the relative contribution of cell-autonomous factors and environmental cues in the control of distinct axon growth patterns, we examined Purkinje cell (PC) axons. The main PC neurites elongate during embryonic life, when they reach their targets in the cerebellar nuclei in vitro their neurite re-extension in conditions of minimal cell-to-cell contact. We first examined neurons that had been maintained for 7 days in vitro (DIV). A large fraction of embryonic PCs exhibited an axon stem with a small or absent terminal ramification  or P0 (arborizing phase) cerebella and evaluated fication , while tfication . These qfication , was sigfication . AltogetTo clarify whether embryonic neurons could not develop the terminal arbour due to an unhealthy state, we quantified PC survival. Consistent with previous reports in vitro) and this difference, 1.5–2.7 the speed of E17 cells, was maintained during the whole period in culture. Terminal ramifications progressively spread at longer time points . However, embryonic neurites elongated throughout the whole period, whereas the postnatal ones reached a steady state from 5 days on  for embryonic cells and of terminal plexus enlargement (arborizing growth) for postnatal PCs. Nevertheless, qualitative observations of the cultured cells at both ages revealed a variety of axonal phenotypes . For insin vitro, all the three classes of axons  were represented in both embryonic and postnatal cultures, but their relative frequencies varied over time , with most cells (more than 80%) belonging to the EL class with a short stem and no tree ) to the percentages of EL axons at different time points , and eventually developed longer stem neurites than their postnatal counterparts .To further characterize the neuritogenic competence of E17 and P0 PCs, we asked whether axons of distinct ages belonging to the same class shared the same growth features. We first compared PCs of the EL class, whose growth is restricted to stem neurite extension. The growth rate of postnatal EL axons was particularly fast just after plating, but decreased considerably during the following days . On thei1, 243 = 55.67 P<0.001) and extended considerably larger trees , whereas their stem axons resulted only slightly longer than embryonic ones . To further characterize the dynamics of arborizing growth, we analyzed stems and trees separately. In both embryonic and postnatal neurons the high growth speed of AR axons was predominantly due to the fast extension of the terminal tree , with no differences between embryonic and postnatal cells . Furthermore, although the overall growth speed of EL axons of both ages was slower than that of AR axons , the elongation of the stem neurite in AR cells proceeded more slowly than that of EL PCs .Within the AR class, postnatal axons were growing faster , but a conspicuous fraction reached stage 3  a significant fraction of EL PCs persists in embryonic cultures up to the longest time examined, indicating that they are unable to switch to the AR mode; ii) the growth speed of embryonic and postnatal axons is consistently different throughout the entire period; iii) the axonal features of embryonic and postnatal AR cells are significantly different. These considerations suggest that embryonic PCs do not acquire the growth properties of postnatal neurons by unfolding a purely cell-autonomous program, but require extrinsic instructive information.t test E17 vs P0 cultures at 7 DIV, P = 0.005, P = 0.015, respectively). Therefore, to ask whether the growth pattern of E17 PC axons could be modified by the postnatal milieu in toto, we prepared co-cultures of E17 and P0 cells . In these cultures, postnatal PCs maintained their characteristic pattern of axon growth (not shown), while embryonic neurons . Over the examined time, the frequency of embryonic EL cells in the co-cultures decreased following a kinetic similar to that seen in pure embryonic cultures , while EL PCs showed increased growth speed  and stem elongation, reaching values typical of P0 cells .Postnatal cues did not alter the overall features of co-cultured E17 AR PCs, which remained clearly distinct from their P0 counterparts . To expose embryonic PCs to an environment highly enriched of GCs, E17 cerebellar cells were plated onto GC monolayers. In these cultures, the density of GCs was 808.33±30.05 GCs/mm2 . Embryonic PCs showed a modest tendency to dendritic maturation (2 = 8.57 P = 0.01). At 14 DIV, EL PCs were only 7% , which are a predominant component of the postnatal cerebellum and well-known regulators of PC maturation turation , but disturation . The evo only 7% , while t only 7% . The dif only 7% .Because of the specific culture conditions of this experiment, the embryonic PCs were growing in contact with GCs . In addiin vitro. Embryonic neurons perform a predominantly elongating growth, carried out at relatively slow speed (40±1.13 µm/day). They can switch to the arborizing mode, but about one third appears totally unable to develop a terminal tree within the examined period. Postnatal PCs grow axons at a higher speed (80±2.59 µm/day) and, within 14 days in vitro, all turn to the arborizing phenotype, characterized by the fast expansion of a wide terminal plexus. Embryonic neurites can be induced to spread arbors by GC-derived signals, but their phenotype is not affected by the postnatal milieu in toto. On the whole, certain features of PC neurite extension  appear more related to the neuron age than to the axon growth mode and are scarcely sensitive to culture conditions, indicating that the neuritic pattern expressed in vitro is largely determined by intrinsic stage-specific neuronal properties.To investigate the mechanisms that regulate the different phases of axonal morphogenesis, we compared the development of E17 and P0 PC axons in vitro, embryonic neurons mostly extend their stem axon, while postnatal ones soon turn to arbor formation, indicating that they are endowed with distinct age-dependent inclination to carry out specific growth patterns. In agreement with previous observations about dendritic maturation in vitro, which is much better for embryonic than neonatal neurons. Hence, this different capability cannot be attributed to the health of the cultured neurons. On the other hand, the specific neuritic patterns could be determined by the different environmental conditions provided by embryonic and postnatal cultures. Previous studies on dissociated PCs indicate that exposure to specific substances can stimulate neuritic extension, but do not affect the overall growth pattern in vitro PCs spontaneously reinitiate neuritic development according to a preferential elongating or arborizing pattern that reflects the embryonic or postnatal age when they were dissociated.PC axons navigate towards the deep nuclei during embryonic development and develop their terminal trees during early postnatal life in vitro suggests that this transition follows an intrinsic developmental clock. Nevertheless, the growth pattern and dynamics of E17 AR axons are overtly different from those of their P0 counterparts, and many embryonic PCs are still unable to form terminal branches at 14 days in vitro, long past the age of terminal plexus formation in vivo. These observations indicate that the complete transition to the postnatal pattern of axon growth requires specific interactions that are lost when embryonic PCs are isolated from their native environment.The diverse axon growth capability of E17 and P0 neurons indicate that during the last days of embryonic development PCs undergo crucial changes for the acquisition of the postnatal arborizing pattern. The existence of embryonic PCs able to develop a terminal arbor Although E17 PCs are responsive to the P0 environment , the lack of acquisition of the postnatal neuritic pattern indicates that efficient instructors are not acting in the P0 cultures. As potential candidates for this task we tested GCs, whose promoting activity on PC differentiation is well-recognized in vitro, albeit functional, is not likely to be the only event responsible for inducing the switch to arbor growth mode. Rather, the peculiar features of embryonic AR neurons indicate that, although E17 PCs are already sensitive to heterochronic cues, acquisition of full-blown arborising growth depends on multiple extrinsic signals and endogenous cellular processes. Indeed, between E17 and P0 PCs undergo several key steps in their maturation, including the arrival of olivary afferents Despite the clear-cut action of GCs on PC axon phenotype, in the pure E17 cultures many PCs spontaneously engage into arbor formation, implying that they had already received instructive information at the time of dissociation. During late embryonic development GC precursors progressively accumulate on the cortical surface to form the external granular layer Our study shows that postnatal PC axons grow faster than embryonic ones. This feature is different from other CNS neurons, such as retinal ganglion cells in vitro undergo simultaneous extension of both stem axon and terminal tree. However, their fast growth rate is mostly sustained by arbor expansion, whereas stem elongation is sluggish and consistently slower than that observed in EL neurites. The latter observation does not match the situation in vivo, where stem elongation is slower during embryonic than postnatal life  and Green rats, which overexpress the enhanced green fluorescent protein (GFP) under the control of the β-actin promoter . From the cerebellum of these animals PCs were dissociated at E17 or P0, whereas P6 rat pups were used for cerebellar granule cultures. All procedures were in accordance with the European Communities Council Directive of 24th November 1986 (86/609/EEC), the NIH guidelines and the Italian law for care and use of experimental animals (DL116/92), and were approved by the Italian Ministry of Health and the Bioethical Committee of the University of Turin. Embryos were obtained by caesarean section from rat dams anesthetized by intraperitoneal injection (i.p.) of ketamine and xilazine . P0 pups were cryoanesthetized in melting ice, while P6 pups were anaesthetised by i.p. injections (see above).5 cells/coverslip, 12 mm diameter, Marienfeld GmbH & Co.KG, Lauda-Konigshofen, Germany). Cells were maintained under controlled conditions (37°C and 5% CO2) for up to two weeks, with a medium change after the first week in vitro. At 7 days in vitro AraC  was added to P0 cultures to limit the proliferation of glial cells. Cerebellar cells dissociated at E17 from Green embryos were co-cultivated with Wistar P0 cerebellar cells at a 1∶9 ratio . In other sets of experiments, Green E17 cerebellar cell suspensions were co-cultivated with GCs (E17+GC), which were obtained from P6 Wistar pups following pubished protocols 5 cells/coverslip) and, after 1 hour at 37°C, 1×104 E17 cells from Green rats were seeded on this layer. In this condition, virtually all PCs had contacts with other cellular components. Therefore, to avoid contact-mediated effects and analyse isolated PCs exposed to GC environment, we carried out additional experiments in which coverslips containing pure E17 cerebellar cultures were flipped onto GC monolayers (E17+GCflip) Embryonic and postnatal cerebellar cultures were obtained according to previously established protocols in vitro using 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.4 for 40 minutes and then rinsed 3 times in PBS. For PC identification, cultures were incubated overnight at room temperature with a rabbit polyclonal antibody directed against calbindin , a calcium-binding protein specifically expressed in PCs in the cerebellum The cultures were fixed at 1, 3, 5, 7 and 14 days Morphometric analysis of PC axons was performed by means of Neurolucida software  connected to a Nikon E-800 microscope via a color CCD camera. The material was also examined with a Leica TCS SP5 confocal microscope and digital images were adjusted for contrast and assembled with Adobe Photoshop 6.0 .For each experimental condition, a sample of PC, ranging from 40 to 90 cells, with no or minimal contact with other cells, were randomly selected from 3–4 different experiments and their axons carefully reproduced and measured using the Neurolucida software. To define the elongating or arborizing growth mode, morphometric analysis of PC neurites focused on the axon compartments most relevant to distinguish these growth modalities: i) the axon stem, defined as the longest process that originates from the cell body, and ii) the terminal arbor, including all processes emerging from 2 equally thick and symmetric branches budding off at acute angles from the distal end of the axon stem The degree of maturation of PC dendrites was examined in E17, P0, and GC+E17 cultures at 14 DIV . Distinct stages of dendrite differentiation were scored following Baptista et al. 2 each) localized at central and peripheral sites of the coverslips as defined by two orthogonal coverslip diameters. PCs were identified on the basis of anti-calbindin positivity and only cells extending neurites were considered for survival analysis. For each culture type, PC survival was calculated as percentage of living PCs at 7 DIV over the number of PCs at 1 DIV. With the same sampling method the cell type composition of the cultures was evaluated at 1 and 7 DIV on 3 coverslips from 3 distinct experiments. We counted the total number of cells (DAPI staining), the number of neurons , and the number of Purkinje cells . Analysis of DAPI-positive cells negative for neuronal markers yielded the number of non-neuronal cells. A separate set of coverslips of all experimental sets at 7 DIV was devoted to estimate the number of GCs, identified as described above.E17 and P0 PC density was calculated in 5 quadrants  for frequency analysis. For statistical analysis tree ratio percentages were treated according to the arcsine transformation. In some experimental conditions, EL or AR PCs were not detected at 1 or 14 DIV respectively. In these cases statistical comparisons did not include the missing time points. Effects were considered statistically significant when P<0.05. Data fitting was performed with the free Curve Expert 1.3 software.All data are presented as averages±SEM. Statistical evaluation was performed with SigmaStat software package , applying the Student's


Childhood respiratory allergies, which contribute to missed school days and other activity limitations, have increased in recent years, possibly due to environmental factors.In this study we examined whether air pollutants are associated with childhood respiratory allergies in the United States.2.5), PM ≤ 10 μm in diameter, sulfur dioxide, and nitrogen dioxide and summer exposure to ozone, controlling for demographic and geographic factors.For the approximately 70,000 children from the 1999–2005 National Health Interview Survey eligible for this study, we assigned between 40,000 and 60,000 ambient pollution monitoring data from the U.S. Environmental Protection Agency, depending on the pollutant. We used monitors within 20 miles of the child’s residential block group. We used logistic regression models, fit with methods for complex surveys, to examine the associations between the reporting of respiratory allergy or hay fever and annual average exposure to particulate matter ≤ 2.5 μm in diameter (PM3 levels  and increased PM2.5 . These associations persisted after stratification by urban–rural status, inclusion of multiple pollutants, and definition of exposures by differing exposure radii. No associations between the other pollutants and the reporting respiratory allergy/hay fever were apparent.Increased respiratory allergy/hay fever was associated with increased summer O3 and PM2.5 compared with children with lower exposures.These results provide evidence of adverse health for children living in areas with chronic exposure to higher levels of O Children in the United States are generally healthy; > 80% are in excellent or very good health according to the 2006 National Health Interview Survey (NHIS) . HoweverA possible factor affecting respiratory allergic symptoms is exposure to ambient air pollution, particularly traffic pollutants. Reviews of the effects of air pollution on allergies have concluded that pollutants likely exacerbate effects of allergens among those with existing susceptibility, rather than initiating allergies among people without existing allergies e.g., . D’AmatoNo comprehensive studies have examined associations between allergic symptoms and air pollution among a nationwide sample of U.S. children. From Europe and Asia, increases in indices of air pollution have been found to exacerbate childhood allergic symptoms in some studies  but not 3, sulfur dioxide, and nitrogen dioxide] from the U.S. Environmental Protection Agency , On Agency . With bon Agency , understThe NHIS is a large nationally representative survey of the civilian noninstitutionalized U.S. population . Very brIn 1999–2005, about 35,000 households were sampled each year, yielding almost 100,000 total respondents each year. In addition to the core family questionnaire each family member answers, within each family with children, a sample child was selected for additional questions on health and health care . ResponsA total of 73,198 children 3–17 years of age provided information for the sample child questions in the 1999–2005 NHIS. Of these, a small number are missing data for one or more of the NHIS variables described below, leaving 72,279 eligible for this study.The Air Quality System (AQS) database provides annual averages of air monitoring data—ambient concentrations of criteria and hazardous air pollutants at monitoring sites, primarily in cities and towns . The AQS3, fine PM , respirable PM , NO2, and SO2 from the AQS using the annual summary web pages (3 during the summer (May through September) for these linked data files using daily measures. We calculated summer O3 exposures separately to obtain more comparable measures throughout the United States than annual average O3 exposures—although differing monitoring periods are used across geographic areas, summertime is the most frequently monitored period , usual source of care , health insurance , family income relative to federal poverty level , child’s race/ethnicity . We separated the Mexican or Mexican-American subgroup from the other Hispanic subgroups because of the heterogeneity among these subgroups . For theWe also included county-level geographic level covariates: median income from the 2000 U.S. Census, an index of urban–rural status , and regAsthma is an important childhood condition related to the reporting of allergies and air pollution exposures and, in the United States, related to many of the covariates listed above . To examEnvironmental tobacco smoke affects the respiratory health of children. Unfortunately, the NHIS does not collect smoking information from all adults, just from one sampled adult per family. As a result, our smoking information for a child’s exposure can only be dichotomized as yes versus unknown. Data on the smoking status of children is not collected in the NHIS.We evaluated possible associations between air pollution and childhood respiratory allergy/hay fever using logistic regression models. We used statistical software for surveys SUDAAN;  because We fitted unadjusted and adjusted models separately for each pollutant. We tested interaction terms to determine whether associations differed by characteristics described above. We fitted additional models to examine the impact, if any, of current asthma; because the relationship between allergies and asthma among children is complex , we deciAll characteristics shown in 10 and PM2.5, allowing an approximate estimate of the coarse PM exposure by the difference (PM2.5–10 = PM10 levels – PM2.5 levels). In all linkage groups, respiratory allergy/hay fever reporting was slightly lower than in the overall sample of children, ranging from 18.3% for children linked to NO2 to 19.0% for children linked to PM2.5. There are small demographic differences and larger geographic differences among the linkage subsets.The number of children linked to air monitoring data varies by pollutant . Of the 10 and SO2 and between PM2.5–10 and SO2. Correlations between pollutants were, for the most part, similar whether we used the subset of 32,137 children linked to all pollutants for all calculations or used different subsets, depending on available data, for each calculation.3 were associated with increased reporting of respiratory allergy/hay fever. PM2.5 was associated with reporting of these conditions after including geographic indicators in the models. Other gaseous pollutants were not associated with increased respiratory allergy/hay fever and, in some cases, were negatively associated. After controlling for multiple pollutants .Because including geographic variables had a large impact on the associations, we examined whether effects were similar by urban–rural status by including interaction terms in the regression models. In single-pollutant models, associations were significantly different by urban–rural status for summer O3 and PM variables differed by these factors. Model results with interaction terms indicated that associations did not differ significantly (using p < 0.05 as criterion) by race/ethnicity, sex, or age category for either PM or summer O3. Associations did not differ significantly by poverty status for PM variables, but the associations between summer O3 exposure and respiratory allergy/hay fever for children in poverty categories > 200% of poverty were stronger than for those < 200% of poverty: <100% poverty, adjusted odds ratio (AOR) = 1.05 [95% confidence interval (CI) = 0.95–1.16]; 100% to < 200%, 1.13 (1.01–1.27); 200% to < 400%, 1.28 (1.17–1.39); 400% or higher, 1.25 (1.15–1.35).Given the differences in reporting respiratory allergy/hay fever by child’s race, family poverty status, and child’s age and sex, we examined interactions (products of these factors and the pollutants) to determine whether associations for summer O2 to 26,898 for PM2.5. Results for summer O3 from a model adjusted for demographic and geographic variables remained statistically significant , if slightly weaker than the OR shown in 2.5 were elevated but lower than those shown in 3 and PM2.5 (r > 0.95); for the other pollutants, correlations between the 20-mile and 5-mile variable were at least 0.85. Although the 20-mile and 5-mile exposure variables, calculated from many of the same monitors, are not independent and hence statistical inferences from these correlations are inappropriate, the correlations give an indication of the similarity of the measures.Because the 20-mile exposure radius may be inadequate for assigning exposure for the gaseous pollutants, we replicated the full models using the subsets of children within 5 miles of pollution monitors. Fewer than half of the children with 20-mile exposure variables were available for the 5-mile analyses, ranging from 14,034 for SOWe fit models without controlling for survey year, with similar results (data not shown). Including survey year would be considered appropriate for the control of temporal changes that may have affected reporting of respiratory allergies, such as access to health care; on the other hand, year is also correlated with air pollution levels, and including year would be considered overcontrol if, for example, changes in reporting over time were attributed to the corresponding temporal changes in air pollution. Because we cannot distinguish the two scenarios, we examined results both ways.3 with those using the annual averages of the daily 8-hr running O3 averages provided by the U.S. EPA. These averages were, by design, lower than the summer averages but highly correlated (r = 0.80) despite the differing O3 reporting periods throughout the United States, the use of average daily measurement to calculate the summer-O3 variable, and the use of daily 8-hr averages to calculate the annual O3 variable. Corresponding to the high correlation, the association between annual O3 exposure and respiratory allergy/hay fever adjusted for demographic and geographic factors  was similar to that reported for summer O3 .In further tabulations of our study sample, of the children with respiratory allergy/hay fever, > 30% had been told at some time that they had asthma; in contrast, only 9% of children without allergies or hay fever had been told they had asthma. Adjustment for asthma in the logistic regression models did not change the findings for either summer O2.5 or O3 (data not shown).Finally, we fit models that included adult smoking (yes vs. unknown). In the eligible sample, 20.3% of children lived in a family with a known smoker. Although adult smoking in the family had an independent effect on respiratory allergy/hay fever, its inclusion in the models did not affect associations between respiratory allergy/hay fever and either PM3 and reporting of respiratory allergies and hay fever among U.S. children. These findings were stronger in urban than in less urban areas and persisted when controlling for family income, parental education, family structure, child’s age, median county-level income, access to care, and current asthma status. The association between O3 and respiratory allergy/hay fever was weaker among children living in the less metropolitan counties, which may suggest variation in types of allergens or other factors related to level of urbanization  that modify the effects of O3 on respiratory allergies/hay fever. Furthermore, that the association became stronger as family income rose suggests one of two scenarios: a) Real differences exist and there is something about more privileged children that increases their susceptibility; or b), parents in lower-income groups are underreporting.We found a persistent association between increased levels of summer O3 exposure were most robust, we also found a strong association between PM2.5 exposure and reporting of respiratory allergy/hay fever after controlling for geographic variables. Effects of PM2.5–10 on the reporting of respiratory allergy/hay fever were weaker than the effects of PM2.5 and varied somewhat geographically.Although the findings for increased reporting of respiratory allergy/hay fever with increased O2 or SO2 when derived by spatial averages within 20 or within 5 miles of a child’s residence was associated with reporting of childhood respiratory allergy/hay fever. Although these findings may indeed reflect no association, there is some evidence that metrics for these gaseous pollutants based on ambient monitoring are less representative of exposure than are similar metrics for O3 or PM  for very young children , which may not cover the exact recall period for each respondent. These annual exposures, however, are correlated such that exposure for the calendar year is probably similar to exposure for the previous year; averaged over counties, the correlations between adjacent average exposures were high . In addition, we have no information on local aeroallergen levels, which would have increased our understanding of the effects of air pollution on reported allergies.We used a data set previously linked to annual exposure data (or summer-O3 (< 500) or PM2.5  monitor and were likely poorly distributed demographically and geographically across the United States.Another limitation is the primary use of a 20-mile radius to define exposure from the NHIS respondent’s residential block group. Ideally, more precise exposure measures would be available for each child, either from personal monitoring or finer spatial scales, which would lead to more precise effect estimates. With this survey, there is a substantial trade-off between criteria for proximity of monitors and the sample size and composition of the available data , 2008b. A related issue is our impaired ability to compare within-area effects of local differences in exposure with between-area effects. The NHIS is not designed for local estimates; although it may be possible to define a local unit of analysis from which to frame the comparison, say, county or metropolitan statistical area, the clustered design does not lend itself to sufficiently varied exposures within most counties to adequately examine this question.3 for cyclical periods over 6 months demonstrated altered airway architecture with fewer and narrower small airways, disordered smooth muscle orientation, and hyperplastic bronchiolar epithelium  exceeded the average levels found in our study, these studies present evidence of permanently compromised lung structure and growth for early or chronic exposure to pollutants.Children may be especially sensitive to effects of inhaled toxins and pollutants. Compared with adults, they have higher metabolic rates and minute ventilation rates . Most imithelium . In a prithelium . In addiactivity . Althoug3 and PM2.5 among asthmatic children in New England. The Children’s Health Study from Southern California (There have been several studies of children’s respiratory health and air pollution from the United States. Although these studies focused more on allergies and other respiratory symptoms, they highlight the importance of air pollution for children’s health. For example, two multisite studies primarily related to asthma, the National Cooperative Inner-City Asthma Study  and a stlifornia  has repolifornia , school lifornia , and poolifornia  among chlifornia  and amonlifornia  reported3 and fine PM are associated with childhood respiratory allergies. The weight of the findings to date support detailed monitoring of air quality as high levels of exposure are increasingly being demonstrated as unsafe to public health and costly in terms of morbidity, lost school and work days, and lower quality of life.The results of this study agree with the findings in the literature: exposures to O