The filoviruses, Ebola (EBOV) and Marburg (MARV), cause a lethal hemorrhagic fever. Human isolates of MARV are not lethal to immmunocompetent adult mice and, to date, there are no reports of a mouse-adapted MARV model. Previously, a uniformly lethal EBOV-Zaire mouse-adapted virus was developed by performing 9 sequential passages in progressively older mice (suckling to adult). Evaluation of this model identified many similarities between infection in mice and nonhuman primates, including viral tropism for antigen-presenting cells, high viral titers in the spleen and liver, and an equivalent mean time to death. Existence of the EBOV mouse model has increased our understanding of host responses to filovirus infections and likely has accelerated the development of countermeasures, as it is one of the only hemorrhagic fever viruses that has multiple candidate vaccines and therapeutics. Here, we demonstrate that serially passaging liver homogenates from MARV-infected severe combined immunodeficient (scid) mice was highly successful in reducing the time to death in scid mice from 50–70 days to 7–10 days after MARV-Ci67, -Musoke, or -Ravn challenge. We performed serial sampling studies to characterize the pathology of these scid mouse-adapted MARV strains. These scid mouse-adapted MARV models appear to have many similar properties as the MARV models previously developed in guinea pigs and nonhuman primates. Also, as shown here, the scid-adapted MARV mouse models can be used to evaluate the efficacy of candidate antiviral therapeutic molecules, such as phosphorodiamidate morpholino oligomers or antibodies. Filoviridae consists of two genera called ebolavirus (EBOV) and marburgvirus (MARV) that are considered significant public health threats due to their very high morbidity and mortality rates , human-to-human transmissibility, and environmental stability. Due to these characteristics, and the fact that the filoviruses have a low infectious dose [<1 plaque-forming units (pfu)] and can be easily produced to >108 pfu/ml in vitro or in vivo [The family  in vivo , the fil in vivo ,6. Initi in vivo .The first known filovirus outbreaks occurred in simultaneously in both Germany and Yugoslavia in 1967 when laboratory workers became infected from blood and tissues of MARV-infected African green monkeys imported from Uganda ,9. SubseHuman-derived Marburg viruses  are not lethal to immmunocompetent adult mice. Previously, an Ebola Zaire mouse-adapted virus was developed by performing 9 sequential passages of Ebola Zaire '76 virus in suckling mice followed by two sequential plaque picks. The resulting virus was uniformly lethal to mice after intraperitoneal inoculation . PatholoA viable lethal mouse model for Marburg virus is critical to the filovirus vaccine research program to understand the immune mechanisms that need to be induced, or avoided, by vaccination. Furthermore, a mouse model would speed the testing and evaluation of new Marburg therapeutic candidates. This effort is currently impeded due to limitations in the numbers of guinea pigs that can be evaluated at one time  and the large amounts of compounds that must be synthesized or purified for testing in guinea pigs, which are 20–50× the size of mice. The purpose of this work was to select a marburgvirus that caused death within a similar timeframe as monkeys or humans (7–10 days) in severe combined immunodeficiency (scid) mice. To accomplish this goal, we repeatedly passaged the liver homogenates of MARV-infected scid mice and then recorded their time to death. Once we identified rapidly lethal mouse-adapted viruses, we characterized the models by immunology and pathology studies. These scid mouse-adapted viruses will be used to explore the virulence factors associated with marburgvirus infection. Furthermore, the scid models of MARV infection will be particularly useful for screening candidate therapeutics for their ability to directly diminish viral replication in the absence of adaptive immune responses.Human-derived (wild-type) and mouse-adapted MARV-Musoke, -Ravn, and -Ci67 virus stocks were propagated no more than three passages in Vero or VeroE6 cells. The human-derived (wild-type) and mouse-passaged MARV-Musoke, -Ravn, and -Ci67 plaques were counted by standard plaque assay on Vero cells . MARV-inad libitum. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.BALB/c severe combined immunodeficient (scid) mice, aged 4 to 8 weeks, of both sexes, were obtained from National Cancer Institute, Frederick Cancer Research and Development Center . Mice were housed in microisolator cages and provided autoclaved water and chow The general approach to adapt MARV to mice was based on virus passage in scid (BALB/c background) mice to avoid usage of suckling mice to develop a lethal mouse-adapted Marburg virus. The goal was to isolate the viral population that was capable of migrating to target tissues/organs  at the earliest time point. Each group consisted of 10 mice that were inoculated intraperitoneally (IP) with 1000 pfu of Marburg virus . Two mice were euthanized on day 7, the livers removed, pooled, and homogenized in 10 ml of PBS. The liver homogenates were blindly passed (200 μl IP) and used to infect new mice to evaluate lethality of the next virus passage. Lethality was monitored in the remaining eight mice of each passage. The supernatants of the liver homogenates from each passage were introduced onto Vero cells to determine the viral titers by plaque assay .For the characterization studies, scid mice were injected IP with ~1000 pfu of 'scid mouse-adapted' MARV-Musoke, passage (P)10; Ravn P(10); or Ci67, P(15) diluted in PBS. After challenge, mice were observed at least twice daily for illness and death and in some experiments, daily weights were determined for each infected group.For mice, blood samples were obtained under anesthesia by cardiac puncture. Viremia was assayed by traditional plaque assay . HematolFour animals from each group were randomly chosen for euthanasia on 2, 4, 6, and 8 days postchallenge for gross necropsy. A full complement of tissues from each mouse was fixed in 10% neutral buffered formalin and held in the BSL-4 laboratory for >21 days. The tissues were embedded in paraffin, sectioned for histology, and stained with hematoxylin and eosin for routine light microscopy or were stained by an immunoperoxidase method , using a mixture of two mouse monoclonal antibodies against MARV nucleoprotein (NP) and glycoprotein, or by the TUNEL method to detect apoptotic cells within the tissue samples.Two groups of 10 scid mice were each administered 1 ml of convalescent sera from guinea pigs that had survived either EBOV or MARV infection. The antibodies were administered IP 1 h after challenge. Both pools of antisera had 80% plaque reduction-neutralization titers of >1:160 against the homologous virus, but <1:20 against the heterologous virus. Alternately, another group of 10 scid mice were administered IP with 1 mg of a mixture of four MARV-specific phosphodiamidate morpholino oligomers (PMOs) targeting the AUG start site of VP24, VP35, NP, and L  1 h after challenge. A control group received saline  alone. The mice were challenged with 1000 pfu of 'scid-adapted' MARV-Ci67 and monitored for survival.Previously, an Ebola Zaire mouse-adapted virus was developed by performing 9 sequential passages of Ebola Zaire '76 virus in suckling mice . We chosWe next intended to characterize the rapidly lethal 'scid-adapted' mouse models of MARV-Musoke, -Ravn, and -Ci67 via serial sampling studies of infected scid mice. It was of particular interest to determine if the infection caused similar laboratory, immunological, and pathological responses in mice, as was observed in MARV-infected guinea pigs and nonhuman primates. Within 3–4 days after injection with the 'scid-adapted' MARV strains, mice developed anorexia, a hunched appearance, and exhibited decreased grooming. Some mice also appeared to have blood in their urine and many mice developed hind-limb paralysis after 'scid-adapted' MARV infection (data not shown).6 pfu/ml in the serum at days 6–8  in screening potential anti-MARV therapeutics, we treated scid mice that were infected with 'scid-adapted' MARV-Ci67. Since the scid mice do not have functional B or T cells and cannot mount an adaptive response to clear the virus, we only expected to see a delay in the mean time-to-death and not a survival benefit in these experiments. In the first portion of this experiment, 1 ml of pooled sera from convalescent guinea pigs that were previously infected with EBOV-Zaire or MARV-Musoke was administered IP 1 h after challenge to the MARV (Ci67)-infected scid mice. The scid mice that were treated with MARV convalescent sera had a MTD of >23 days Figure . This wain vivo. Therefore, we passaged the viruses until the time to death was consistently in the range of 7–10 days. These more virulent 'scid-adapted' viruses will allow for more rapid and efficient testing of candidate prophylactic and therapeutic treatments against multiple MARV isolates.In previous studies, scid mice became ill and died within 3–4 weeks after inoculation with ZEBOV ('76), Sudan EBOV, or GP-adapted MARV-Ravn, but not with the other viruses . HoweverInitial serial sampling studies to characterize the pathology of these more virulent, scid-adapted MARV strains indicate similarities to the filovirus disease observed in other models. After parenteral challenge, the incubation period for MARV is 2 to 6 days, with death typically occurring between 7 and 11 days after infection in both guinea pigs and nonhuman primates -30. InitRodents infected with filoviruses appear to have slightly different coagulopathic responses than filovirus-infected nonhuman primates ,26-30,32Sequence comparisons of the original wild-type and more virulent scid-adapted MARV are required. Based on previous reports with mouse and guinea pig-adapted EBOV ,33,34, win vivo efficacy studies. Furthermore, guinea pigs require much larger cages, limiting the number of animals within a study and are also much more difficult and dangerous to handle under BSL-4 conditions than mice, requiring at least two laboratory personnel for treatments and challenges. A delay in time to death in this newly developed scid mouse model of MARV infection will indicate a positive result that should be followed up in the more intensive and expensive guinea pig studies. Thus, this novel MARV mouse model will allow for faster and more efficient in vivo screening of potential MARV prophylactics and therapeutics.This scid mouse model of MARV infection has obvious uses as a model for analysis of therapeutics and candidate antibodies. It will be much more efficient for the purposes of quickly screening lead compounds and neutralizing antibodies than guinea pigs or nonhuman primates. Before the development of this novel scid mouse model of MARV, a large quantity of antiviral compound and/or antibodies was required to achieve relevant physiological levels in guinea pigs, which are 20–50 times larger than mice, for the purposes of initial


Identification of insulin resistance (IR) in the general population is important for developing strategies to reduce the prevalence of non-insulin-dependent diabetes mellitus (NIDDM). We used the original and a modified version of the Quantitative Insulin Sensitivity Check Index , and the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) to divide non-diabetic normotensive adults into high- (HIR) and low-insulin-resistant (LIR) subgroups to investigate similarities and differences in their characteristics.Three hundred fifty-seven healthy adults aged 18-50 years were recruited randomly from health centers in Jeddah in a cross-sectional study design. Anthropometric and demographic information was taken. Insulin, glucose, lipid profile and free fatty acid were determined in fasting blood samples. M-QUICKI, HOMA-IR and QUICKI were calculated. Reported cut-off points were used to identify HIR subjects, who were then matched for age and sex to others in the study population, resulting in 3 HIR and 3 LIR subgroups.P=.01) with HOMA-IR and QUICKI values. Increased adiposity was the common characteristic of the three HIR subgroups. HIR subgroups identified using M-QUICKI (97 subjects) and HOMA (25 subjects), but not QUICKI (135 subjects), had statistically different biochemical characteristics compared to corresponding LIR sub-groups.Two hundred nine subjects satisfied the selection criteria. M-QUICKI correlated significantly (Adiposity, but not sex, is a risk factor for IR in the studied population. Further studies are needed to choose the most appropriate index for detecting IR in community-based surveys. Insulin resistance underlies abnormalities of glucose, lipid and blood pressure homeostasis.46There is an increasing prevalence of insulin-resistance-related disorders in Saudi Arabia.71012In humans, the “gold standard” for assessing insulin resistance is the euglycemic hyperinsulinemic clamp (IS clamp) because it directly measures insulin action on glucose utilization under steady-state conditions. However, this technique is difficult and can only be used for a small number of subjects.168The aim of this study was to employ this modified version (M-QUICKI), the original one, and the “Homeostasis Model Assessment of Insulin Resistance” (HOMA-IR) to separate selected non-diabetic adults into high- (HIR) and low-insulin-resistant (LIR) subgroups, using earlier reported cut-off points, in order to investigate whether this division produces similar results and whether it leads to a difference in well-known anthropometric and biochemical characteristics between LIR and HIR subgroups.http://www.analystsoft.com/en/products/biostat/)and selecting the power of the study as 0.9 (90%). The calculated sample size was found to be 205. According to population density, a sample size of 209 was then calculated for each center, after rounding fractions. Exclusion criteria included the following: reported diabetes , endocrine disorders, hypertension, reported dyslipidemia and coronary heart diseases. Hypertension was defined as a systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg26In a cross-sectional study, healthy subjects aged 18-50 years were recruited randomly from individuals visiting health centers during the period between July 2005 and January 2007. Six health centers  were chosen randomly. Based on an earlier study of insulin resistance in Saudi diabetic individuals,Selected subjects were given an appointment for blood collection while fasting. Collected samples were immediately placed on ice prior to processing. Glucose was determined first in separated serum, and subjects showing hyperglycemia were excluded. Remaining samples were divided into aliquots and frozen at –70°C for later determination of lipid profile, insulin and FFA.www.wako-chemicals.de), with intra- and inter-batch CV being 5.2% and 9.8%, respectively.Glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides (TGs) were estimated using automated enzymatic methods . The coefficient of variation was <2% and <5% for intra- and inter-batch, respectively, in all cases. Insulin was estimated in one batch using the electro-chemiluminescence immunoassay (ECLIA) on Modular Analytics E 170 (Elecys module) immunoassay analyzer supplied by Roche Diagnostics GMbH, Germany. (CV was 9.7%.) All measurements were carried out at the university hospital biochemistry laboratory. FFAs were estimated manually in serum using an enzymatic method  was used to analyze the data. Significance was assigned at P<.05.Descriptive statistics such as mean (standard deviation) for normally distributed data or median and interquartile range (IQR) for non-normally distributed variables were calculated for all parameters in each of the six resulting subgroups. Statistical analyses were performed using the unpaired Three hundred fifty-seven subjects were recruited. Only 209 subjects  satisfied the criteria and provided required samples. The demographic and anthropometric characteristics of the selected group are presented in Using the QUICKI and a cut-off point of <0.357,P=.76) since it included 47 males (34.8%) and 88 females (65.2%). Due to limitations of sample size, matching was not done on a one-to-one basis, but was done keeping in mind age and the ratio of the sexes. This resulted in exclusion of some of the HIR subjects, giving a total number of 121 subjects in the HIR subgroup  and 74 subjects in the LIR subgroup . The calculated age means (SD) did not differ significantly between the two subgroups . However; when the Chi-square test was applied, the distribution between the different classes of BMI was significantly different in the two subgroups (P<.01), and the percentage of subjects with abdominal obesity was also significantly higher in the HIR subgroup (P=.047). On the other hand, more than 50% of the subjects in both subgroups reported a family history of diabetes, with no significant difference in the percentage between the two subgroups (P=.67).The division between sexes in the HIR subgroup was not significantly different from that in the group as a whole . Most had a normal lipid profile, with total cholesterol level being ≥5.2 mmol/L in 42 individuals (34.7% of the entire subgroup) compared to 39.1% in the LIR subgroup. LDL-cholesterol level was ≥3.36 mmol/L in 38 subjects (31.4%) compared to 40.5% in the LIR subgroup, and HDL-cholesterol was ≤1.09 mmol/L in 18 subjects (14.9%) compared to 16.2% in the LIR subgroup. Furthermore, only 20 subjects (16.5% of the HIR subgroup) had higher-than-acceptable plasma triglycerides  compared to 18.9% in the LIR subgroup. Moreover, the majority of subjects in the HIR subgroup  had a glucose value <6.0 mmol/L and/or insulin and FFA values in the lowest quartile of the calculated range for the studied population.P=.76).Using the modified QUICKI and a cut-off point of <0.419,20P=.01) with HOMA values (r =–0.756) and with QUICKI values (r=0.758).Matching for age and sex could be done for 90 IR subjects only. The HIR group had significantly higher mean weight, higher percentage of obese individuals and higher percentage of subjects with abdominal obesity . In addiP=.79). Twenty-three of these subjects were also identified by modified QUICKI to have HIR. All of these individuals had the well-recognized characteristics of IR as diagnosed by the IS clamp technique, such as obesity (BMI ≥30 kg/m2) and abdominal obesity. All of them had a family history of diabetes. Twenty-two of them (84.6%) had high total and LDL-cholesterol, fasting glucose >6.0 to <7.0 mmol/L, and insulin, TG and FFA values in the upper quartile of the calculated range for the studied population. When means or medians of anthropometric and biochemical characteristics of the HIR and LIR subgroups divided according to this index and matched for age and sex (results not shown) were compared statistically and significant differences (P<.001) were found in all cases.Using the HOMA index and a cut-off point of >3.8,Only 22 subjects were identified by all the three indices. These were all obese, with abdominal obesity, abnormalities in lipid profile, blood glucose >6.0 mmol/L, insulin and FFA levels in the upper quartile of the calculated range for the studied population.Approximately 42% of the randomly recruited population had blood glucose values ≥7 mmol/L and had to be excluded according to our criteria. Therefore, the prevalence of diabetes in our region of the country could be much more than reported. Screening for insulin resistance and management of the condition would help prevent, or at least delay, the onset of diabetes.P=.001). The high percentage of hypercholesterolemia places our population at high risk of cardiovascular disease, with the males having a higher risk considering their higher LDL and lower HDL levels and the fact that the median triacylglycerols value was significantly higher (P=.021).Our calculated percentages for overweight and obese individuals in both sexes were lower than those reported earlier by Alsaif et al.Our use of different indices to identify individuals with HIR in our population gave widely differing results. Despite the fact that HOMA-IR and QUICKI both use fasting insulin and glucose levels to calculate insulin resistance and both correlate reasonably with the clamp technique,1617Another reason for the suspected underestimation by HOMA could be the fact that this method reflects hepatic IR only, not IR at peripheral tissues.32Theoretically, the above could also be applied to QUICKI, and subjects with IGT are expected to be missed. However, compared to HOMA-IR, QUICKI is reported to have the advantage of being applicable to wider ranges of insulin sensitivity,31On the other hand, using the modified QUICKI and the cut-off point reported by Perseghin et alInsulin resistance in adipose tissue will lead to elevated plasma FFA. Therefore, inclusion of FFA into the QUICKI formula can be beneficial and can increase its detection power by including those subjects with peripheral IR, especially in view of the following: (1) increased fasting FFA concentration could reflect insulin resistance earlier than hyperglycemia since lipolysis is more sensitive to insulin than glucose utilization;2021The superior ability of modified QUICKI to identify HIR individuals in Saudi subjects is also strongly suggested by the finding that the means or medians of almost all biochemical parameters, except HDL-cholesterol, were significantly higher in the LIR subgroup. Thus, the identified HIR subgroup using this index had all the well-recognized characteristics of insulin resistance as diagnosed by the gold standard method, unlike that identified using QUICKI.Even though it is difficult with the available data to decide which of the three indices gives a better estimation of insulin resistance in Saudi non-diabetic normotensive individuals, our data show that when the three indices were used to identify HIR individuals, division between sexes was not significantly different from that in the group as a whole. Therefore, it can be suggested that sex is not a risk factor for insulin resistance in the studied population.2) have postprandial hyperinsulinemia and relatively low insulin sensitivity,2) populations, suggesting an inherited component to insulin resistance.2 and apparently contributes to a high prevalence of type 2 diabetes and premature CVD.Furthermore, increased body fat content was noted when all three indices were used to identify HIR individuals, as reported earlier.


Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. MicroRNAs (miRNAs) are highly conserved, noncoding small RNAs that regulate the expression of broad gene networks at the posttranscriptional level.miR-222, miR-21, and miR-146a) related with oxidative stress and inflammatory processes in 63 workers at an electric-furnace steel plant.We evaluated the effects of exposure to PM and PM metal components on candidate miRNAs  and after 3 days of work (postexposure). Relative expression of miRNAs was measured by real-time polymerase chain reaction. We measured blood oxidative stress (8-hydroxyguanine) and estimated individual exposures to PM1 (< 1 μm in aerodynamic diameter), PM10 (< 10 μm in aerodynamic diameter), coarse PM (PM10 minus PM1), and PM metal components  between the baseline and postexposure measurements.We measured miR-222 and miR-21 (using the 2T−ΔΔC method) was significantly increased in postexposure samples . In postexposure samples, miR-222 expression was positively correlated with lead exposure , whereas miR-21 expression was associated with blood 8-hydroxyguanine  but not with individual PM size fractions or metal components. Postexposure expression of miR-146a was not significantly different from baseline  but was negatively correlated with exposure to lead  and cadmium .Expression of Changes in miRNA expression may represent a novel mechanism mediating responses to PM and its metal components. Epidemi effects . Foundry effects , potenti effects , the und effects .Inhaled PM pollutants have been shown to produce systemic changes in gene expression, which can be detected in peripheral blood of exposed individuals . ExpressmiR-222, miR-21, and miR-146a, have been implicated in disease mechanisms that may be related to PM exposure, such as oxidative stress , a central mediator for PM-related inflammation and health effects  measured in blood DNA.In the present study, we investigated the effects of PM exposure on miR-222, miR-21, and miR-146a measured in blood RNA from foundry workers with well-characterized exposure to a wide range of PM levels. To clarify the mechanisms activated by PM exposure, we evaluated whether the expression of a) a baseline sample collected in the morning of the first day of a workweek (after 2 days off work) before the beginning of any work activity (time 1); and b) a postexposure sample collected at the same time on the fourth day of work, after 3 consecutive days of work (time 2). Written informed consent and approval from the local institutional review board IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico were obtained before the study.We recruited 63 healthy male workers , in a steel production plant in Brescia, Northern Italy. These workers were free of cancer, cardiovascular disease, and pulmonary disease, and all of them had been working in their current job position for at least 1 year. To discriminate short- and long-term effects of PM, we obtained blood samples for DNA methylation analysis at two different times: 10) and ≤ 1 μm (PM1) measured using a GRIMM 1100 light-scattering dust analyzer . Concentrations of coarse particles were calculated from these measures as the difference between PM10 and PM1. We measured PM metal components on the PM10 fraction of PM mass through multielemental analysis by means of inductively coupled plasma mass spectrometer . We measured arsenic, cadmium, lead, manganese, and nickel concentrations using the Total Quant method, and chromium concentrations using the Dynamic Reaction Cell (DRC) method with ammonia  endogenous control . Real-time PCR was performed in triplicate, including no-template controls. The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The relative gene expression was calculated via a 2t−ΔΔC method , modified for miRNA extraction [see Supplemental Material (doi:10.1289/ehp.0901300)]. We used specific TaqMan MicroRNA Assays  to detect and quantify mature miRNAs as recommended by the manufacturer, using ABI Prism 7900HT sequence detection systems (Applied Biosystems). In the reverse transcription (RT) step, 10 ng total RNA was employed in RT reactions  using reagents from the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and specific miRNA primers provided with the TaqMan MicroRNA Assays. Real-time polymerase chain reaction (PCR) was performed using TaqMan MicroRNA Assays together with TaqMan Universal PCR Master Mix on an Applied Biosystems 7900 Sequence Detection System (95°C for 1 min and 40 cycles of 95°C for 15 sec and 60°C for 30 sec) . Normalit method . Data art was used in the statistical analyses.We measured the content of 8-OH-dG in mtDNA using quantitative real-time PCR to amplify different fragments of mtDNA on each sample with and without hOGG1 (human 8-oxoguanine DNA glycosylase 1) pretreatment, as previously described by http://www.microrna.org/microrna/home.do) to predict the target genes of miR-222, miR-21, and miR-146a. A total of 1,056 target genes were annotated for miR-222, 1,065 for miR-21, and 1,038 for miR-146a. Kyoto Encyclopedia of Genes and Genomes  pathway searching was performed by mapping the predicted target genes from the KEGG human database. A total of 150 KEGG pathways were annotated for miR-222, 153 for miR-21, and 160 for miR-146a.We used the miRNA target prediction software miRanda , a literature search tool providing gene and signal transduction pathway mining within the National Center for Biotechnology Information’s PubMed database  and postexposure (time 2). We evaluated the association of PM mass and PM metal component levels with miRNA expression measured in postexposure samples using simple linear regression models and multivariable models. The adjusting variables were selected a priori based on the assumption that potential confounders had to be related to inflammation or oxidative stress generation. Age, body mass index (BMI), smoking, and number of cigarettes per day are all potential determinants of inflammation, oxidative stress, or both. In addition, we adjusted for percent granulocytes to control for possible shifts in leukocyte differential count.The Student’s paired As a sensitivity analysis, we also fitted duration of smoking in the model described above. The results of the models including duration of smoking were very similar to those including number of cigarettes per day.p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SAS .To compare the magnitude of the associations of miRNA expression with different exposures, we calculated standardized regression coefficients. Outliers were excluded from regression analysis by dropping observations with studentized residuals that exceeded +3 or −3. We checked regression assumptions by performing diagnostic tests for each model, including the Shapiro–Wilk test to verify normality of residuals and the White test to verify the homogeneity of variance of the residuals. For miRNAs that did not show differences in their expression between time 1 and time 2, we also used mixed models that regressed PM and metal exposures against all measures of miRNA expression, regardless of whether they were measured at baseline or postexposure samples . A two-sThe characteristics of the 63 study subjects are summarized in miR-222 expression was significantly increased in blood samples taken after 3 days of work (time 2) compared with the measurement at time 1 .miR-21 expression was not significantly associated with any of the measures of exposure to PM and PM metal components . In these models, we evaluated the association between the exposure and miR-146a, assuming that there were no short-term effects from the exposures . In these models, miR-146a expression showed a negative significant association with lead exposure . miR-146a expression also showed a negative significant association with cadmium .miR-21 expression and 8-OH-dG , which was confirmed in multivariable regression . miR-222 and miR-146a were not associated with 8-OH-dG , lead , and cadmium , whereas in current smokers we observed no significant association. In noncurrent smokers, miR-146a expression was down-regulated in response to lead  and cadmium , whereas in current smokers we observed no significant association.In noncurrent smokers, miR-222 was up-regulated in association with lead , whereas miR-146a expression was down- regulated in association with lead . We found no significant associations among individuals between 46 and 65 years of age. The complete analyses stratified by smoking and age are reported in Supplemental Material, Tables S3 and S4 (doi:10.1289/ehp.0901300).The results stratified by age showed that in subjects 25–45 years of age http://www.genome.jp/kegg), a pathway analysis database, to the target genes identified for miR-21, miR-222, and miR-146a using miRanda. The enriched pathways we identified appeared to be largely overlapping . Among the top-ranked pathways, we found those related to general functions  and others with more specific functions, including a high proportion of pathways related to oxidative stress and inflammation [miR-222, miR-21, or miR-146a) and the general pathway key words in the same sentence. We found two pathways related to miR-222, seven related to miR-21, and six related to miR-146a , we observed stronger associations among younger subjects. Identification of sets of individuals who have enhanced responses to PM may suggest possible mechanisms of physiologic assault, and provide data that can be used for more detailed risk assessment (Results of the present study show that associations of metals with sessment .In the present study we investigated a population with well-characterized exposure to PM and PM metal components that allowed for contrasting subjects over a wide range of different exposure levels. Because of the limited number of study subjects, it is possible that the associations observed were due to chance. However, the occupational exposure and relatively controlled environment of a foundry provide a good setting for evaluating these mechanistic questions and reduce bias and chance findings. Our study was based on subjects working in several areas of the same factory but did not include a different population of subjects without exposure to PM. Limiting our investigation to individuals who have all been working in the same facility avoided potential concerns related to the selection of external referents who might have differed from the exposed population in terms of socioeconomic factors and other characteristics determining hiring into the plant . HoweverIn summary, our findings suggest that air particles, particularly those rich in lead and cadmium, are able to modify miRNAs expression. Further studies are required to determine the role of such alterations along the pathways determining the effects of PM on human health.


Macrophages are the dominant phagocyte at sites of wound healing and inflammation, and the cellular and acellular debris encountered by macrophages can have profound effects on their inflammatory profile. Following interaction with apoptotic cells, macrophages are known to switch to an anti-inflammatory phenotype. Activated platelets, however, are also a major component of inflammatory lesions and have been proposed to be pro-inflammatory mediators. In the present study, we tested the hypothesis that macrophage interaction with activated platelets results in an inflammatory response that differs from the response following phagocytosis of apoptotic cells.Human monocyte-derived macrophages (hMDMs) were co-incubated with autologous activated platelets (AAPs) and the platelet-macrophage interaction was examined by electron microscopy and flow cytometry. The cytokines TNF-α, IL-6, and IL-23 were also measured during LPS-activated hMDM co-incubation with AAPs, which was compared to co-incubation with apoptotic lymphocytes. Cytokine secretion was also compared to platelets pre-treated with the gluococorticoid dexamethasone.Macrophages trapped and phagocytized AAPs utilizing a mechanism that was significantly inhibited by the scavenger receptor ligand fucoidan. LPS-induced macrophage secretion of TNF-α, IL-6, and IL-23 was inhibited by co-incubation with apoptotic cells, but enhanced by co-incubation with AAPs. The platelet-dependent enhancement of LPS-induced cytokines could be reversed by pre-loading the platelets with the glucocorticoid dexamethasone.The interaction of human macrophages with autologous platelets results in scavenger-receptor-mediated platelet uptake and enhancement of LPS-induced cytokines. Therefore, the presence of activated platelets at sites of inflammation may exacerbate pro-inflammatory macrophage activation. The possibility of reversing macrophage activation with dexamethasone-loaded platelets is a promising therapeutic approach to treating unresolved inflammation. A major function of macrophages is phagocytosis of cellular and acellular debris during inflammation and wound healing, and the activation response of macrophages following phagocytosis can be varied depending on the local extracellular environment -6. The iDuring their differentiation from primary monocytes, macrophages acquire specialized receptors and machinery for recognizing and clearing both apoptotic and infected cells . In cleaPlatelets are anucleate cells which play an integral role in maintaining vascular integrity. Within their 8-10 day lifespan, platelets can become activated either in the circulation or during adherence at a site of injury, and during this process they become targeted for destruction by macrophages . The proCells that have become apoptotic as part of their normal life cycle are recognized and cleared by phagocytosis in a manner that usually inhibits pro-inflammatory responses -27. AlthHuman monocytes were isolated and cultured using techniques similar to those previously described ,31. Brieg to generate platelet-rich plasma (PRP). PRP was pelleted by centrifugation for 10 min at 800 × g and the platelet pellet subsequently washed 2 times in citrated saline (pH 6.8). A portion of the platelet samples were degranulated by incubating 1 ml of platelets  with 10 μl of 10 μM calcium ionophore A21387 (Sigma) for 15 min with rocking at room temperature, followed by three centrifugational washes with citrated saline. For phagocytosis experiments, platelets were fluorescently labeled with Cell Tracker Green CMFDA (Invitrogen) as previously described [Platelets were isolated from whole blood collected into acid-citrate-dextrose (ACD) from healthy human donors and centrifuged for 15 min at 500 × escribed . After tFor analysis of surface P-selectin and phosphatidylserine, platelets  were first incubated in either citrated saline or serum-free RMPI media for 1 hr at 37°C. A portion of the platelets were activated by including thrombin  in the incubation reaction. To detect surface expression of activation markers in each platelet treatment group, a 10 μl aliquot of platelets was stained with either FITC-anti-CD62P (Biolegend) or FITC-Annexin-V (Biolegend) for 30 min at room temperature, after which the cells were fixed and analyzed immediately by flow cytometry. Flow cytometry analysis was performed using a CyAn flow cytometer (Beckman Coulter) and the Summit analysis software.6 dex-platelets were added to each well of macrophages in a 24-well plate.Dexamethasone-loaded platelets were prepared by incubating 1 ml of platelets  in citrated saline with 5 ul of dexamethasone  for 15 min on a rocker at room temperature. Platelets were then washed three times with citrated saline to remove unbound dexamethasone. For subsequent experiments, 5 × 10To prepare apoptotic cells, PBMCs were isolated as above, and following monocyte-adherence to gelatin-coated flasks the non-adherent lymphocytes were collected. Cells were rendered apoptotic (Annexin-V positive) by UV-irradiation for 10 min followed by overnight incubation in RPMI + 10% FBS at 37°C + 5% CO2.Thirty minutes prior to the start of each experiment, 7-day old MDMs were washed 3 times with PBS and incubated with 500 μl fresh RPMI media. In some experiments the media was supplemented with 10% autologous human serum. A 25 μl aliquot of fluorescently labeled platelets  was added to each well of macrophages. Platelets and macrophages were co-incubated for 45 min.For SEM analysis, the co-cultures were washed once with PBS and fixed in 2% paraformaldehyde and 0.5% glutaraldehyde. The samples were then processed as previously described  and examLatrunculin , used as a pan-phagocytosis inhibitor, was added to a portion of macrophage-containing wells 30 min prior to addition of platelets. Fucoidan (Sigma) was added to macrophages at a final concentration of 250 μg/ml 30 min prior to addition of platelets.6) were added to each well in addition to LPS (100 ng/ml). Some samples also received dexamethasone alone at a final concentration of 1 μM. After 24 hrs, supernatants were collected, spun 10 min at 14,000 g, and frozen at -80°C. Cytokines were measured by ELISA using capture and detection antibodies (eBioscience) per the manufacturer's instructions. Cytokines were measured in duplicate and averaged. The amount of protein secreted was normalized to the amount secreted by macrophages treated with LPS alone. Each experiment was performed at least 3 times using 3 different MDM donors. In each experiment, the platelets added were from the same donor as the MDMs. Treatment groups were compared using an unpaired t-test.Each well of MDMs was washed 3 times with PBS and incubated with fresh RPMI + 10% autologous human serum. Activated, degranulated, or dexamethasone-loaded platelets  and autologous platelets, we utilized an Platelet phagocytosis by the macrophages in our co-culture system was subsequently confirmed by TEM and flow cytometry. Macrophages that were co-incubated with AAPs for one hour developed vacuoles, many of which contained contents that were the same size and shape as platelets Figure . These pWe also used flow cytometry to more accurately examine the level of platelet activation in different culture conditions. Platelets were analyzed for expression of P-selectin, an alpha granule component expressed during early platelet activation, and phosphatidylserine, a membrane lipid exposed on the surface of completely (and irreversibly) activated platelets. Incubation in serum-free media alone for 1 hour resulted in an approximately ten-fold increase in P-selectin expression but did not induce surface expression of phosphatidylserine  by preparing in serum-free media or "irreversibly activated" (with phosphatidylserine exposure in addition to P-selectin) by treatment with the calcium ionophore A23187 .The inflammatory response of the hMDMs was assessed by measuring the levels of TNF-α, IL-6, and IL-23 after incubation with autologous primed platelets, autologous activated platelets, or control apoptotic leukocytes in the presence of LPS for 24 hours. As an additional control, we analyzed "platelets-only" cultures using the same media and incubation times as the platelet-macrophage co-cultures and were unable to detect any TNF-α, IL-6, or IL-23 in platelets alone . We therefore conclude that the cytokines secreted in this system are macrophage-derived, and in each experiment the cytokine levels were normalized to the amount of cytokine secreted by hMDMs incubated with LPS alone.When compared to LPS stimulation alone, macrophage co-incubation with apoptotic cells inhibited LPS-induced secretion of all three pro-inflammatory cytokines Figure . These dBased on the knowledge that platelets can bind glucocorticoids via glucocorticoid receptors , we testin vitro system. In this system, uptake of freshly isolated platelets is dependent on actin polymerization, but occurs independently of any soluble serum factors. Additionally, phagocytosis of platelets is enhanced with platelet activation.Phagocytosis is an important means of clearing both immunologically compromised and apoptotic cells. Monocyte-derived macrophages are efficient phagocytes in organs of the reticuloendothelial system and within injured tissues; however, the process of platelet clearance by macrophages is poorly understood. This work has demonstrated phagocytosis of fresh autologous activated platelets (AAPs) by monocyte-derived macrophages using an entirely human-derived in vitro that phagocytosis of aged platelets is mediated by scavenger receptors [Previous studies on platelet phagocytosis have focused on modified platelets such as chilled platelets, opsonized platelets, and aged platelets -46, eacheceptors . Therefoeceptors -50.Because phagocytosis correlates with platelet activation, we would expect no phagocytosis to occur in the presence of quiescent platelets. However, RPMI media alone causes platelet activation Table , probablThe macrophage response following phagocytosis of cells expressing surface phosphatidylserine is usually immunosuppressive ,27,53,54In contrast to the effect of apoptotic cells, activated platelets enhanced pro-inflammatory cytokine secretion from LPS-activated macrophages. The cytokines measured in the current study, TNF-α, IL-6 and IL-23, are significant readouts because they are known to be secreted by macrophages, but not platelets, and they play important roles in mediating pro-inflammatory responses. Interestingly, the pro-inflammatory cytokine secretion was also enhanced by platelets with surface phosphatidylserine exposure. The finding that degranulated platelets, washed free from their secreted proteins, also enhanced LPS-induced macrophage cytokine secretion suggests that a secreted platelet factor is not likely to be responsible for this effect. However, a secreted platelet factor could exert the observed effect if it remained bound to the platelet surface after secretion from the platelet. Because the platelets remained in the co-incubation for the entire experiment (24 hrs), the possibility also exists that the inflammatory consequences of platelet-macrophage interactions occur independently of phagocytosis. Cell contact itself could be responsible for the observed effects. Nonetheless, we have shown the pro-inflammatory effect of platelets does occur in conditions which favor platelet uptake Figure .Recent studies have highlighted additional roles of platelets beyond hemostasis, particularly with respect to platelet-mediated effects on inflammation ,57,58. TPro-inflammatory cytokines secreted by macrophages can also exert effects on surrounding cells and tissues. For example, IL-6 and IL-23 stimulate T-cells for induction of Th17 immune responses, which are operant in autoimmune diseases such as inflammatory bowel disease, lupus, psoriasis and arthritis -67. We sGlucocorticoids such as dexamethasone can exert powerful immunosuppressive effects on leukocytes and are thus an attractive therapy for modulating inflammation . After sWe have shown here that the interaction of human macrophages with autologous platelets results in scavenger-receptor-mediated platelet uptake and enhancement of LPS-induced cytokine secretion. Given the presence of activated platelets together with macrophages during the response to injury and during inflammation, activated platelets at sites of inflammation most likely exacerbate the macrophage response. The presence of platelets must therefore be carefully considered when studying the cellular interactions occurring in inflammatory lesions.We have also presented evidence here that platelets can be engineered to exert anti-inflammatory effects on macrophages. Given the emerging role of platelets in inflammatory diseases, the possibility of reversing macrophage activation with dexamethasone-loaded platelets is a promising therapeutic approach to treating unresolved inflammation.The authors declare that they have no competing interests.CMS conceived of the study, participated in its design and coordination, performed phagocytosis assays and cytokine measurements, and drafted the manuscript. WDH participated in the design of the study, coordinated the selection and participation of blood donors, and processed the blood samples. THF was the principal investigator on this project and provided guidance and advice on the experiments and manuscript. All authors read and approved the final manuscript.


Alternative splicing of exons in a pre-mRNA transcript is an important mechanism which contributes to protein diversity in human. Arrays for detecting alternative splicing are available using several different probe designs, including those based on exon-junctions. In this work, we introduce a new method for predicting alternatively skipped exons from exon-junction arrays. Predictions based on our method are compared against controls and their sequences are analyzed to identify motifs important for regulating alternative splicing.Ab initio motif finding algorithms are applied to identify several motifs that may be relevant for splicing during development.Our comparison of several alternative methods shows that an exon-skipping score based on neighboring junctions best discriminates between positive and negative controls. Sequence analysis of our predicted exons confirms the presence of known splicing regulatory sequences. In addition, we also derive a set of development-related alternatively spliced genes based on fetal versus adult tissue comparisons and find that our predictions are consistent with their functional annotations. This work describes a new method for analyzing exon-junction arrays, identifies sequence motifs that are specific for alternative and constitutive splicing and suggests a role for several known splicing factors and their motifs in developmental regulation. One of the essential processes is pre-mRNA splicing, in which the spliceosome complex recognizes splice sites (SS) in the precursors of mRNA following transcription, removes noncoding introns and joins neighboring exons to form mature messenger RNA that can be further translated into protein sequence. Both 5' splice sites . e.g.,  have beee.g., , arrays e.g., ,38, exone.g., ,39 or boe.g., ,40,41. Me.g., ), and the.g., ,42,43.de novo motif finding algorithms. Enrichment analysis of gene ontology annotation supports the functional roles of the predicted development-related alternative splices and suggests a new scheme for identification of process-specific alternative splicing.In this paper, we propose a new statistical approach for identifying alternatively spliced exons from exon-junction arrays, and predict a large set of alternatively skipped exons. This is achieved using the most comprehensive exon-junction array analysis to date , which myi, j, k from two dye-swapped arrays for each exon-junction probe j of gene k in tissue i, where i is one of the 52 tissues surveyed and j = 1, ..., nk for gene k, which has nk probes bridging nk+1 exons. Note that intensity patterns of exon-junction (EJ) probes alone cannot distinguish between intron retention or alternative 5' or 3' splice site usage, and hence we only focus on the effective detection of exon skipping in the 8618 genes containing 5 exons or more (nk ≥ 4 on the array). For each gene, we fit a linear model with terms for probe and tissue effects. Because of the large number of effects that need to be estimated, we must restrict the analysis to genes where there are enough data points. We found that a minimum of 5 exons was an adequate cutoff that avoids over-fitting in our statistical analysis but does not filter too many genes. For each gene, 5 exons corresponds to 4 exon-junctions (4 data points per tissue) and consequently 3 adjacent exon-junction pairs.The pre-processed array data described in  were obtyi, j, k: (i) probe-specific effect describing sequence binding affinity, (ii) tissue-specific effect resulting from differential expression, and (iii) alternative splice (AS) effect determined by exon inclusion or alternative splice site selection. A simple approach to recover the variance components of individual signal contribution is to use linear models on the properly transformed data (EJ probe intensities), essentially assuming that the transformed intensity is the sum of the probe, tissue, AS effect and white noise. A skipped exon will result in the two spanning EJ probes to be switched "off". This supports an exon-skipping score that measures the magnitude and concordance of AS effects in adjacent EJ probes spanning the same exon. Identifying exons that are alternatively skipped in some of the tissues surveyed is then equivalent to the detection of scores that deviate across tissues. In summary, our data analysis method can be described as a three-step procedure:If we treat exon inclusion as a simplified binary "on" or "off" event in each tissue, then conceptually, there are three main signal sources, other than noise, for the observed EJ probe intensity 1. Estimate the variance stabilizing transformation  for the ri, j, k represent AS effects adjusted for probe- and tissue-specific effects.2. Fit a linear model on the transformed data to estimate probe and tissue effects. The residuals of the fit ri, j, k and test for large deviations of this score across tissues.3. Summarize exon-skipping events at the exon and gene level by defining an exon-skipping score based on the residuals e.g., [ad hoc thresholding of the residuals ri,j,k to define AS scores (step 3). We considered several alternatives to this approach. First, the original method of [Note that , among oe.g., ), also uethod of  only exaethod of  to stabiIn particular, at each stage of the three-step procedure, we considered several alternative methods for the same objective: applying the logarithm or arcsinh-based variance stabilizing transformation (VSN) function for stabilizing variances in step 1; using the standard least square fit (mean) or robust fit (median or median polish) for linear model parameter estimation in step 2; and measuring deviations from normality of residuals in step 3 using the non-parametric Kolmogorov-Smirnov (KS) test, a weighed non-parametric Kolmogorov-Smirnov test (WKS), parametric Wilk-Shapiro test (WS)  or simplUsing the control data sets and receiver operating characteristic (ROC) curve analysis, we found that the VSN transformation performs better than log-transformation as expected (data not shown), and that the fitting procedures in Step 2 were indistinguishable in terms of prediction errors (data not shown). Figure -4). Several of the highest scoring examples using the WKS method, such as UBA3 (ubiquitin-activating enzyme 3 isoform 1), MAPKAP1 (mitogen-activated protein kinase associated) and PPM1B (protein phosphatase 1B isoform 1) were identified as "cassetteExon" gene entries from UCSC but were low scoring using the CV method, which only uses data from a single exon-junction. This illustrates that the combined product score based on neighboring exon-junctions can predict known examples which are missed by single junction methods. In summary, given the original linear model in [To further validate these results, we examined the WKS scores on another set of positive controls; the positive RT-PCR results in  and the model in , we deriUsing a score cutoff that balances the rate of true positive and false positives in our original two controls, we identified a set of alternatively spliced genes and corresponding exons (see Methods). We then determined the lowest ranking genes and exons according to our score to obtain an equivalently sized set of constitutive spliced exons. In total we predicted 8433 alternatively skipped exons and 8113 constitutive exons. An analysis of the Gene Ontology (GO) terms  for our To identify sequence motifs associated with the regulation of alternatively skipped exons, we adapted the contrast-kmer-based RESCUE-ESE algorithm for splicing  and the A direct contrast of alternatively skipped exons (AE) versus constitutively spliced exons (CE) in addition to differences in exons versus flanking introns gave 6 motifs associated with the 5' splice site of AE  as in the RESCUE-ESE analysis for constitutive splicing, the number of significant kmers reduced drastically. The only significant AE motif associated with weak splice sites is the well known motif GAAGA from 13 and 6 non-redundant hexamers in the 5' and 3' analysis respectively Figure .We also focused the contrast analysis on constitutively spliced exons to select for ESEs relevant for constitutive splicing using CE versus AE and exons versus introns comparisons and found 23 motifs for the 5' analysis and 12 motifs for the 3' analysis. The majority of motifs detected are CG-rich with diverse consensus patterns, most likely reflecting the coding bias of CE . Table We applied similar contrast analysis to identify intronic motifs associated with AE by selecting for kmers over-represented in introns flanking AE versus CE-associated introns and in introns versus exons. We found A/T rich motifs associated in introns flanking AE and pyrimidine tract-like motifs and G-triplets in introns flanking CE  are relevant for development, we analyzed a comprehensive gene expression data set from the Gene Expression Atlas [To investigate whether on Atlas , which con Atlas . We extron Atlas . Figure on Atlas . In parte.g., liver), developmental stages  and disease cell lines . Therefore, it is possible to identify AS involved in specific biological process by examining splice patterns between tissue pairs. For instance, genes that show different splicing profiles in fetal versus adult brain tissues may be indicative of functional roles in the regulation of brain development through splicing control. By comparing fetal versus adult tissues for several different organs, we adapted our gene-level exon-skipping score to obtain alternatively spliced genes that are associated with development.These results imply that some SR proteins may be involved in a general developmental response. To explore this hypothesis, we further investigated the exon-junction array, which has extensive results from different tissues . Define d = rij - ri'j as the difference between two tissues, i and i', in exon j. Extreme values (both positive and negative) for d indicate differences in exon-skipping levels between the tissues for that exon. We calculated a gene-level tissue pair difference score as the KS-statistic of d across all exons relative to a simulated null model assuming normally distributed residuals rij to gauge the differences in exon-skipping patterns between two tissues. Genes with Bonferroni-adjusted p-value < 0.05 were selected as candidate genes for development-related alternative exon-skipping in brain, liver, and lung tissues . For example, for our lung-developmental genes  and identified several motifs that are highly specific. For example, the known exonic sequence enhancer CACC was discovered in the constitutive exons but not in the alternative exons. Another known enhancer, GAAGAA, appears to be more specific to alternative exons than constitutive exons. Furthermore, we found that the occurrence of GAAGAA is biased towards exons with weak splice sites, while the other identified motifs associated with alternatively skipped exons do not have splice strength specificity. The sequence analysis also shows that, although originally developed for gene expression data [Following the array analysis, we examined our sequence predictions for dual purposes; to validate our analysis of the exon-junction array and to predict novel splicing enhancers and silencers. The results from the sequence analysis provide a further source of validation for the quality of our alternative and constitutive exon predictions. Using randomization trials, we found that our predictions were enriched in sequences that were not contained in random sets of sequences. We also identified several sequence signals that are consistent with the experimental literature . We made gene predictions for three tissue types  but only looked at the intersection to focus on general developmental alternative splicing, which was motivated by observed expression changes in specific SR proteins. Our final predictions for development-related alternative splicing were consistent with functional annotation and literature searches. In our sequence analysis of the development-related predictions, we found that a form of the GAAGAA motif may also have a role in alternative splicing during development. Furthermore, the changes in gene expression between fetal and adult tissues for several SR proteins that bind to this motif provide further evidence for their roles in developmental regulation.A useful feature of the Rosetta array is that both fetal and adult samples were included for three different tissues. We used this to develop a method for predicting genes with pairwise tissue differences in exon-skipping and applied our procedure to explore both the Because of the array design, our work focuses on the detection of exon-skipping. However, these data can also be used to predict and analyze other types of alternative splice events. In particular, if alternative splice site selection or intron retention occurs, we would observe variation in a single exon-junction across tissues, but not necessarily in consecutive junctions, as observed with exon-skipping. Therefore, a product summarization step would not be necessary, but we could adapt our procedure in other ways to predict alternative splice selection and intron retention. We will, however, not be able to discriminate between these other types of splice events because of the nature of the array design.A recent direction taken by several groups is to use sequence conservation across multiple species to aid in the search for enhancers and silencers -25,66,67j and tissue i is estimated by the residual ri, j, k from a linear model fit. To identify whether exon-skipping has occurred in some tissues, we considered four exon-skipping scores that measure the deviations of the residuals across tissues. All calculations described in this and all other Methods sections are performed in the software R  unless otherwise noted.Within each gene, the alternative splice (AS) effect for every probe j, pi, j, k = ri, j, kri, j +1, k. For the exon-level score, residual products across tissues i for exon j are examined, and for the gene-level score, the residual products across tissues i and exons j for gene k are examined. To obtain p-values based on the two-sample KS test, we compared the observed residual products with randomly sampled residual products from the null hypothesis that that they are a product of normally distributed random variables with mean 0, ri, j, k ~N, where σ is sampled from the empirical distribution for all genes.One score is based on the two-sample Kolmogorov-Smirnov (KS) test statistic of the residual products of adjacent exon-junction (EJ) probes spanning exons wkKSk) test statistic. The linear weight function is constructed to adjust for the overall expression signal strength and results in increasing the weight of more highly expressed genes. It is defined as yi, j, k is the average background subtracted intensity. To obtain p-values, observed values of WKS were compared with randomly sampled WKS statistics based on the product of randomly sampled weights, from a uniform distribution within the empirical range of calculated weights for all genes, and randomly sampled KS statistics, sampled from the theoretical distribution of the KS statistic.Exploratory data analysis showed greater variability for genes with low overall expression signal and thus we considered a more appropriate exon-skipping score, the two-sample weighted Kolmogorov-Smirnov (WKS = Another score is based on the coefficient of variation (CV), a statistic that measures large variations and is defined as the standard deviation divided by the mean. For exon-level score, we calculated the CV of the residual products across tissues, approach  which reri, j, k ~X where X is distributed as N. As before, the tests are performed across exons or genes to obtain the exon- or gene-level scores respectively.Finally, we considered a score based on a direct test for normality using the Wilk-Shapiro test (WS) . In thisxi, j, k, for each exon probe set. To identify genes that are likely to be constitutively spliced, we rank genes according to their expression variability across the 11 tissues and across corresponding core exon probesets, A collection of sample Affymetrix Human Exon 1.0 ST array data (from ) was useThe positive control (AS) consists of 164 EST-supported alternatively spliced genes that were placed on the arrays in Le et al., 2004 .th percentile according to their exon-level KS-statistic, which resulted in 9178 predicted exons. The KS-statistic is used for the exon-level score because the WKS-statistic has a gene-level correction for expression variability that is the same for all exons in a gene. Using the UCSC Genome Browser collection of RefSeq sequences (July 2003), 8433 exons were identified along with their neighboring introns (n = 13813) by sequence matching. Multiple occurrences of neighboring introns were removed. For the GO term analysis, we used a more stringent cutoff and only examined genes in the 95th percentile according to their WKS-statistic (n = 431). GO term enrichment was determined using the Benjamini-Hochberg correction for controlling the false discovery rate at the .001 level.To determine a set of alternatively spliced genes, we used a cutoff for the gene-level WKS-statistic that produced a true and false positive rate of ~70% and ~25% respectively on the controls. This cutoff resulted in 3912 predicted alternatively spliced genes. Of the exons in these genes, we then extracted exons in the 75th percentile of genes according to the gene-level WKS-statistic and extracted the exons within these genes that scored below the median exon-level KS-statistic. Using the UCSC Genome Browser collection of RefSeq sequences, 8113 of these exons were identified along with their neighboring introns (n = 10081). Multiple occurrences of neighboring introns were removed.To obtain a similar sized set of constitutive genes and exons so that the sequence comparisons would be balanced, we evaluated the lowest ~15We performed a search for over-represented kmers in our exonic and intronic sequences using the RESCUE-ESE method as described in . Brieflyi.e., subtracting the contribution from the previously selected kmer(s)). We masked the splice sites by removing 5 bp at the 5' and 3' end of each exon. The output lists all significant kmers with p-values adjusted using the Bonferroni correction.In addition, we searched for novel sequence motifs utilizing the whole data set without the need to dichotomize exons into AE and CE by applying the correlation-based method REDUCE  to the sTo look for motifs in the development-related AS exon sets, we examined the genes that had appropriate keyword hits (see below). Within these genes, sequences were extracted for exons predicted to be AE based on our exon-skipping score. De novo motif finders MEME  and BioP, with the "annotate" and "XML" packages was used to search each abstract of the references listed in the PubMed report for this gene. A hit for a gene was defined if the keyword(s) occurred in at least two of the abstracts for the gene. For each keyword, we performed a Fisher's exact test to evaluate whether the keyword appeared more frequently than expected by chance in our gene set compared to all genes. For the splicing keyword we searched for either "splicing" or "splice".For all genes in the Genome Browser RefSeq transcript list, the software R AS: alternative splice; AE: alternatively spliced exons; CE: constitutively spliced exons; CV: coefficient of variation; EJ: exon-junction; ESE: exonic splicing enhancers; KS: Kolmogorov-Smirnov; WS: Wilk-Shapiro; SS: splice site; ROC: receiver operating characteristic.KK and YHY performed the analysis, contributed to the design of the study and wrote the manuscript. RFY conceived the design of the study, coordinated the work and edited the manuscript. All authors read and approved the final manuscript.This file contains supporting tables: Table S1: Significant GO terms for top scoring alternatively-spliced genes. Table S2: Motifs found in alternative exons within 200 bp of the 5'SS. Table S3: Motifs found in alternative exons within 200 bp of the 3'SS. Table S4: Motifs found in alternative exons within 200 bp of the 5'SS and 3'SS. (weak splice site strength comparison). Table S5: Motifs found in constitutive exons within 200 bp of the 5'SS. Table S6: Motifs found in constitutive exons within 200 bp of the 3'SS. Table S7: Motifs found within 200 bp of the 5'SS of 3' introns flanking alternative exons. Table S8: Motifs found within 200 bp of the 3'SS of 5' introns flanking alternative exons. Table S9: Motifs found within 200 bp of the 5'SS of 3' introns flanking constitutive exons. Table S10: Motifs found within 200 bp of the 3'SS of 5' introns flanking constitutive exons. Table S11: Number of significant kmers in predicted exons and flanking introns and randomly selected exons and flanking introns. Table S12: Significant GO terms for genes with development-associated alternative splicing in brain. Table S13: List of exons predicted to undergo development-related AS. Table S14: Binding sites for SR proteins.Click here for fileThis file contains additional results and methods.Click here for fileThis file contains one supporting figure: Figure S1: Differences in log-intensities (y-axis) between fetal and adult tissues for SR proteins listed in [isted in . There aClick here for file


This study examines the prognostic significance of human papillomavirus (HPV) in patients with locally advanced oropharyngeal squamous cell carcinoma (SCC) treated primarily with surgery or definitive radiotherapy.One hundred and ninety-eight patients with Stage 3/4 SCC were followed up for recurrence in any form or death from any cause for between 1 and 235 months after diagnosis. HPV status was determined using HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry. Determinants of recurrence and mortality hazards were modelled using Cox's regression with censoring at follow-up dates.n=110), definitive radiotherapy-alone (n=24) and definitive radiotherapy with chemotherapy (n=47) groups, patients with HPV-positive cancers were one-third or less as likely to have loco-regional recurrence, an event or to die of any cause as those with HPV-negative cancers after adjusting for age, gender, tumour grade, AJCC stage and primary site. The 14 patients treated with surgery alone were considered too few for multivariable analysis.Forty-two per cent of cancers were HPV-positive (87% type 16). HPV predicted loco-regional control, event-free survival and overall survival in multivariable analysis. Within the surgery with adjuvant radiotherapy (HPV status predicts better outcome in oropharyngeal cancer treated with surgery plus adjuvant radiotherapy as well as with definitive radiation therapy±chemotherapy. It is now accepted that human papillomavirus (HPV) is an aetiological agent of up to 60% of oropharyngeal squamous cell carcinomas (SCCs)  and therRadiation therapy has an important role in the management of oropharyngeal cancers, either as definitive therapy or as an adjuvant therapy after surgery. Altered fractionation and concurrent chemotherapy have been developed to maximise disease control .There is increasing use of HPV status to guide treatment of oropharyngeal SCC , and two patients were missing adequate follow-up data (a 46-year-old man with a Stage 4 tonsillar SCC and a 68-year-old man with Stage 4 tonsillar SCC), leaving 198 patients. The study was approved by the ethics committees of Sydney South West Area Health Service . Most of the demographic and clinicopathological data were retrieved from the database of the Sydney Head and Neck Cancer Institute at Royal Prince Alfred Hospital, which spans the study period. Department of Radiation Oncology and Anatomical Pathology databases and hospital records were used to verify and input missing data as required.Patients were followed up for the occurrence of an event, defined as recurrence in any form or death from any cause, for between 1 and 235 (median 26) months after diagnosis. Patients who did not experience an event before the end of follow-up were known at that date to be alive and free of recurrence.μm sections of formalin-fixed paraffin-embedded cancer, using an HPV E6-based multiplex real-time PCR assay (MT-PCR) modified from the method of Evidence that HPV is transcriptionally active or localised to the nuclei of tumour cells is needed to establish causality in head and neck cancer . OverexpExpression of p16 was carried out by semiquantitative immunohistochemistry. Antigen retrieval was carried out using Target Retrieval Solution pH9  in a microwave oven for 10 min on high setting. After cooling, slides were placed in an autostainer (Dako). Endogenous peroxidase was blocked using 0.3% hydrogen peroxide in Tris-buffered saline (TBS); sections were incubated with primary antibody  (one out of 200) for 30 min, washed in TBS, treated with the EnVision Flex Dual Link horseradish peroxidase/DAB visualisation system (Dako) and then counterstained with haematoxylin. Staining was evaluated by three observers, including at least one pathologist (CSL). Typically, strong diffuse p16 staining was seen in the nucleus and cytoplasm of cancer cells with the proportion essentially all or none . Weak fot-test for the continuous variable age and chi-squared tests for categorical variables. One-way analysis of variance and chi-squared tests were used to compare patient characteristics in four treatment groups: surgery alone; surgery with adjuvant radiation therapy; definitive radiation therapy alone; and definitive radiation therapy with chemotherapy.Associations between HPV status and clinicopathological characteristics were assessed using a two-sample Survival analyses were conducted for the outcomes of loco-regional recurrence, event-free survival and overall survival, with time to each outcome calculated from the date of diagnosis. An event was defined as recurrence in any form or death from any cause, with only the first event taken into account. Patients without events were censored at the date of last follow-up. Univariate associations between HPV status and time to loco-regional recurrence, an event or death from any cause, were summarised for all patients and separately for each treatment group using Cox's proportional hazards models. Unadjusted survival curves were obtained using Kaplan–Meier estimates.Overall multivariable survival models were constructed with inclusion of HPV status and other known or potential correlates of outcome: age, gender, tumour grade, AJCC stage, primary site and treatment modality. Multivariable survival models were constructed separately for the three radiation therapy treatment groups, adjusting for HPV status, age, gender, tumour grade, AJCC stage and primary site where possible. Multivariable analyses were not carried out for the surgery-alone group because of the small numbers (14).We assessed whether the effect of HPV differed by treatment type by using interaction terms between HPV status and treatment , and adjusting only for age, tumour grade, tumour stage and site to avoid potential overfitting problems owing to small numbers. Adjusted survival curves by HPV status and treatment were obtained for the three treatment groups using Cox regression models, again adjusting only for age, tumour grade, stage and site. All analyses were conducted using the SAS System for Windows  and Stata Statistical Software .Forty-two per cent (83 out of 198) of the cancers were HPV-positive. Human papillomavirus type 16 accounted for 87% (72 out of 83) of the HPV-positive cases. Human papillomavirus types 35 and 18 were the most common other HPV types. Five cancers positive for HPV type 16 also contained a second HPV type . Twenty-one (11%) cancers tested HPV DNA positive/p16 negative and three (2%) cancers were HPV DNA negative/p16 positive . The 21 vs 62.6 years, P<0.0001), more likely to have primary disease in the tonsil (P=0.003) and had higher grade cancers (P=0.002). Patients with HPV-positive cancers were also more likely to have Stage 4 than Stage 3 cancers (P=0.03), and this was driven by more advanced nodal disease (P=0.02).The patients’ characteristics are summarised in The choice of treatment modality was based primarily on clinician and institution preference. Of the 195 patients analysed, 14 were treated with radical surgery alone . One hundred and ten patients were treated with surgery with adjuvant radiation therapy (five with concurrent chemotherapy and five with induction chemotherapy) . FourteeThe 35 patients who received concurrent chemotherapy and definitive radiation therapy (mainly 5-fluorouracil or 3-weekly cisplatin) were treated between 1990 and 2006; all but three of these patients were treated after 1996. These 35 patients were equally divided between the HPV-positive and -negative groups. Over the study period, there was a change in definitive radiation dose scheduling from 60 Gy in 30 fractions over 6 weeks in the early 1990s to 70 Gy in 35 fractions over 7 weeks from 1999. The dose of adjuvant radiation therapy also increased over the study period from 50 Gy in 25 fractions to 60 Gy in 30 fractions in more recent years. All patients were treated with multiple photon fields using standard fractionation. Patients receiving surgery alone were older than other patients and patients receiving definitive radiotherapy with chemotherapy were more likely to have tumours located in the base of tongue .One patient was missing loco-regional recurrence status, yielding 194 patients for analysis. Loco-regional recurrence occurred in 54 (28%) of 194 patients: in four of the 14 (29%) patients treated with surgery alone, 24 of the 109 (22%) patients treated with surgery and adjuvant radiation therapy, 10 of the 25 (40%) patients treated with definitive radiotherapy alone and 16 of the 47 (34%) patients treated with definitive radiation therapy with chemotherapy. Recurrence occurred at the primary site in 30 patients and in the regional nodal area (with disease controlled at the primary site) in 24 patients. Sixteen patients developed distant metastasis as the first site of recurrence, none of whom had simultaneous loco-regional recurrence.Univariate analysis of data from all patients showed that HPV-positive cancers were less likely to recur loco-regionally than were HPV-negative cancers . This asP=0.7 for interaction between HPV status and treatment group in the adjusted analysis. After adjustment for age, tumour grade and tumour stage, the risk of loco-regional recurrence associated with HPV-positive cancers relative to HPV-negative cancers was about one-third  in the surgery with adjuvant radiation therapy group, one-eighth  in the definitive radiation therapy-alone group and one-quarter  in the definitive radiation therapy with chemotherapy group  patients treated with surgery alone, 48 of the 110 patients (44%) treated with surgery and adjuvant radiation therapy, 16 of the 24 (67%) patients treated with definitive radiotherapy alone and 26 of the 47 (55%) patients treated with definitive radiation therapy and chemotherapy. Univariate analysis of data from all patients showed that patients with HPV-positive cancers were less likely to suffer an event than those with HPV-negative cancers . This asP=0.4 for interaction between HPV status and treatment group in the adjusted analysis. After adjustment for age, tumour grade and stage, the risk of an event associated with HPV-positive cancers relative to HPV-negative cancers was about one fifth  in the surgery with adjuvant radiation therapy group, about one-seventh  in the definitive radiation therapy-alone group and about one-third  in the definitive radiation therapy with chemotherapy group  treated with surgery alone, 38 of the 110 patients (35%) receiving surgery with adjuvant radiation therapy, 13 of the 24 patients (54%) receiving definitive radiotherapy alone and 24 of the 47 patients (51%) receiving definitive radiation therapy with chemotherapy. Univariate analysis of data from all patients showed that patients with HPV-positive cancers were less likely to die of any cause than those with HPV-negative cancers . This asP=0.3 for interaction between HPV status and treatment group in the adjusted analysis. After adjustment for age, tumour grade and stage, the risk of death from any cause associated with HPV-positive cancers relative to HPV-negative cancers was almost one-tenth  in the surgery with adjuvant radiation therapy group, almost one-tenth  in the definitive radiation therapy-alone group and about one-third  in the definitive radiation therapy with chemotherapy group , with the surgery and adjuvant radiotherapy group having the lowest risk of loco-regional recurrence and the surgery-only group having the highest risk.On the basis of multivariable analyses of all patients adjusting for HPV status, type of treatment was the only clinical characteristic that showed evidence of an association with loco-regional recurrence (P=0.0001), site of cancer (P=0.04) and type of treatment (P=0.0003) after adjusting for HPV status. Poorer outcomes were seen in Stage 4 patients, patients with cancer in sites other than tonsil and patients receiving surgery only.Multivariable predictors of event-free survival were identified as stage (P<0.0001), site of cancer (P=0.001) and type of treatment (P=0.05). Again, poorest outcomes were seen in Stage 4 patients, patients with cancer in sites other than tonsil and patients receiving surgery only.After adjusting for HPV status, clinical characteristics that showed evidence of associations with overall survival were identified as stage (P for interaction >0.3 in each case).When stratified by HPV status, outcome-adjusted survival against each end point was consistently highest for surgery with adjuvant radiotherapy, intermediate for definitive radiotherapy with chemotherapy and lowest for definitive radiotherapy alone . (PatienRecent studies have suggested that the favourable outcome associated with HPV in oropharyngeal cancer is owing to an increased sensitivity of virus-related cancers to radiation therapy or chemoradiotherapy (As most recent studies have focused on HPV and outcome following definitive radiotherapy or chemoradiotherapy, the interest of this study centres on patients with surgery as primary treatment. Our results suggest that outcomes in patients with locally advanced HPV-positive cancers treated with surgery and adjuvant radiotherapy were as good as, and possibly better than, those treated with definitive radiotherapy with or without chemotherapy. This also appeared to be the case for HPV-negative cancers, and the effect of HPV status on outcome did not appear to differ materially between these treatment groups. In their study of patients with all stages of disease, P-values for interaction make any inference from this difference very uncertain. Moreover, we cannot exclude selection bias relating to performance status, co-morbidities, smoking and alcohol exposure that might affect the HRs (The seemingly lower HRs for outcome of HPV-positive cancer relative to HPV-negative cancer in the definitive radiotherapy-only group than in the definitive radiotherapy with chemotherapy group were unexpected. However, the wide CIs about the HRs and the high The heterogeneity of disease within the head and neck region is well recognised. Although oropharyngeal SCCs are regarded as a relatively homogeneous group, studies with the statistical power to examine site within the oropharynx have found that patients with base of tongue SCCs have the worst prognosis (The proportion of oropharyngeal cancers attributable to HPV has varied from 19% to more than 60% across different studies . Our oveTesting for HPV status of oropharyngeal SCCs is increasing in clinical practice, but there is no level 1 or 2 evidence to guide treatment based on HPV status. We conclude that patients with HPV-positive locally advanced oropharyngeal SCC have a more favourable prognosis than HPV-negative cancers, regardless of whether they are treated with radical surgery plus adjuvant radiation therapy or by an organ-preserving approach using definitive radiation therapy with or without chemotherapy. The possibility that type of treatment, as well as HPV status, may influence outcome warrants investigation in randomised controlled trials.


A follow-up study of 1844 renal transplant patients in Italy showed a 113-fold increased risk for Kaposi's sarcoma. Kaposi's sarcoma risk was higher in persons born in southern than in northern Italy. Significant increases were also observed for cancers of the lip, liver, kidney and for non-Hodgkin's lymphoma. Renal transplant patients experience higher rates of cancer, particularly virus-related cancers, than the general population. The overall cancer risk is three- to four-fold elevated, but some specific cancers show greater increases . For nonThe prevalence of infection with KS-associated herpesvirus )  or central Italy (Rome). Of these, 1340 received their transplants at Niguarda Hospital, Milan, between 1972 and 2003 and 504 at Policlinico Gemelli Hospital, Rome, between 1970 and 2000. As with other investigations, we excluded patients who survived less than 10 days after transplant, those with a pretransplant history of cancer, or who developed cancer in the 30-day period after transplantation (Person-years (PY) at cancer risk were measured from the date of first renal transplant and ended upon at tumour diagnosis, death, or date of last follow-up visit, whichever occurred first. For the purposes of this analysis, periods of graft failure were not considered. For each type of cancer observed, the expected number  was computed using incidence rates from all population-based cancer registries of Italy for the 1988–1992 period . IncidenThe median age of the 1844 renal transplant patients was 42 years (interquartile range: 31–51 years). In 15 940 PY of observation (median follow-up time: 7.0 years), 104 cases of cancer, other than nonmelanoma skin cancers, were diagnosed. Standardised incidence ratios according to cancer type or site, and sex are shown in The SIR for all cancers was 1.8 (95% CI: 1.5–2.2), and was of similar magnitude in the two sexes. The SIR for KS was 112.6 but it was greater among women  than men . A 6.6-fold increased SIR (95% CI: 3.7–11.0) was seen for NHL. Other cancers with significantly elevated SIRs included: carcinomas of the lip , liver , and kidney . The SIRs for lip and liver cancers were more elevated in women than men, whereas the excess risk for native renal cancer was found in men only . StandarAmong renal transplant patients, KS was more frequent  in persons aged 50 years or older at transplant than in those younger than 40. Kaposi's sarcoma was nonsignificantly more common in men than in women, whereas it was nearly six-fold more frequent (95% CI: 1.6–19.7) in patients born in southern Italy, as compared to those born in northern Italy . A stronMost of these 1844 patients (87%) received their transplant after cyclosporin came into widespread use (from 1983 onwards), and one-third after the introduction of the newer immunosuppressive drugs (from 1997 onwards). All 23 KS cases were diagnosed in 1983 or later inpatients who were treated with cyclosporin-containing regimens. No significant difference in KS IRRs was seen according to the calendar period of transplant .Our study provides estimates of cancer incidence for renal transplant recipients in northern or central Italy and IRRs for KS. The overall increase in cancer risk we found (1.8-fold) is somewhat lower than that reported from previous investigations in transplant patients (With respect to the increased risks for NHL and renal cancer, our results agree with previous investigations (This study also revealed that renal transplant patients in Italy have a four-fold increased risk of liver cancer, an excess risk not generally seen in transplant patients elsewhere (In conclusion, our findings in patients who underwent renal transplantation in northern or central Italy provide further evidence of the strong association between organ transplant and virus-related cancers. Our observations on post-transplant KS confirm the need for HHV-8 screening protocols and for guidelines on the clinical management of HHV-8 infection in transplant centres, at least in areas endemic for KS (