Subclinical lung function alterations can sometimes be discovered in asthmatic patients under clinical control. This study aimed to identify the burden of asthmatic children with subclinical airways abnormalities who may benefit from an adjustment in asthma therapy. 134 6-to-17-year-old asthmatic children were enrolled. Of them, 98 presented apparently under clinical control disease and all performed spirometry before and after bronchodilation: 17 (17.3%) had a positive bronchodilation test, in addition to significantly lower lung function indexes as compared to those with under-control asthma who had a negative bronchodilation test. These patients were randomized and re-evaluated: patients (n=8) receiving an adjustment in their therapy showed an improvement in lung function tests and quality of life indexes as compared to 7 without therapy adjustment. In conclusion, a substantial number of apparently-under-control asthmatic children show airways alterations that can be improved by adjusting their therapy, which also seems to enhance their quality of life. Asthma is the most frequent chronic disease in childhood and its prevalence is increasing ,2, but o1) measures the best lung function that can be achieved by bronchodilator therapy on the day of the visit [1 indicates bronchial obstruction.Lung function testing and the measurement of bronchodilation responsiveness are recommended for the diagnosis of asthma and for its monitoring over time . The poshe visit ; a subopSeveral studies confirm that lung function can be altered even in patients whose disease is judged to be under control according to their symptoms referral and the use of reliever medications ,7,9. ColIt seems that taking into account only the symptoms as referred by the children or their parents in order to judge asthma control is insufficient -9. The p134 children  aged 6 to 17, who had been consecutively referred to the Allergology. Outpatient section of our clinic between November 2005 and October 2006, were included, all having a diagnosis of allergic asthma which had been established previously.1; 7) absence of bronchospasm at auscultation. Therapy prior to the visit was recorded.All the patients underwent a clinical examination to assess their asthma severity according to the GINA guidelines , and the1 before and after bronchodilation expressed as a percentage change relative to the baseline values (deltaFEV1): the test was considered positive when deltaFEV1 was ≥12% [1 ≥12% instead of ≥15% to increase the sensibility of the test.All the patients performed a spirometry by means of a Multispiro SA/100 spirometer . The best of a minimum of three recordings was chosen and a bronchodilation test was done by the administration of 200 mcg of dry salbutamol powder and repetition of the spirometry 15 minutes later. Spirometric indices were expressed as percentage of the predicted values (Polgar revisited) . The reswas ≥12% ,12, 13. Parietaria judaica) and perennial  allergens involved in allergic asthma .A Skin Prick Test was performed to establish possible sensitization to the main seasonal  .Data were examined by means of SPSS 13.0 for Windows . Student’s t-test and linear regression were used. Results were considered significant for a p value ≤0.05.This research was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from parents before the children were included in the study.1 ≥12%. This allowed us to divide our population into three different groups, one including patients with their asthma under control and with a negative bronchodilation test, the second consisting of children with controlled asthma but a positive bronchodilation test and the third including patients with uncontrolled asthma. Table 1 describes the main clinical and functional characteristics of our population. Table 2 shows the results of spirometry, bronchodilation test, Skin Prick Test and PAQLQ scores.According to our parameters, the asthma was under control in 98 children (73.1%), but not in the remaining 36 (26.9%). Of the 98 children with controlled disease, 17 (17.3%) had a positive bronchodilation test, meaning a deltaFEV1 and the ratio between FEV1 and the forced vital capacity (FVC) differed significantly (p≤0.001 and p≤0.005 respectively) between patients with apparently under control asthma (with a positive or negative response to the bronchodilation test) and patients with not-controlled asthma.Firstly, differences in spirometric data between different groups in our population were examined. Results of t-test showed that FEV1 and FEV1/FVC differed significantly (p≤0.05 and p≤0.01 respectively) between patients with asthma under control and a positive or negative response to the bronchodilation test. No significant differences were found in FVC between these groups. FEV1 and FEV1/FVC were not significantly different (p=0.180 and p=0.785 respectively) between patients with asthma under control but a positive bronchodilation test and patients with uncontrolled asthma.Moreover, FEVAnalysis of the differences in HRQL between groups showed that PAQLQ scores were similar in all children with asthma under control independently of bronchodilation test results, but a statistically significant difference was found between the scores of children with controlled asthma and a positive bronchodilation test, and children with uncontrolled disease (p≤0.01).3).Examination of the allergic condition in the population did not highlight differences between the three groups regarding positive Skin Prick Test to the main allergens involved in allergic asthma  had an adjustment in their asthma therapy, consisting of the introduction of or increase in inhaled corticosteroids (ICS), which have been shown to provide an improvement in airway responsiveness and asthma control . Remaini1 after salbutamol ≥12% .Fifteen children were re-evaluated after 1 to 4 months, 8 of whom had had their therapy adjusted and 7 had not. Control of spirometry showed that 7 (87.5%) of the 8 children with a change in their therapy had a negative response to the bronchodilation test and 1 (12.5%) had a positive response, whereas among the patients with no change in asthma therapy 3 (42.9%) had a negative bronchodilation test, and the remaining 4 (57.1%) had an increase in FEVThe same group of patients completed the PAQLQ again at the second visit. Median values showed an improvement in HRQL in those children who had been treated with ICS , and a worsening in children whose therapy had not been modified (6.73 and 6.39), but differences are not statistically significant. The examination of the subscales of the questionnaire indicates that 5 out of 8 children who had been treated with ICS had an improvement in the “activity limitations” domain, whereas only 2 out of 7 children who had not been treated with ICS showed this enhancement.The main goal in the management of the asthmatic child is to obtain control of the disease, but subclinical alterations can be occasionally revealed in the lung function of asthmatic patients under apparent clinical control. No specific guidelines could be found about the approach to this problem, even if several studies reported such discrepancies, and the fact that asthma control is poorly judged when symptoms alone are considered has already been stated ,8,16-19.Korhonen  in 1999 evaluated the treatment policy for asthmatic children in the area of Kuopio, Finland . PEF, spOur study shows a prevalence of 17.3% of positive bronchodilation tests in a group of asthmatic children who were in a symptom-free period, indicating an occult bronchial obstruction. The lung function of these children was more similar to that of children with uncontrolled asthma than to the one of those with controlled asthma and a negative bronchodilation test.The reasons why these children are asymptomatic despite having pulmonary function impairment can be multiple. First of all, an underlying, but still subclinical, pathological process in the airways could be hypothesized, similarly to what happens in some patients with apparently outgrown disease -22. SecoWe believe that patients with a positive response to the bronchodilation test even in the absence of symptoms should be regarded as undertreated. Our data on lung function after treatment figure  show an The administration of the PAQLQ did not reveal any differences in HRQL between children with under-control asthma but a different response to bronchodilation. All of them, however, had a significantly better HRQL than children with uncontrolled disease. Although HRQL evaluation does not help to identify patients in need of further therapy, it is interesting to note that controlled patients with a positive bronchodilation test have some characteristics  in common with those well controlled and not respondent to bronchodilation, while they are more similar to uncontrolled children for other features (lung function).However, PAQLQ re-administration in the 15 children who repeated spirometry because of the discrepancy between clinical and functional data indicates an improvement in HRQL in those who underwent ICS therapy or had an augmentation in the dosage. Their activity limitations seem to be the most concerned, because 5 children out of 8 improved in this domain of the questionnaire.The first studies which identified such discrepancies between lung function and clinical control were conducted 20 to 30 years ago. Even if since then new drugs have been developed, asthma has been widely studied and its diagnosis and treatment have generally improved, the point of asymptomatic airways alterations is still up-to-date. Adequate treatment prevents pulmonary function from worsening: in persistent asthma, ICS suppress airway inflammation, reduce airway hyperresponsiveness, improve asthma control and prevent symptoms . PersistUndertreated patients are exposed to a worse progression of asthma severity and the bronchodilation test, even in the absence of Nitric Oxide measurements, seems to identify those children who are in need of a reassessment of their therapy. Moreover, our results indicate that the pharmacological treatment of an occult bronchial obstruction helps improving HRQL.In conclusion, we believe that it is advisable to perform spirometry in asthmatic children in order to provide adequate therapy for their actual lung condition, which can be revealed by the bronchodilation test. Establishing asthma control solely on the basis of clinical parameters could be insufficient. In the absence of Nitric Oxide measurements, spirometry is a simple examination which is normally performable in every pneumologic or allergologic outpatient clinic and allows an evaluation of the bronchial obstruction status of the lung of the asthmatic school-aged child without any further, expensive investigation.


Both epidemiologic and strain-related factors may contribute to large clusters of tuberculosis patients. Mycobacterium tuberculosis are described as clustered. Cluster size may depend on patient or strain characteristics. In a 7-year population-based study of tuberculosis in Karonga District, Malawi, clusters were defined by using IS6110 restriction fragment length polymorphism, excluding patterns with <5 bands. Spoligotyping was used to compare strains with an international database. Among 682 clustered patients, cluster size ranged from 2 to 37. Male patients, young adults, and town residents were over-represented in large clusters. Cluster size was not associated with HIV status or death from tuberculosis. Spoligotypes from 9 (90%) of 10 large cluster strains were identical or very similar (1 spacer different) to common spoligotypes found elsewhere, compared with 37 (66%) of 56 of those from nonclustered patients (p = 0.3). Large clusters were associated with factors likely to be related to social mixing, but spoligotypes of common strains in this setting were also common types elsewhere, consistent with strain differences in transmissibility.Tuberculosis patients with identical strains of  Many studies have investigated risk factors for clustering, but relatively little is known about the determinants of cluster size , or they are more virulent and are therefore more likely to give rise to secondary cases within the period studied  based on the ISEpidemiologic differences can be explored by examining risk factors for cluster size. Giordano et al.  strains from patients with smear-positive tuberculosis in 1998 or 1999, as examples of strains that had apparently not spread in the population; and from all positive cultures from 2002. Previously identified spoligotypes were defined as widespread if the international database described them as both “ubiquitous” and “recurrent,” “common,” or “epidemic.”Spoligotyping . Cluster size was divided into 4 groups , and assOver the study period, 1,248 cases of culture-positive tuberculosis were diagnosed in patients in Karonga District. RFLP results were available on 1,194 isolates from 1,044 patients. After we excluded 25 isolates because laboratory error was suspected (Cluster size varied from 2 to 37. The determinants of cluster size are shown in >15 people) were found in at least 4 of the 6 geographic areas of the district, and most were found throughout the district. The distributions of the 4 largest clusters are shown in the All of the large cluster strains (>15 people) were compared with the international database , respectively. The spoligotype for strain kps121, spoligotype129, was not similar to any widespread types.The spoligotypes from the RFLP-defined large cluster strains were compared with spoligotypes from patients with positive cultures in 2002, and from patients with smear-positive tuberculosis and unique RFLP patterns in 1998 through 1999. Overall, 9 (90%) of 10 of the large cluster strains had spoligotypes that were identical to, or only 1 spacer different from, previously described widespread spoligotypes. For the patients from 2002, this proportion was 90 (71%) of 126 (p = 0.3 when compared to the large cluster strains), and for the smear-positive unique strains, it was 37 (66%) of 56 (p = 0.3 compared to the large cluster strains).All the spoligotypes that were found in the RFLP-defined large cluster strains were also found among (RFLP-defined) unique strains. Seventeen of the unique strains had spoligotype 59, and 2 others had closely related patterns ; 1 had spoligotype 21, and 1 had a closely related pattern; 4 had spoligotype 53, and 2 had closely related patterns; and 6 had spoligotype 129. Of the 56 patients from 1998 to 1999, none had Beijing spoligotypes, but we have previously described strains with Beijing spoligotypes and unique RFLP patterns in this population  had spoligotype 59, and 10 more had closely related patterns; 11 (9%) had spoligotype 21; 8 (6%) had spoligotype 53, and 2 had closely related patterns; 7 (6%) had the Beijing spoligotype; and 8 (6%) had spoligotype 129. The 36 isolates with spoligotype 59 had 23 different RFLP patterns with a similarity coefficient of 63%.This study suggests that both epidemiologic and strain-related factors may contribute to large cluster size. In large clusters young adults, male patients, and those living in the town were over-represented, all factors likely to be associated with increased social mixing. Similar associations with age and sex have been found previously, in the United States and Denmark. In Denmark the largest cluster was particularly predominant in the capital city  and cluster size, but most patients had sputum smear–positive disease. There was also no statistically significant association with degree of smear positivity (not shown). An overall association with infectiousness would not necessarily be expected: the infectiousness of the first cases of a cluster may be important in determining size, but the first cases for the large clusters, which were found throughout the period of study, are not identifiable. There was no significant association with isoniazid resistance, but only 39 (6%) patients had resistant strains. Isoniazid resistance has been associated with reduced clustering and reduced generation of secondary cases . Virulent strains could lead to large clusters if virulence were associated with increased transmission rates or increased rates of disease after infection (Evidence that strain characteristics may have contributed to cluster size comes from the finding that the spoligotypes of most of the common RFLP-defined strains in this study were identical to, or only 1 spacer different from, widespread spoligotypes already described. Unique RFLP-defined strains from smear-positive patients in the early part of the study were used as a comparison group. Smear-positive case-patients were chosen to maximize the likelihood of transmission occurring; early cases were used to allow time for secondary cases to have been identified if they had occurred. These unique strains were less likely than the large cluster strains to have spoligotypes that were closely related to widespread types, but this difference was not statistically significant, and the spoligotypes that were found in the large cluster strains were also found among the unique strains. Interestingly, strain kps121, which was the only large cluster strain with a spoligotype not closely related to a widespread previously described type, was also the 1 large cluster strain that was clearly decreasing in the Karonga population.The finding of large cluster strains with previously described widespread spoligotypes may suggest that these strains are particularly transmissible or particularly likely to cause disease. Other possibilities are that they are older in evolutionary terms, and thus have had more time to become widespread, or that we are seeing a founder effect in some populations with subsequent spread following human migration patterns. Spoligotype 59 was common in the Malawi population in all groups of patients, clustered and unique, and was associated with a wide diversity of RFLP patterns, which suggests that it may be a longstanding strain in this area. It was also the most common spoligotype found in studies in Zimbabwe and Zambia (


Performing linkage and association analyses on a large set of correlated data presents an interesting set of problems. In the current setting, we have 3554 expression levels from lymphoblastoid cell lines in 194 individuals from 14 three-generation Utah CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees. We formed multivariate expression phenotypes from six sets of genes. These consisted of a set of genes identified by the data providers as showing common linkage to a region of chromosome 14, as well as five other sets suggested by ontological evidence. Using principal-component analyses, we generated seven quantitative phenotypes for expression levels from these six sets of genes. We performed quantitative genome linkage screens on these traits using the expression traits from the third generation of each pedigree. As expected, the strongest linkage signal was achieved when the trait under analysis was the composite of the expressions of genes previously showing linkage to chromosome 14. In particular, this trait produced a LOD score of 5.2 on chromosome 14. The trait also produced LOD scores over 3.5 on chromosomes 1, 7, 9, and 11; this suggests that these genes may be controlled by additional genetic factors on the genome. Subsequent association analyses on the first two generations of these pedigrees identified two polymorphisms on chromosome 11 as significant after correcting for multiple tests. These results suggest that principal-component analyses are useful for the analysis of pleiotropic loci. Furthermore, we have identified two single-nucleotide polymorphisms that may influence the expression of multiple genes linked to chromosome 14. The Genetic Analysis Workshop 15 data set consists of 3554 expression levels from lymphoblastoid cell lines in 194 individuals from 14 three-generation Utah CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees. In earlier work [Principal-components analysis (PCA) is a technique for reducing multi-dimensional data sets into lower dimensions for analysis. The goal is to capture as much variation as possible of the higher dimensional data set in a lower dimensional set. Because microarray expression data typically produces thousands of correlated observations for each array, PCA is a natural analytic technique. Several other groups have shown the utility of PCA for the decomposition of expression data -4; here In microarray data, PCA provides orthonormal bases for the expression array profiles and the gene transcriptional responses. The details are well described elsewhere .Our goal was to perform PCA on several different subgroups of expression phenotypes and then perform linkage analysis on the component scores. We performed the PCA in the R language . In ordeWe investigated the set of expression phenotypes linked to chromosome 14 by Morley et al. . The celFor the strongest linkage signals, we downloaded data from the HapMap project for the CEPH families for SNPs within the two-LOD support . We perfFor a number of phenotypes, SOLAR reported an estimated heritability of 1.0. Further investigation revealed that the individual expression levels as well as the component scores often showed an intraclass correlation (ICC) of more that 50% in sibships. Heritability is often estimated as twice the intraclass correlation in sibships; this would imply a heritability greater than one. This is clearly nonsensical, so we suspect that this indicates a shared environment with an effect on multiple expression levels. We performed a PCA on the 471 genes that had ICC in sibships greater than 0.5 (the "high heritability" genes). We also performed further tests to determine whether these genes contained an over-representation of ontological categories. In particular, we examined the 10 most represented ontological categories in the high heritability genes, and then performed 10,000 re-samplings of 471 genes from all 3554 to determine whether any of these 10 categories were over-represented.We first examined the reliability of the PCA. Using the entire set of phenotypes, we found that the components generated from 14 unrelated individuals had a 98.7% correlation with the components generated from all 110 third-generation individuals. Furthermore, 10,000 repeated samplings of 14 unrelated individuals produced an average correlation of 96.1% with the 14 individuals initially chosen. We took this as evidence that the components were reliable, even using a relatively small number of individuals; we then used the same 14 individuals throughout. The PCA analysis of each of the six categories of gene expression then produced 14 principal components. Recently, Raiche et al. [The VC analysis of the separate phenotypes linked to chromosome 14 yielded fewer significant linkage peaks than originally reported; this was not unexpected because the methodology differed. In particular, the original analyses used a modification of the Haseman-Elston (HE) method ; under sp = 9.8 × 10-6), so we included age in all SNP analyses. We identified 761 nonsynonymous SNPs in these regions, so we set our initial significance threshold to a conservative level of 0.05/761 = 6.6 × 10-5. One SNP, rs10458896, is significant at this threshold . The corresponding q-value is 0.034; this is the only q-value less than 0.05. This is a non-synonymous SNP in KIF18A, a kinesin family member on chromosome 11. In the analysis of all the SNPs, we found 143,798 SNPs in these regions. We set our significance threshold to 0.05/143798 = 3.5 × 10-7. One SNP in an intergenic region on chromosome 11, rs10768321, is significant at this threshold with a p-value of 5.8 × 10-8 and a q-value of 0.008; again, this is the only q-value less than 0.05.For the association analysis, we focused on the five regions with linkage signals over 3.5 for the traits linked to chromosome 14, including the linked region on chromosome 14. We found the first principal component of these traits was strongly associated with age , "nucleotide binding" (p < 0.0001), "ATP binding" (p = 0.0001), and "RNA binding" (p = 0.0002).For the analysis of the top ten ontological categories represented by the 471 "high heritability" genes, we computed empirical As expected, PCA of traits with common linkage to a region on chromosome 14 does show a very strong signal in this region. Several other novel linkage signals appear, including two for the ontological category "cell-cycle." In principle, one would expect some genes from the same ontological category to be controlled by the same master regulators; this has been demonstrated in yeast . HoweverWe also note that the association analyses were performed on a set of individuals related to, but distinct from, the individuals used in the linkage sample. In particular, some individuals from the first two generations of the Utah CEPH pedigrees have been genotyped by the HapMap project. For the linkage analyses, we used only the phenotypes of the third generation. Because the linkage analysis was restricted to a single generation, we only used age as a covariate in the association analyses. A combined linkage and association analyses would be possible if the significant SNPs, rs10458896 and rs10768321, were genotyped on the third generation of the CEPH pedigrees. We would expect that the linkage signal on chromosome 11 would be reduced in a SOLAR analysis including these SNPs as covariates if the SNPs "explain" some of the linkage signal.The "high heritability" genes present a conundrum. We speculate that there may be shared environmental factors that influence many expression phenotypes; these factors would elevate the correlation in sibships and produce high heritability estimates. Because four ontological categories  are significantly over-represented in these high heritability genes, further investigation into potential environment factors modifying these functions may be of interest. These high heritability estimates do not occur when the other generations are included in the analyses; the inclusion of parents and potentially cohort effects reduce the heritability estimates.Performing PCA on a set of genes linked to chromosome 14 increases the LOD score; this cannot be considered stronger evidence of linkage because we anticipate a similar result if the clustering was due to chance. However, the linkage on chromosome 14 is supported by the analysis of all 3554 expression phenotypes where the largest peak is in the same region. We also find strong evidence for other loci on chromosomes 1, 7, 9, and 11 that control the genes linked to chromosome 14.Association analyses were successful in identifying two polymorphisms on chromosome 11 regulating the set of genes displaying linkage to a region of chromosome 14. The association analysis was performed in two steps; first, analysis of only non-synonymous SNPs and then analysis of all SNPs in the regions of interest. The analysis of non-synonymous SNPs yielded a polymorphism on chromosome 11 in KIF18A significant after correcting for the number of non-synonymous SNPs. The analysis of all SNPs in the region yielded a SNP in an intergenic region on chromosome 11 significant after correcting for the total number of SNPs in the regions of interest. Further investigation into these SNPs may show a role in the regulation of a large number of genes.We identified a set of genes that apparently had heritability greater than one. Shared environmental factors may increase the intraclass correlation within sibships. We found four ontological categories  that are significantly over-represented in these high-heritability genes; further investigation into potential environment factors modifying these functions may be of interest.The author(s) declare that they have no competing interests.


To assess the risk of developing Type-1 diabetes among children who were exposed to maternal bereavement during the prenatal or 1-year preconception period.st 1979 through December 31st 2004, and their next of kin. Altogether, 39,857 children were exposed to bereavement during their prenatal life. The main outcome of interest was hospitalization for type-1 diabetes .We identified N = 1,548,746 singleton births born in Denmark between January 1We found the strongest association for type-1 diabetes among children exposed to traumatic father or sibling deaths ; the association was mainly seen for girls .We found evidence to suggest that female fetuses exposed to severe prenatal stress are at increased risk for developing type-1 diabetes. Diabetes is one of the most common chronic diseases among children; the increase in frequency of type-1 diabetes in youth has been among the most concerning aspects of the evolving epidemic. Incidence of type-1 diabetes varies with age, for boys it peaks at 12–13 years, and girls around 9–12 years Certain genetic factors have been associated with type-1 diabetes; but the observed changes in incidence over time cannot be attributable to susceptible genes alone, only in combination with environmental factors. Functions of isolated genes include insulin production and metabolism, protection from β-cell apoptosis, immunity and other unknown factors The link between environmental and lifestyle factors such an increased weight and height st 1979 through December 31st 2004, N = 1,548,746  to identify singleton births in Denmark born January 1st 2004, whichever came first.Information on birth outcomes, such as gestational age at birth and birth weight, were obtained from the Danish Medical Birth Registry which has been computerized since 1973. Annual information on maternal education, residence, and income was retrieved from the Fertility Database at Statistics Denmark, available since 1979. The date of conception was estimated by the date of birth minus gestational age, and the prenatal exposure period included from 12 months before conception until the birth of the child. We categorized children as exposed to bereavement during prenatal life if their mothers lost a child, husband, sibling or parent during the prenatal period, ascertained via the death registry; all the remaining children were included in the unexposed cohort. Cohort members were followed from birth until first diagnosis of diabetes, death, emigration, or December 31th revision, from 1979 to 1993 (249) and 10th revision, from 1994 onwards (E10). We also looked for additional cases from the Pharmaco-epidemiological Prescription Database, in operation since 1995, using the ATC codes for insulin analog drugs (A10A), though there was almost 100% overlap with these cases and those found in the hospital register. If an individual has a diagnosis for both type-1 and type-2 diabetes, they were classified as type-1 since the later is more often misclassified than the former. Also, type-1 diabetes is a more serious disease than type-2 and therefore probably better diagnosed.Diabetic status of children and their parents was ascertained from the National Hospital Registers, which holds information on all discharges from Danish hospitals since 1977; outpatients have been included in the register since 1995. Diagnostic information was based on the Danish version of the International Classification of Disease (ICD) 8st, 2nd, 3rd, and 4th quartile); maternal education ; parental diabetes status ; marital status ; gestational age at birth ; birth weight ; sibling order ; calendar year  and offspring's sex . Age, residence, calendar period, maternal education, maternal income and parental marital status were treated as time-dependent variables as they were extracted from the birth year of the offspring.We estimated incidence rate ratios (IRRs) from birth using log-linear poisson regression models and used person-years as the offset variable. The analysis was performed using PROC GENMOD in SAS version 9.1.3. Unadjusted and adjusted models were generated. Adjusted estimates of IRRs (aIRR) included maternal age ; residence ; income ; and death from other causes. This study was approved by the Institutional Review Board at the University of California at Los Angeles. Individual written consent was not obtained from study participants as there was no record linking the subjects to identifying information.see We identified 39,587 children exposed to bereavement during or before their prenatal life. Children in our study were followed between 2 to 27 years. Exposed and unexposed children were comparable on most background characteristics although mother's of bereaved children were slightly older and had more children, something we expected to see among those who had lost a child see table 1see We found that children exposed to bereavement during their prenatal life were more likely to be hospitalized with a type-1 diabetes diagnosis later in life see table 2To the best of our knowledge, this is the first population based study that has examined the link between prenatal stress exposure and diabetes in the offspring of humans. We found evidence to suggest that female fetuses exposed to maternal bereavement during the preconception and the prenatal period are at an increased risk for developing type-1 diabetes. We observed higher IRRs for traumatic deaths, especially those of a father or sibling. Since we do not have data on all stress exposures or biomarkers of stress hormones we cannot quantify the exposure contrast between the exposed and unexposed. However, we believe that the exposure contrast between the two groups is substantial as bereavement due to death of close family member is one of life's most stressful events. The difference seen for girls and boys could be due to chance or could be related to modification by sex hormones during prenatal life.The main strengths of our study are the large cohort size, long follow-up time and high quality of data. Completeness of type-1 diabetes has been demonstrated in previous studies The etiology of type-1 diabetes and the timing of a causal window is not clearly understood, though the disease is postulated to have a multiple stages. It is possible that bereavement is one of the environmental factors that when combined with a genetic factor initiates the disease process shortly after conception. Type-1 diabetes is a rare disease in childhood and if stress exposure can cause the disease one would not expect common stressors to play a role. The disease could on the other hand be caused by a common cause if that cause produces susceptibility for a second hit later in life; should this be the case one would expect low to moderate association for the prenatal exposure if data were not stratified on the second set of causes. The association between prenatal bereavement and type-1 diabetes was observed mainly among female children, but not among male children, exposed to traumatic father or sibling deaths. Previous studies have demonstrated important sex differences in glucocorticoid programming of the HPA axis and metabolic physiology, but how these differences contribute to differential risk for type-1 diabetes is unknown This study supports the view that type-1 diabetes may have causes that operate in fetal life. We have focused on stress as one of many possible exposures but are aware that stress may just be an upstream cause of other factors of importance such as infections, or changes in dietary habits. Stress exposure may impact the immune system and thus increase the susceptibility of infections. If stress is not a direct cause of diabetes it would explain the rather weak association we find.While one cannot avoid bereavement as a potential stressor during pregnancy one may be able to avoid other sources of stress, or mitigate the effects via therapy. This study and other studies suggest that exposure to severe stress during the critical period that surrounds pregnancy may have life-long effects on the offspring. We did not find a strong link between bereavement and diabetes when the death was non-traumatic or that of a grandparent or maternal sibling, suggesting that only the most severe stress exposures have a programming effect. A small sample human study with a positive finding has been published on markers of glucose metabolism also suggested a link between stress and diabetes The findings of this study may have public health implications especially in settings where sources of intense stress are increasing, such as in areas of political unrest, migration, conflict or environmental disasters. Future research should consist include a large enough sample size to assess affects of timing of exposure and also corroborate the findings in this study.


This model likely can be used to predict thymocyte evolution following therapeutic strategies designed for recovery of the thymus in diseases coursing with thymus involution, such as some primary immunodeficiencies, acute infections, and malnutrition.Intrathymic T cell development is an important process necessary for the normal formation of cell-mediated immune responses. Importantly, such a process depends on interactions of developing thymocytes with cellular and extracellular elements of the thymic microenvironment. Additionally, it includes a series of oriented and tunely regulated migration events, ultimately allowing mature cells to cross endothelial barriers and leave the organ. Herein we built a cellular automata-based mathematical model for thymocyte migration and development. The rules comprised in this model take into account the main stages of thymocyte development, two-dimensional sections of the normal thymic microenvironmental network, as well as the chemokines involved in intrathymic cell migration. Parameters of our computer simulations with further adjusted to results derived from previous experimental data using sub-lethally irradiated mice, in which thymus recovery can be evaluated. The model fitted with the increasing numbers of each CD4/CD8-defined thymocyte subset. It was further validated since it fitted with the times of permanence experimentally ascertained in each CD4/CD8-defined differentiation stage. Importantly, correlations using the whole mean volume of young normal adult mice revealed that the numbers of cells generated in silico with the mathematical model fall within the range of total thymocyte numbers seen in these animals. Furthermore, simulations made with a human thymic epithelial network using the same mathematical model generated similar profiles for temporal evolution of thymocyte developmental stages. Lastly, we provided  T lymphocytes play an important role in the adaptive immune response and their development is fundamental in the process of defense against pathogens as well as tumor cells. These lymphocytes are able to provide immune memory and to respond to hundreds of thousands of different pathogens through specific T cell receptors (TCR) −CD8−) progenitors into the thymus by cortex-medulla junction (CMJ); 2) migration of immature DN cells, from the CMJ towards the subcapsular zone (SCZ) of the thymic lobule, with subsequent proliferation and differentiation of double positive (DP) (CD4+CD8+) cells; 3) reorientation of the migratory process, now towards the medulla, positive selection, and generation of single positive  cells; 4) further interactions of SP thymocytes with epithelial and dendritic cells in the medulla for complete development (negative selection) to ensure central tolerance; and 5) egress of mature SP T cells to peripheral lymphoid organs.Intrathymic T cell differentiation is a complex sequence of biological events, comprising cell proliferation, differential membrane protein expression, gene rearrangements, and massive programmed cell death versus CD8 lineage commitment signal-duration” model postulates that long duration TCR signals instruct DP thymocytes to differentiate in CD4SP T cells and short duration TCR signals instruct DP thymocytes to differentiate in CD8SP T cells At the end of the differentiation route, cells are committed to the T lymphocyte lineages CD4 or CD8. The moment and the exact mechanism of intrathymic lineage commitment, as well as the mechanisms leading to a 2∶1 CD4/CD8 T cell ratio in the thymus remain unknown. Signals elicited by binding of the T cell receptor (TCR) and the CD4/CD8 co-receptor to type I or type II major histocompatibility (MHC)/peptide complex determine the positive and negative selection events, as well as the control of commitment towards the differentiation into CD4 or CD8 single positive cells. It has been shown that a Notch receptor Interactions between thymocytes and the thymic microenvironment are bi-directional: If on one hand cell-cell interactions determine the fate of developing thymocytes, on the other hand they also control the architecture of thymic microenvironments, thus characterizing a “crosstalk” between the lymphoid and the microenvironmental compartments within the organ In recent years, mathematical modeling and computational simulation assumed an important role in the sense of completing the experimental modeling to study a variety of biological events, including those discussed above. In this respect, mathematical modeling has been applied to study some specific issues related to T cell development, as for example, a) to compare the intrathymic development of bone marrow precursors, derived either from young or old donors versus young bone marrow, leading to the notion that further experiments should be performed to better tackle this issue The mathematical model used to study the effect of aging on bone marrow cells colonizing the thymus A second mathematical approach, also applying ordinary differential equations, focused on the naïve T cell compartment (corresponding to the recent thymic emigrants) defined by the presence of TREC formed during T cell receptor gene rearrangement instructive” theory of thymic lineage commitment The first mathematical model to study thymocyte subset dynamics was introduced by Ramit Mehr and co-workers, aiming to capture the essential features of T cell differentiation and selection As regards thymocyte subset dynamics, a single experimental system of transient perturbation of T lymphocyte homeostasis was evaluated in combination with a mathematical model, to quantify dynamics of mouse T cell differentiation −/− and CCR9−/− knockout mice. The model also predicted that competition between thymocytes for interaction space and cell dissociation rate would determine the T cell development and the lineage commitment to CD4 or CD8 The mathematical model introduced by Sol Efroni and co-workers was different in that it also took into account the spatial information in thymocyte development, being based on reactive animation Overall, the data summarized above indicate that the use of mathematical models in the study of the mechanisms governing thymocyte migration may provide new clues for designing therapeutic strategies targeting T cell precursors and to predict abnormalities of such process.−CD8−), double positive (CD4+CD8+) and single positive (CD4+CD8− and CD4−CD8+) thymocytes, as well as epithelial cells, and the influence of chemokines in the cell migration. This model, different from the others discussed above, uses in the simulations, epithelial networks obtained from experimental data  to induce thymocyte differentiation and migration.We introduced herein a mathematical model based on cellular automata The parameters of our mathematical model, described in details in the We first applied the our cellular automata model to approach the production and the diffusion of chemokines in the digital lattice, including the spatial gradient responsible for thymocyte migration. The relevance of the study with chemokines lies on the fact that it can help us to determine relevant molecules involved in thymocyte migration, in respect to each stage of thymocyte development. The spatial distribution for each chemokine depends of the epithelial network used With the automata cell model we can observe the migration and development of a given thymocyte from the initial stage, where only DN cells exist in the lattice, until a steady state where we find thymocytes in all stages of development adopted in the model  see the. The thyAccording to our model, in normal conditions the first cell divisions occur at ∼2 days, when the DN cells, guided by CXCL12 gradients  reach thap = 5.6 days required for apoptosis in the model has not yet been achieved. When DP cells initiate apoptosis (15% of DP cells/day), around day 9, the number of single positive thymocytes decrease and stabilizes .In the course of DP cell migration, positively selected cells evolve to CD4SP or CD8SP thymocytes and continue migration towards the medulla, as shown in abilizes .InterestIt should be pointed out that the population of the thymocytes in each developmental stage is strikingly similar to that observed experimentally, the average total number of thymocyte obtained from the mathematical model is approximately 8587.2 cells, where 5% of the thymocytes are in the DN stage, 80% in the DP stage, and 15% in the SP stage 3, that corresponds to ∼0.0042% of the normal adult mouse thymus volume: ∼55 mm3, as assessed by nuclear resonance imaging and volume displacement Although the mean total thymocyte numbers obtained from the mathematical model (∼8587.2 cells) is smaller than the number of cells experimentally observed (160–320 million cells) cT parameter, which defines the average number of successive intT interactions of a given DP cell with the same epithelial cell needed for differentiation into CD4SP thymocytes or, otherwise in the CD8SP cell subset. The CD4/CD8 ratio increases with time after the fifth day (when the first SP thymocytes appear), from zero to reach a peak around 3 next two weeks of simulation (∼15 days). From this point on it decays, reaching a stationary value around 2, after 70 days of simulation (cT = 1.53 gives the CD4/CD8SP ratio of 2∶1 (2.09±0.06)  depends on several factors, such as the orientation of the thymocyte during migration, the spatial distribution of the epithelial cell in the neighborhood of that particular thymocyte, the number of thymocytes and their spatial distribution in the neighborhood, etc.In the present model, the CD4SP/CD8SP thymocyte ratio is controlled by the mulation . This be09±0.06) . Of noteWe also verified in the mathematical model presented herein that the time of permanence of each thymocyte in the lattice is very close to that observed biologically The distribution of permanence time for the total thymocytes and for each differentiation stage  can be seen in γ-radiation dose to induce thymus regression and to study the expression and function of extracellular matrix receptors in distinct thymocyte subsets emerging during the short-term autoctonous repopulation of the organ. As shown in rd day of post-irradiation, when the repopulation of the mouse thymus starts. As seen in The results of our mathematical model are in agreement with other experimental results and reproduces experiments of mouse sub-lethal irradiation In these simulations, the relative numbers of CD4 and CD8 SP thymocytes increave above the normal values. This is likely explained by the fact that at the beginning of simulation (first week), the spatial competition between thymocytes is relatively small and DP thymocytes have not undergone apoptosis by lack of interaction with the epithelial profile, then there is the overshoot of single positive thymocytes. After the first week of simulation, the great number of thymocytes in the lattice increase the spatial competition and after a time Tap, DP cells can die by apoptosis by lack of interaction with the epithelial profile (15% of DP cells/day), then the number of single positive thymocytes decrease and stabilizes thereafter.To compare the results obtained with the mouse epithelial network, we also evaluated the dynamics of the model with a human epithelial network derived from data obtained experimentally in our laboratory, by immunolabeling a histological section of a normal child thymic tissue with a pan-cytokeratin antibody. In this epithelial network we observed the same density of occupation, as depicted in cT also follows a decay in a power law, as we found using the mouse epithelial network, but with the exponent a = −20.5. A 2∶1 ratio using the human epithelial network was achieved for cT = 1.39, thus smaller than that obtained with the mouse network.The temporal evolution of thymocytes obtained with the human epithelial network, Overall, the results derived from the mathematical model indicate that the thymus architecture  may also be important to thymocyte lineage commitment and may influence the CD4/CD8 cell ratio.We further evaluated the putative influence of the thymus architecture using an epithelial network that was generated randomly, as shown in After 200 days of simulation, the average number of thymocytes in the lattice was around 16950 cells, two times the number of cells obtained with the mouse epithelial network. The large amount of thymocytes in the lattice can be explained by the absence of large clusters of the epithelial network, which increase the availability of epithelial cells to interact with thymocytes and decrease the apoptosis by lack of stimulus. Interestingly if at day 50 of simulation is reestablished the normal mouse epithelial network, the selection process and proportions of thymocyte populations normalize after seven weeks.The small number of CD8SP thymocytes in the lattice (∼28 cells) can also be explained by the absence of large clusters of epithelial cells, with a consequent increase in the probability of a given DP thymocyte to interact with the same site in its neighborhood and to promote the differentiation into CD4SP thymocytes.The mathematical model herein developed for thymocyte migration and differentiation is in agreement with experiments that evaluated thymus repopulation from double-negative cells, following sub-lethal irradiation in mice versus CD8 lineage commitment. This can be determined by the duration of the TCR signal: Long duration signals favor development of CD4SP cells and short duration signals rather favor differentiation into CD8SP cells In our mathematical model, we also approached the CD4 Since the simulations were performed using a 2-D lattice, the results derived from a 3-dimensional version of the model may present some differences in respect to thymocyte moving interactions and divisions. Considering that a 3-D sponge-like epithelial network has one more degree of freedom, we could expect changes in the thymocyte spatial competition and in CD4/CD8 ratio. Nevertheless, it its worthy to point out that the final numbers of thymocytes obtained when we took into account the whole volume of the organ, fell within the range actually seen in the thymus stimuli, and this is important for normal T cell differentiation. More recently, we further developed this concept, proposing that thymocyte migration is actually derived by a resulting migration vector, which in turn appears as a consequence of various migration vectors (each one corresponding to a given ligand/receptor pair interaction) that can differentially contribute to generating the resulting migration vector Previous studies showed that chemokines produced by thymic microenvironmental cells, together with extracellular matrix proteins , have a pivotal role in thymocyte migration, and data suggest a combinatorial role for these molecules in this migratory process In any case, the cellular automata-based mathematical model for thymocyte differentiation and migration could be useful in evaluating abnormal thymocyte differentiation/migration and in predicting thymocyte evolution following therapeutic strategies designed to recover the thymus in diseases coursing with thymus involution, such as some primary immunodeficiencies, acute infections, and malnutrition http://www.intel.com) (see the basic program on http://g2.sourceforge.lattice), labplot (http://labplot.sourceforge.lattice/) and the xmgrace (http://plasma-gate.weizmann.ac.il/Grace).The computational language used in the mathematical model implementation was FORTRAN 90  = [0, 1) that represents the MHC/peptide of the microenvironmental cell that will interact with the thymocyte, as explained below in double-positive cell rules. Finally, the lattice update was made in parallel, whereas all sites were updated simultaneously.The simulations were performed using a two-dimensional lattice representing a section of the thymic lobule (cortex and medulla) derived from actual C57BL/6 mouse thymus histological cross section as seen in S and its migration is influenced by gradients of concentration created by the diffusion of different chemokines produced by the epithelial network. Specific sites overlapped by the epithelial network produce K amounts of chemokine in each lattice update. The thymocyte moves preferentially towards the site of its neighborhood in which the concentration of the chemokines is higher. In this case, the site is chosen to move with probability:The thymocyte moves in each lattice update with speed Ci′,j′ represents the concentration of the chemokines in the site , the sum run over the Moore's neighborhood of  (first and second neighbors), the actual site of the thymocyte. If another thymocyte exists in the site  chosen for the movement, the thymocyte stays immobile in the site  until the next lattice update, when then again a new site  is chosen for the movement. Which chemokine source will guide the thymocyte movement depends on the developmental stage of each cell.Where movp will be launched. Thus, the migratory movement in the two-dimensional lattice is preferentially between the sites overlapped by epithelial profiles.Along with thymocyte migration, if the chosen site for migration is overlaid by the epithelial network, an additional chance ∇2 = 0) solved by finite differences The diffusion of the chemokine is obtained using the Laplace's equation  (first neighbors). The chemokines in each lattice updating also undergo enzymatic cleavage d of their concentration in each site. Since in this mathematical model the chemokine sources do not change, the values adopted for the concentration to each chemokine in the lattice are those reached in the steady state. The rules for emigration and for each thymocyte stage in the model are described below. Should we state that, after pre-designing this mathematical model, we checked whether it would fit experimental data on thymus replenishment following sublethal γ-ray radiation in vivo conditions.The sum run over the Newman's neighborhood of site  is chosen, representing its TCR receptor in the interaction with the epithelial network. Positive selection or apoptosis of a given DP cell will take place when:To each new DP cell, a random variable DPN = N interactions of the DP cell (i) with the sites overlapped by epithelial network, occurring during its migratory process.Where, i) or in Newman's neighborhood, and have a duration time of intT. After the intT, the DP cell that had stopped, starts the migratory process again, allowing to occur new interactions with the epithelial network, including the very last one.The interactions between a given DP thymocyte and the epithelial network occurs in its own site (N(i) is smaller than θ1, high affinity exists between the DP cell TCR(i) receptor and the MHC/peptide of the corresponding epithelial cell, resulting in thymocyte death by apoptosis. If ΔN(i) is larger than 2θ, a low affinity exists between the DP cell TCR(i) receptor and the MHC/peptide of the epithelial cell, also resulting in the thymocyte death by apoptosis. The positive selection and differentiation in the SP cell occurs when the DP cell TCR(i) receptor interacts with an intermediate affinity. We can also identify ΔN(i) as avidity. Indeed, the TCR is probed with N molecules MHC plus peptide. In the present model, if the DP cell does not succeed in interacting with N sites overlapped by the epithelial network before a time apT, then the thymocyte will also die by apoptosis due to lack of stimuli.If ΔBiologically, if the TCR has a very low affinity with the MHC/peptide complex, the thymocyte dies by apoptosis, due to lack of interaction. Similarly, if the TCR has a high affinity with the MHC/peptide complex, the thymocyte also dies by apoptosis, and this largely prevents the development of auto-reactive T cell clones, and thus autoimmune diseases. We should point out that, despite the fact that the generation of TCR specificities involves a complex process gene rearrangement N in equation 3 only determines the number of DP cells undergoing positive selection. Those positively-selected DP cells exhibiting a given number of successive interactions intT with the same site on average larger than cT, will differentiate in the CD4SP cell and otherwise in the CD8SP cell. The SP cell, either CD4 or CD8, continues the migration towards the medulla, induced by the CCL19 and CCL21 chemokine gradients. Within the medulla SP cells also undergo a selection process after SPN interaction with epithelial cell profiles, similar to what happens with DP cells (equation 3). This negative event ensures the complete thymocyte development as well as central tolerance.In the present model we consider that the duration of the TCR-induced signal determines the lineage commitment of each given T cell When studying thymocyte egress, we initially considered that the post-selection SP cells are guided through the S1P gradient and then leave the thymus, crossing the endothelial barrier at the cortex-medulla junction Movie S1Thymocyte migration and development in the mice epithelial network. Computational simulation of thymocyte migration and development in the lattice overlapped by mice epithelial network. Legend of colors: black for the epithelial network, red for DN cells, orange for DP cells, blue for CD4SP cells, and cyan for CD8SP cells. The black color is shown for the site with a thymocyte and a portion of the epithelial network.(3.46 MB MOV)Click here for additional data file.Movie S2Thymocyte migration and development in the human epithelial network. Computational simulation of thymocyte migration and development in the lattice overlapped by human epithelial network. Legend of colors: black for the epithelial network, red for DN cells, orange for DP cells, blue for CD4SP cells, and cyan for CD8SP cells. The black color is shown for the site with a thymocyte and a portion of the epithelial network.(2.23 MB MOV)Click here for additional data file.Movie S3Thymocyte migration and development in a mathematical epithelial network. Computational simulation of thymocyte migration and development in the lattice overlapped by a random epithelial network mathematically generated. Legend of colors: black for the epithelial network, red for DN cells, orange for DP cells, blue for CD4SP cells, and cyan for CD8SP cells. The black color is shown for the site with a thymocyte and a portion of the epithelial network.(4.20 MB MOV)Click here for additional data file.File S1FORTRAN90 PROGRAM(2.21 MB GZ)Click here for additional data file.


C. elegans development were incomplete, and little is known about how these patterns change as a function of genetic background. We used microarrays that comprehensively cover known and predicted worm genes to compare the landscape of genetic variation over developmental time between two isolates of C. elegans. We show that most genes vary in expression during development from egg to young adult, many genes vary in expression between the two isolates, and a subset of these genes exhibit isolate-specific changes during some developmental stages. This subset is strongly enriched for genes with roles in innate immunity. We identify several novel motifs that appear to play a role in regulating gene expression during development, and we propose functional annotations for many previously unannotated genes. These results improve our understanding of gene expression and function during worm development and lay the foundation for linkage studies of the genetic basis of developmental variation in gene expression in this important model organism.Gene expression is known to change during development and to vary among genetically diverse strains. Previous studies of temporal patterns of gene expression during  C. elegans genome was sequenced C. elegans genome likely have no obvious phenotypes when singly deleted, as there have been several genome-wide RNAi screens for function Microarray analysis of gene expression has provided considerable insights into biological processes. The data generated through this method allows predictions of function to be made for previously uncharacterized genes based on the observation that genes with similar function often show a common expression pattern. Thus, genes of unknown function that are expressed similarly to a set of genes whose function is known can be putatively assigned to a gene ontology (GO) term C. elegans development has been studied extensively, many of the studies were done in the pre-genomic era. In the past ten years, several groups have completed microarray time courses examining various aspects of C. elegans development Drosophila data that showed nearly every gene changing over developmental time, the early studies identified relatively few genes that showed significant variation based on stage Although a priori knowledge of the number of clusters into which the data should be divided.Additionally, in using microarray data from a time course, it is important to recognize the temporal nature of the data. Most time series data is currently analyzed using methods that were initially developed for static data, and while these methods have provided biologically meaningful insights, they assume that each of the data points is independent C. elegans, N2 (Bristol) and CB4856 (Hawaii) C. elegans genes, and applied methods specifically developed to analyze time courses and to discover regulatory motifs in order to identify developmentally regulated genes and to divide the data into biologically meaningful clusters. By studying a wild isolate in addition to the laboratory strain we are able to identify the types of genes that vary over development and to assess the amount of natural variation in expression levels present in the C. elegans population.In this study we compared transcript levels across development between two genetically divergent isolates of C. elegans development on a genome-wide scale we used Agilent 4×44k C. elegans oligo microarrays to measure expression of 13,474 genes at 6 different developmental time points  in two different strains (N2 and CB4856). We analyzed the data using a two-way ANOVA to identify the relative impact of strain, stage, and strain by stage interaction on the observed transcript levels. 12,390 (91.9%) transcripts were found to have a significant stage effect, 2,797 (20.8%) were found to have a significant strain effect, and 283 (2.1%) were found to have a significant strain by stage interaction term , Duf-19 (p = 2.4e-5), major sperm proteins (p = .0035), protein tyrosine kinases (p = .000953), protein tyrosine phosphatases (p = 6.21e-15) and serine/threonine kinases (p = 4.03e-7). Genes containing Math-BTB or DUF-19 domains are preferentially deleted in CB4856 when compared to the N2 genome, relative to other gene families Many of these gene families have been implicated in innate immunity Of these genes, none are highly expressed during the egg stage . Every oThe Short Time series Expression Miner (STEM) was used to cluster the microarray data due to its ability to take into account the temporal nature that is inherent in a developmental time course The clusters recapitulate known biology. Clusters 2–5 and 7 show significant enrichment for genes that are in the gene ontology (GO) category of multicellular organismal development, which is the most significantly enriched GO-term, as well as embryonic development ending in birth or hatching. Clusters 2 and 4 are also enriched for the GO-terms larval development (sensu nematoda) and post-embryonic development. The general developmental role can be broken down further. Cluster 2 shows enrichment for genes related to hermaphrodite genitalia development and sex differentiation. This cluster shows a peak in the egg stage as well as the L3 and young adult stages. It is known that the cells involved in the development of the gonad are born in the embryo, differentiate during L3 and have matured by the time egg laying begins in the young adult stage C. elegans early lineage specification Combining current knowledge with the clustering results can also help to uncover potentially novel biology. Clusters 3, 4 and 7 are all enriched for cell-cycle response genes and for terms related to development. However, their different profiles may indicate different functions within the larger class of genes relating to the differential timing of developmental programs. Cluster 7 is the most complex of the three clusters, with many GO-terms associated with the genes in the cluster, ranging from M phase to intracellular organelle part. The genes belonging to cluster 3 appear to be mostly responsible for the early embryonic cell divisions and patterning as they are also enriched for P-granule function and pole plasm location, both of which are extremely important in One surprising observation from this data is the prevalence of GO-terms related to neuronal development or function that appear in the developmentally organized clusters, especially clusters whose expression peaks after the embryonic stage. 80 new neurons are born during the larval stages of development—mainly during the L1 and the L2 larval stages, although the PDA neurons are born during L3 and several neurons that are born in the embryonic stage have been shown to reverse polarity in the larval stages In order to identify motifs important for developmental regulation of expression levels, the genes belonging to the significant STEM clusters were analyzed using FIRE C. elegans and C. briggsae. The conservation score measures the fractions of 7-mers that are better conserved than the motif, i.e. a conservation score of 1 means that the motif is better conserved than all 7-mers. For the data without the young adult stage and using only the significant clusters from STEM that were enriched for GO-terms pertaining to development, 19 motifs were found—11 from the 5′ promoter region and 8 from the 3′ UTR. Of the 19 motifs found in the smaller dataset, 7 were also found in the larger one. Some of the motifs that were seen in the smaller dataset, but not the larger, could have been obscured through the inclusion of many more genes not directly related to development. As much of worm biology is still unknown, the exclusion of clusters not directly relating to development could also obscure relevant biology. This is shown by the fact that several known developmental motifs are found in the larger dataset but not in the smaller.59 motifs were found in the complete dataset—14 motifs from the 3′ UTR and 45 motifs from the 5′ end. Of these motifs, nearly half are highly conserved (conservation index >.9) between C. elegans NR2A4 family C. elegans NHRs that are most homologous to the vertebrate HNF4 are classified as belonging to the supplementary nuclear receptor (supnrs) group I, which consists of 24 members The FIRE analysis is able to recapitulate known biology. For example, the HNF-4 motif was found in both analyses. HNF-4 is the hepatocyte nuclear factor 4, a nuclear hormone receptor that is the vertebrate homolog to the The E2F motif was found in the dataset that did not include the young adult time point. E2F proteins have been shown to be important for cell-cycle progression and development in mammals C. elegansC. elegans, EGL-43, is necessary for the AC/VU cell fate specification The GATA-1 motif found in the larger data set belongs to the GATA transcription factor ELT-2 that is known to regulate most intestinal development in C. elegans and vertebrate development MicroRNAs (miRNA) are known to play a large role in C. elegans genomes, most of whose targets and binding sites remain unknown, and many of which have developmental phenotypes when deleted In addition to the miRNA binding sites, several other motifs were identified in the 3′ UTR. There are over 500 RNA-binding proteins that have been annotated in the Of the motifs identified by FIRE, there are seven new 5′ motifs and two novel 3′UTR motifs found in sets of genes that are significantly enriched for terms relating to development, including: positive regulation of growth, development, embryonic development, development (sensu metazoa), organ development, reproduction, hermaphrodite genitalia formation, cuticle components, and signaling and ion channels , 3, S4. When compared to the yeast genome, the worm genome is severely underannotated with regard to gene ontology. Of the roughly 9,000 genes that are placed into significant clusters by the STEM algorithm, over 2,600 genes currently have no GO-annotations of any kind in the most current release of the ontology (4/20/08). Using clustering and motif analysis to divide genes into sets of putatively functionally related genes, and applying prior knowledge regarding the function of the known motifs, it becomes possible to propose annotations for previously unannotated genes. The motif analysis is useful because it can be used to divide clusters into smaller classes with more narrow functions. We propose general annotations for these 1,568 previously unannotated genes .For example, cluster 7 is composed of genes responsible for reproduction and development, but also genes responsible for mismatch repair and other cell-cycle functions that require DNA binding. While both functions are closely related, they represent distinct cellular processes. By using motif analysis, we are able to separate the functions in genes with no known function.Ceh-44, which is the C. elegans homolog, is known to function in neuronal development and is enriched in larval neurons and thus the genes that possess this motif in their promoter regions are likely involved in neuronal development We propose that those genes in cluster 7 with the motif [AT]CG[AG]A[AGT]TA[ACT] are involved in nucleotide binding, while those with the motifs [CGT]CG[AC]GA[AT][CG][AGT], .[AG]ATCGAT[AT], .[ACT]A[CT]GCGC and .CGA[AC]G[AC]A[ACT] are involved in processes that are related to reproduction . It is pAn additional 338 genes have a significant motif in their promoter region, but the genes with these motifs have no known coherent function.C. elegans. Over 90% of the genes were found to vary significantly over developmental time, and 71% of the genes were placed into clusters that represent patterns of differential expression over time. 20% of the genes display significant variation due to strain. A small but significant fraction of the genes display strain by stage interaction effects; that is, their pattern of expression over time differs between the two isolates.In this study, we have identified the relative contribution of strain, stage, and strain by stage interaction on gene expression across development between two genetically divergent isolates of C. elegans and the gene classes that have been implicated are among the fastest evolving gene families in the C. elegans genome C. elegans immune system is genetically hard-wired, large amounts of natural variation are needed in order to respond to the broad range of pathogens that a worm might encounter during its lifetime Of the 283 genes with strain by stage variation, 58 have been directly implicated in innate immunity. Additional genes in this set belong to families involved in innate immunity, but the genes themselves have not been picked up in a screen. These genes are likely involved in innate immunity, but have not yet been identified because only a small subset of possible pathogens have ever been tested on Diverse expression patterns are observed in the genes that are involved in innate immunity; however, none of the genes are expressed at a high level during the egg stage. This could be because the eggshell provides better protection against pathogens than the cuticle of the larval and young adult worms, thus the embryonic worm does not have to commit resources to pathogen defense. Additionally, the major means of pathogenesis appears to be through ingestion Motif analysis of the 5′ promoter region and the 3′ UTR of the genes in each of the clusters led to the discovery of novel motifs that may have functional roles in development. Two novel motifs, [ACG][AG]CTTA[GT]A from the 5′ region and [AG]A[CT]A[AG]AT[CT] from the 3′ UTR, are enriched for the same GO-terms, namely structural constituent of the cuticle, ion/anion transport, and phosphate transport. Because 3′ UTRs tend to be targets for miRNAs, and miRNAs tend to be negative regulators of their targets, it is possible that these two motifs represent a mechanism for both positive and negative regulation of the same process.C. elegans nervous system Uncovering the phenotype of previously identified miRNAs is an open field, as the identification of miRNAs through sequencing has outpaced the study of their function. In our analysis we identified the binding site for miR-238/239. Currently, there is no known function for these miRNAs. However, their targets are enriched for receptor activity and neurotransmitter binding. It has been previously shown that miRNAs are responsible for at least one case of left/right patterning in the C. elegans genome is unannotated with regard to function or GO-term category. In our set of roughly 9,000 genes that cluster using the STEM algorithm, over 2,500 have no known function or GO-annotation. Although annotation using small scale, directed experiments is often more accurate that using large scale data, many of the unannotated genes will likely have no obvious function or their function would have been identified in one of the many RNAi screens in C. elegans. By combining clustering with motif analysis we were able to separate the function of large clusters, which should provide a more accurate annotation for these genes. We have proposed general GO-terms for 1,568 previously unannotated genes.Much of the C. elegans. Many of the genes identified belong to the innate immune system. Because the innate immune system is hard-wired, genetic diversity must be present within the species to allow for the varying pathogen exposures based on environment. As these genes are expressed even in the absence of the pathogen, they may also serve another developmental function. In addition, we show that by combining clustering with motif discovery, biological coherence of clusters can be increased, aiding large-scale annotation efforts.This work provides new insights into the type of genes that differ between natural isolates of Caenorhabditis Genetics Center . Strains were maintained according to established procedures Wild-type N2 (Bristol) and CB4856 (Hawaii) worms were obtained from the Hermaphrodite-only worms were grown on 10 cm plates of Nematode Growth Medium agar (NGM) seeded with 1 mL of OP50 and kept at a constant temperature of 20°C. Care was taken to ensure that worms on the plates remained unstarved for more than three generations before using the populations for RNA extraction. Worms were synchronized as previously described The protocol was adapted from C. elegans 4×44k oligo microarrays for each array, according to the manufacturer's protocol. Samples were loaded into each array randomly on the slide, so that each slide did not contain more than one sample from each time point. The slides were scanned using an Agilent DNA microarray scanner and the data was extracted using Agilent Feature Extractor (version 9.5).RNA was labeled using the Low RNA Input Fluorescent Linear Amplification Kit  according to the manufacturer's instructions. The reference used is a 50∶50 N2∶CB4856 combination of RNA isolated from separate plates of hermaphrodite only mixed-stage populations. Four replicates of each time point were completed. For each time point two of the experimental samples were labeled with Cy3 while two were labeled with Cy5. 850 ng of an experimental sample and of the reference were hybridized to Agilent C. elegans gene ontology was downloaded from the Gene Ontology Consortium on April 20th, 2008 The array data was uploaded to the Princeton University Microarray Database (PUMAdb) for processing. The data was collapsed by SUID, using the average value of each probe. Spots were considered good data if intensity was well above background and the feature was not a nonuniformity outlier Only genes with greater than 80% good data were kept for further analysis. Missing values were imputed using KNN-impute in the MultiExperiment Viewer Table S1Supplementary (0.06 MB XLS)Click here for additional data file.Table S2Supplementary (0.06 MB DOC)Click here for additional data file.Table S3Supplementary (0.12 MB DOC)Click here for additional data file.Table S4Supplementary (0.37 MB DOC)Click here for additional data file.Table S5Supplementary table 5(0.18 MB XLS)Click here for additional data file.Table S6Supplementary table 6(0.24 MB XLS)Click here for additional data file.


This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts.Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals’ eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses.This treatment resulted in advanced cortical cataracts that developed 5–11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes.2 with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O The in vivo behavior of functional cells and their descendents after receiving oxidative damage from reactive oxygen species (ROS) is of major biologic importance. Equally important are the agents and mechanisms involved in producing that damage as well as its cell and tissue signature and its repair. We have explored the sequence of these events in a single layer of lens epithelial cells (LECs) on the surface of the front half of the mouse lens that migrate and differentiate to become the internal lens fiber cells and then the lens fibers. For the source of oxidative damage, we have used soft X-rays. The literature contains ample evidence that several types of radiation, such as heavy charged particles ,2, neutrThe first set of 12 irradiated and 12 control C57BL/6 female mice were two months old at the time of the primary radiation experiment and were followed for timing and extent of cataract development by monthly slit lamp examinations for 11 months after the irradiation. A second group of 12 irradiated plus 12 control animals that were also two months of age, similarly irradiated, and of same sex and strain was used to provide irradiated and control lenses, respectively, for cell culture of LEC, half for clonal studies and half for metaphase spreads. A third group of such mice were used for antibody studies of 8-hydroxyguanosine (8-OHG) and DNA repair antibody presence 72 h post-irradiation. Lenses were isolated by posterior excision from the extirpated eyeball 2 min after euthanasia of the mice by cervical dislocation. Only females were used to avoid the stress and fighting damage often seen in separately weaned and caged males of this strain. These specific pathogen-free animals were purchased from Harlan Laboratories, Inc  and housed four per cage and fed the standard maintenance Purina mouse diet 5015 . Humane protocols for maintenance and euthanasia from the University of Washington IACUC committee and the national AALAC organization were followed.When used for the comet assay, the quickly extirpated lenses were placed immediately over ice to prevent enzymatic activity. Lenses for immunohistochemistry were immediately placed in Carnoy’s fixative. Lenses for confocal examination were placed sequentially in the Hoechst 33342 and the dihydrorhodamine dyes, which were diluted in Eagle’s minimal essential medium (MEM) immediately after removal, with the total period in these dyes not exceeding 2 h. Controls in which the dyes were followed from the first to the fourth hour showed no change in the dye penetration or in the final confocal readout. Further, the differences seen in dye presence between the previously irradiated lenses and the non-irradiated control lenses did not change over the length of time that they were allowed to remain in the dye solution.A cabinet X-ray machine  was used to irradiate mice placed 13 inches from the source. Delivery of 11 Grays (Gy) of X-rays was at 5 mA, 100 kV over 5.5 min. This is a low energy delivery compared to energy delivered by a 300 kV therapy machine or a Cesium source. Five mice at a time were placed on a circular rotating base with heads to the center and the remainder of the body including the tail covered with 3 mm of lead. Thus, only the heads were exposed to the x-radiation. The settings and delivery were measured by specialists from the University of Washington Environmental Health Division using a Victoreen meter. The mice were anesthetized with a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) diluted with saline 12.6 fold to deliver anesthesia at 0.02 ml/gm of bodyweight. This was injected intraperitoneally (I.P.). just before the irradiation. They remained anesthetized for approximately 30 min. None of these animals in either the control or the head-irradiated groups showed any signs of distress, reduced physical activity, or reduced food intake during the study period from the beginning to the conclusion of the experiment 11 months following the irradiation. There was a general hair loss from the head that was obvious two weeks after irradiation with re-growth of melanin-free hair over the subsequent three to four weeks. Two mice developed sufficient subsequent scar tissue near one eye in each animal that while not covering the eye, the scar tissue prevented an accurate slit lamp reading of cataract development on that side of the face.th month post-irradiation by slit lamp using a Kowa SL14 hand held slit lamp  after dilation with a 3:1 volume mixture, respectively, of tropicanamide  and phenyl hydrochloride , human use ophthalmic solutions that achieved full dilation. The viewer was blinded as to the animal’s previous treatment. The degree of lens opacity was rated by half steps from 0 (completely clear) to 4 (complete opacity of a mature cataract). When lenses were removed from the euthanized animals for the confocal studies, they were first scored for opacity using a dissecting microscope. Thus, in each sacrifice of two mice each in the X-ray and the control groups at each time point at one, three, five, and seven months, the slit lamp cataract readings increased in severity in the irradiated mice as noted in the Results section. The surgically removed lenses were immediately examined under a dissecting microscope and rated separately by two persons without knowledge of the irradiated or control status of the donor mouse. There was no more than a 0.5 difference on the 0 to 4 point rating scale between the live animal slit lamp scoring and that made under a dissecting microscope for those lenses removed surgically for confocal study (notations of 1+ to 4+ indicate that the rating level had reached that stage in either type of examination). Confocal, fluorescent dye readings were done immediately after the dissecting microscope reading. These confocal readings rated the corresponding statuses of the LEC nuclear DNA clearance, the internal lens DNA distribution (both fragmented and in free form), and the ROS presence. These, in turn, corresponded with the live animal slit lamp and the post-sacrifice dissecting microscope readings for increasing lens opacity with all three factors increasing with time beginning at five months post-irradiation. It is noted that the appearance of the individual cataracts increased over time but was nevertheless sporadic with some not appearing until the seventh month or beyond. Thus, the selection of donors for confocal studies was made from among those mice presenting with cataracts as determined by slit lamp examination. However, as no cataracts were present at one month and three months post-irradiation, random animals from each group were selected for confocal study at these times.The irradiated mice and non-irradiated controls in group 1 were examined every 30 days through the 11Groupings of two mice each from the irradiated group and their non-irradiated controls were sacrificed at three, five, and seven months post-irradiation for living lens collections and for immediate exposure to the fluorescent dyes listed below followed by dual laser confocal examination. Sacrifices 72 h post-irradiation were from the third group of mice and were fixed in Carnoy’s fixative and used to prepare 10 μM lens tissue sections using fluorescence antibody studies of the DNA adduct, 8-OHG, and the DNA repair protein, XRCC1. Sacrifice of mice for LEC in vitro culture studies was at 72 h post-irradiation followed by culture as noted below. Mice were first anesthetized then sacrificed by cervical dislocation.The integrated comet alkaline gel migration assay as described in Singh et al.  integratBriefly, a computer-assisted Zeiss 2-photon LSCM  was used on whole lenses immediately after extraction and subsequent 60 min immersion in MEM media containing 10 uM Hoechst 33342 dye for DNA detection and quantitation and 25 uM dihydrorhodamine (DHR) for ROS detection and quantitation. For LSCM settings and dye dilutions see Pendergrass et al. . The souAll antibody staining was by primary and secondary antibody technique of Carnoy’s-fixed, paraffin-embedded serial sections with viewing and photography using a Bio-Rad  computerized microscope with camera attachment. The antibody used for 8-hydroxyguanosine staining  was rabbit polyclonal anti-mouse. The primary antibody used for XRCC1 staining was also rabbit polyclonal anti-mouse from the same source. All secondary antibodies were fluoresceinated goat-anti rabbit, which was also attained from the same source. Control slides substituted non-specific rabbit IgG for the primary antibody also from Alpha Diagnostics. Paraffin block preparations fixed in Carnoy’s fixative were found to be superior for sections and antibody stains when compared to frozen sections. Coomassie blue dye was used on a Carnoy’s-fixed tissue section to illustrate the aggregated protein status in a 4+ cataract.2. Twenty-four h before fixation in Carnoy’s fixative, the cells growing on the coverslips were exposed to 0.01 µg/ml colchicine overnight to accumulate metaphases. The coverslips were mounted on glass slides and the resulting metaphase spreads that were treated by Wright-trypsin G-banding procedures were read by one of us (K.S.) for chromosomal aberrations. To increase the number of metaphase cells scored, we have included in From the second similarly irradiated group, noted above, the lenses from six irradiated mice and an equal number of non-irradiated controls were removed 72 h post-irradiation; the LEC were collected by stripping them as a layer from the surface of the lens with forceps and placing them in a 1% collagenase/dispase and 3% bovine serum albumin (BSA) solution for 30 min at 37 °C. Completion of LEC separation was accomplished with forceps followed by collagenase/dispase dispersal. A control group consisted of mice of the same age and sex that did not receive the irradiation but were otherwise treated similarly. The LEC were then passed through a 70 μm filter, plated in DMEM+10% fetal calf serum, and cultured for a total of 12 days in 60 mm plates  into which glass coverslips has been placed. Metaphase spreads were made from cultured LEC of irradiated mouse lenses removed 72 h post-irradiation and cultured for a total of 12 days under 3% OLEC from six animals, each collected from the irradiated and the non-irradiated subgroups of the second group of irradiated and control mice, as noted above, were separately pooled after sacrifice of the mice at 72 h post-irradiation. The cells were collected and dispersed using collagenase/dispace and grown in an MEM medium containing 10% fetal bovine serum (FBS). After seven days in the original mass plating, they were then freed with collagenase/dispase, washed in phosphate buffered saline (PBS), and singly and sparsely replated in triplicate in a similar medium on 35 mm collagen coated plates for individual cell clonal growth. After nine days in the second plating, the size of the individual clones formed was assayed by counting individual cells in each clone after crystal violet staining. The clone counts were then assayed for the percent of clones in large or small cell content groups using several size classification sets . Several combinations of these clone number classification groups were used to determine whether significant differences in the percentage of the present large or small clones existed between the collections for the irradiated donors and the non-irradiated controls. Total numbers of clones of any size were also recorded.The degree of DNA presence and DHR in the cortex was compared between irradiated and control lenses for fluorescence intensity by confocal readings from several lens depths using the Mann–Whitney non-parametric test.th month post-irradiation, 80% of the irradiated eyes were positive for advanced cataract at which time the experiment was ended. In all instances and at all observational time points, the individual 3.5–4+ cataracts developed within the 30-day observation periods from previous readings of 1.5–2+. Only opacities scored as 3.5+ to 4+ were considered to be cataracts with 3.5+ representing an advanced opacity including the entire lens as viewed by slip lamp and 4+ representing a lens opacity with 100% light reflection . As seen in The typical cataracts that appeared over a 30 day period but only 5-11 months after radiation are shown in The comet assay measures primarily single strand breaks and alkali labile sites if run under alkaline conditions as it was here . AlthougIn spite of the initial recovery of DNA strand breaks, a dysfunctional lens status increased over time as confirmed by confocal viewing of living lenses using fluorescent dyes for nuclear fragments, free DNA presence, and localized ROS presence as well as noting aberrant LEC migration within the lens. These changes increased progressively in the irradiated mice when viewed in this manner. They were not present in the first month but developed minimally by the third month and were present as marked changes in cataractous, irradiated lenses at five and seven months post-irradiation  with abeIn addition to the LSCM studies, we prepared Carnoy’s-fixed paraffin sections in which we used an antibody to 8-hydroxyguanasine (8-OHG) to measure the in situ presence in the lens of this DNA adduct that is a result of oxidative damage ,31,32. TFinally, we prepared clonal cultures separately for the irradiated mice and their non-irradiated control group in the manner noted in the Methods section. This separation measured relative capacity of individual cells from each of the two groups to replicate by producing clones with a larger or smaller number of daughter cells, i.e., to proceed through repeated cell divisions in a specific time period. However, we found neither significant clone size differences nor differences in the total number of clones formed between the irradiated and non-irradiated group (results not shown), thus LEC replication capacity was not affected in the cells surviving the irradiation event.In our previous work, we have shown that the gradual development of age-related cataract (ARC) is characteristic for several strains of untreated mice and rats  with theIn the present study, we describe in head-only x-irradiated mice a delay of several months for cataract development but then within a 30-day period the rapid appearance of cataracts that present with the same physical characteristics as those of ARC with aberrantly migrating and incompletely differentiating LEC, massive ROS presence, and evidence of retained DNA damage. After the x-irradiation, the repair of DNA strand breaks and the appearance of alkaline sensitive sites occurred within 30 min in the surviving LEC as measured by comet assay. However, these cells and possibly their immediate descendants showed in vivo the retained nuclear presence of the DNA adduct, 8-hydroxyguanasine (8-OHG), 72 h later. This was accompanied at this time by attempted or ongoing repair by the single strand DNA repair protein, XRCC1. However, the clonal measurement of in vitro replication potential in this population of LEC was not different from that of non-irradiated control donors two weeks after irradiation, although more of the cells from the irradiated population had abnormalities in chromosomal content. Thus, the initial, nonlethal, radiation-induced oxidative damage is retained in the DNA of successive proliferating LEC descendants, affecting their DNA status and causing abnormal cellular behavior, which results in cataracts months later as seen by both slit lamp and confocal microscope/fluorescent dye viewing.Worgel  reportedWe provide evidence that the X-ray induced oxidative damage manifests itself in progenitor LEC that repair DNA single strand breaks and related alkali sensitive sites  within 3Overall, our findings suggest to us that oxidative damage caused by soft X-rays represents a premature version of the status that develops in age-related cataract and that both are caused by oxidative damage to LEC DNA. In relation to this, we note that in mutant mice in which oxidative damage is reduced or slowed, the development of ARC is significantly less than that those in the same age controls ,36 while2 entry into the lens at surface sites denuded of LEC as shown in Finally, the progression to advanced cataracts some months post-irradiation in the studies of Worgel et al.  and Koda