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Background: Recently published asthma guidelines by the European Respiratory Society and the American Thoracic Society (ERS-ATS) define severe disease based on medication use and control level. These guidelines also emphasize that asthma severity involves certain biomarker phenotypes, one of them being eosinophilic phenotype. The quantification of the influence of eosinophil level toward predicting disease severity can help decision makers manage therapy better earlier., Objective: To develop a risk-scoring algorithm to identify patients at greater risk of developing uncontrolled severe asthma as defined by ERS-ATS guidelines., Methods: Data on asthma patients were extracted from the EMRClaims + database from January 2004 to July 2011. Patients with continuous enrollment 12 months before and after the date of the first encounter with a diagnosis of asthma (index date) with at least 1 blood eosinophil test result in the 12 months after the index date, but before the development of uncontrolled severe asthma or the study end date, were included. Uncontrolled severe asthma was defined as the first date on which all criteria of the ERS-ATS definition were first satisfied in the 12 months after the index date. Age (≥ 50 years vs. < 50 years), race, and sex were measured at index, and the Charlson Comorbidity Index (CCI) score (> 0 vs. 0) was measured in the pre-index period. Elevated eosinophil level was defined as a test result with ≥ 400 cells/µL. The study cohort was randomly split 50-50 into derivation and validation samples. Cox proportional hazards regression was used to develop the risk score for uncontrolled severe asthma using the derivation cohort with independent variables of eosinophil level, age, sex, race, and CCI. A bootstrapping procedure was used to generate 1,000 samples from the derivation cohort. Variables significant in ≥ 50% of the samples were retained in the final regression model. A risk score was then calculated based on the coefficient estimates of the final model. C-statistic was used to test the model's discrimination power., Results: The study included 2,405 patients, 147 (6%) of whom developed uncontrolled severe asthma. Higher eosinophil level and CCI score > 0 were significantly and independently associated with an increased risk of uncontrolled severe asthma in the derivation cohort (HR = 1.90, 95% CI = 1.17-3.08 and HR = 2.00, 95% CI = 1.28-3.13, respectively); findings were similar in the validation cohort. Total risk score was categorized as 0, 2, and 4. All models showed good C-statistics (0.79-0.80), indicating favorable model discrimination. There was a significantly greater number of patients with uncontrolled severe asthma in the risk score segments of 2 and 4 compared with 0 (each P < 0.0001)., Conclusions: A risk stratification tool using peripheral eosinophil counts and CCI can be used to predict the development of uncontrolled severe asthma., Disclosures: This study was funded by Teva Pharmaceuticals. eMAX Health Systems was a consultant to Teva Pharmaceuticals for this study and received payment from Teva Pharmaceuticals for work on this study. Casciano and Dotiwala are employed by eMAX Health Systems. Krishnan, Li, and Martin received payment from eMAX Health Systems for work on this study. Small was employed by Teva Pharmaceuticals at the time of this study. Study concept and design were contributed primarily by Casciano, Krishnan, Small, and Martin, along with Li and Dotiwala. Dotiwala, Casciano, Small, and Li collected the data, along with Martin and Li and Krishnan. Data interpretation was provided by Martin, Casciano, and Li, with assistance from the other authors. The manuscript was written by Li, Casciano, Dotiwala, and Small, with assistance from the other authors, and revised by Dotiwala, Small, Li, and Martin, with assistance from Krishnan and Casciano.

Background: Asthma represents a significant clinical and economic burden to the US healthcare system. Along with other clinical manifestations of the disease, elevated sputum and blood eosinophil levels are observed in patients experiencing asthma exacerbations. The aim of this study was to evaluate the association between blood eosinophil levels and asthma severity defined using Expert Panel Report 3 guidelines., Methods: Patients with asthma diagnosis between 2004 and 2011 were extracted from the EMRClaims+ database (eMAX Health, White Plains, NY) containing electronic medical records linked to insurance claims for over 675,000 patients. The date of first asthma diagnosis was defined as the 'index date'. Patients were required to have at least 1 peripheral eosinophil test (elevated defined as ≥ 400 cells/μL) in the 12 month 'assessment' period following the index date. We classified patients as those with mild asthma and moderate-to-severe asthma based on the pattern of medication use, as recommended by the 2007 National Institutes of Health Expert Panel Report. Logistic regression models were used to determine if patients with moderate-to-severe asthma had increased likelihood of an elevated peripheral eosinophil count, after accounting for demographics and comorbidities., Results: Among 1,144 patients with an asthma diagnosis, 60 % were classified as having moderate-to-severe asthma. Twenty four percent of patients with moderate-to-severe asthma and 19 % of patients with mild asthma had an elevated peripheral eosinophil count (p = 0.053). Logistic regression showed that moderate-to-severe asthma was associated with 38 % increased odds of elevated eosinophil level (OR 1.38, 95 % CI: 1.02 to 1.86, p = 0.04)., Conclusion: Patients with moderate-severe asthma are significantly more likely to have an elevated peripheral eosinophil count than patients with mild asthma.

Background: Asthma is a common chronic condition with an economic burden of almost $56 billion annually in the US. Biologic markers like blood eosinophils, that help predict the risk of exacerbation could help guide more optimal treatment plans and reduce cost. The purpose of this study was to determine whether healthcare resource use and expenditures vary by eosinophil level among patients with asthma., Methods: Patients with a diagnosis of asthma defined by ICD-9-CM code 493.xx between January 2004 and July 2011 were extracted from EMRClaims + database (eMAX Health, White Plains NY). Patients were classified as mild, moderate, or severe by medication use following diagnosis, based on recommendations of National Institutes of Health Expert Panel Report 3. Patients were classified as those with elevated eosinophils (≥400 cells/μL) and normal eosinophil level (<400 cells/μL). Patients were followed for resource use, defined as hospitalizations, ER visits and outpatient visit and associated costs were calculated to assess whether an economic difference exists between eosinophil groups. Non-parametric tests were used to compare resource use and associated cost between elevated and normal eosinophil groups. Multivariate modeling was performed to assess the contribution of eosinophil level on the likelihood of study outcomes among patients with severe asthma., Results: Among the 2,164 patients meeting eligibility criteria, 1,144 had severity designations. Of these, 179(16 %) of patients had severe asthma of which 20 % (n = 35) had elevated eosinophils. Seventeen percent of patients with elevated eosinophils were admitted to the hospital during the follow-up period, significantly greater than patients with normal eosinophil levels (12 %; p = 0.011). Overall, compared to patients with normal eosinophil levels (n = 1734), patients with elevated eosinophil levels (n = 430) had significantly greater mean annual hospital admissions (0.51 vs. 0.21/year, p = 0.006) and hospital costs (2,536 vs. $1,091, p = 0.011). Logistic regressions showed that elevated eosinophil level was associated with 5.14 times increased odds of all cause admissions (95 % CI:1.76-14.99, p = 0.003) and 4.07 times increased odds of asthma related admissions (95 % CI: 1.26-13.12, p = 0.019)., Conclusion: Eosinophil elevation was associated with greater healthcare resource use in patients with asthma.

Background: Elevated sputum eosinophil counts predict asthma exacerbations and responsiveness to inhaled corticosteroids but are impractical to measure in primary care. We investigated the relation between blood eosinophil count and prospective annual asthma outcomes for a large UK cohort., Methods: This historical cohort study used anonymised medical record data to identify primary care patients with asthma aged 12-80 years with 2 years of continuous records, including 1 year before (baseline) and 1 year after (outcome) their most recent eosinophil count. Negative binomial regression was used to compare outcome exacerbation rates and logistic regression to compare odds of asthma control for patients with blood eosinophil counts of 400 cells per μL or less versus greater than 400 cells per μL, adjusting for age, sex, body-mass index, smoking status, and Charlson comorbidity index. The study is registered at ClinicalTrials.gov, number NCT02140541., Findings: Overall, 20 929 (16%) of 130 248 patients had blood eosinophil counts greater than 400 cells per μL. During the outcome year, these patients experienced significantly more severe exacerbations (adjusted rate ratio [RR] 1·42, 95% CI 1·36-1·47) and acute respiratory events (RR 1·28, 1·24-1·33) than those with counts of 400 cells per μL or less. They also had significantly lower odds of achieving overall asthma control (OR 0·74, 95% CI 0·72-0·77), defined as limited reliever use and no asthma-related hospital attendance or admission, acute course of oral corticosteroids, or prescription for antibiotics. Exacerbation rates increased progressively with nine ascending categories of blood eosinophil count as compared with a reference category of 200 cells per μL or less., Interpretation: Patients with asthma and blood eosinophil counts greater than 400 cells per μL experience more severe exacerbations and have poorer asthma control. Furthermore, a count-response relation exists between blood eosinophil counts and asthma-related outcomes. Blood eosinophil counts could add predictive value to Global Initiative for Asthma control-based risk assessment., Funding: Teva Pharmaceuticals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Left ventricular assist devices (LVADs) are increasingly being used as a bridge for cardiac transplantation in patients with decompensated cardiac function. A known complication of these devices is severe and sometimes life-threatening GI bleeding, usually related to the presence of angioectasias. Endoscopy has been generally accepted as safe in patients with cardiac disease and implanted cardiac devices, when it is performed with appropriate monitoring. However, the literature is sparse regarding the safety of endoscopy in patients with LVADs., Objective: Given the potential risks for GI bleeding in this subgroup of patients, our aim was to shed light on the potential safety of endoscopy in these patients., Design: We present our experience with endoscopic intervention for varied sources of GI bleeding in a group of patients with the HeartMate II implantable LVAD., Setting: A tertiary care university-based hospital setting, Tampa General Hospital at the University of South Florida, Tampa, Florida., Patients: Patients with severe cardiomyopathy requiring cardiac support with the HeartMate II implantable LVAD., Interventions: Patients received upper and lower GI endoscopy as dictated by their clinical presentations. One patient received capsule endoscopy as well., Conclusions: We observed that endoscopy, including capsule endoscopy, may be safely performed in these patients with appropriate monitoring.

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process.

Lack of understanding of the mechanism of tissue destruction associated with idiopathic esophageal ulcers (IEUs) poses a diagnostic and therapeutic dilemma for the clinician. The possible role of apoptosis in IEUs, as suggested by endoscopic and histologic observations, was investigated by examination of archival tissues for apoptosis-related DNA fragmentation using in situ nick end labeling (TUNEL). High levels of apoptosis were observed in mucosal cells immediately adjacent to IEUs. Apoptotic cells were virtually absent in normal control tissues, while the edges and bases of lesions and sloughed-off tissues in IEUs in human immunodeficiency virus (HIV)-infected patients showed elevated levels of apoptotic cell death. However, tissue samples from patients with esophageal ulcerations of known etiology showed no apoptosis of mucosal cells. These data support a role for apoptosis in the pathogenesis of IEUs and suggest a mechanism involving HIV-associated bystander killing of uninfected mucosal cells.

Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

Under appropriate conditions (e.g., growth factor withdrawal), the deregulated expression of c-myc in rodent fibroblasts leads to substantial cell death due to apoptosis. To better understand this process, we selected for c-myc-transformed Rat1A fibroblasts that were resistant to growth factor deprivation-induced cell death. One clonal isolate exhibited prolonged survival in serum-free medium and displayed reduced levels of apoptosis-related DNA fragmentation. These cells were also resistant to induction of apoptosis by the protein kinase inhibitor staurosporine. They retained a transformed cell phenotype and expressed the proviral human c-myc allele in an unaltered fashion, strongly indicating that the mutation of a cellular gene other than c-myc accounts for the apoptosis-resistant phenotype. The results of somatic cell hybrid analysis of this cell line are consistent with a recessive mutation. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy.

The loss of telomere sequences during in vitro and in vivo aging suggests that mechanisms affecting telomere length may have important consequences in cellular senescence. In this study, we have found that the activity of single-stranded telomere binding proteins is increased in nuclear extracts prepared from senescent human diploid fibroblasts compared to actively growing cells. Since single-stranded telomere binding proteins are closely related to RNA binding proteins, we examined the binding activity of several major RNA binding proteins to RNA by uv cross-linking. The level of activity was greatly diminished and the overall pattern of uv cross-linked products were altered in extracts prepared from senescent cells. Furthermore, Western analysis revealed a concurrent decrease in senescent extracts of the protein level for many RNA binding proteins, including those which bind to telomere sequence. Although the reduction in the level of these proteins parallels the reduced activity in RNA binding, the paradoxical increased telomere binding activity exhibited by extracts from older cells suggests a complex relationship between these proteins with RNA and DNA. Moreover, the reduced RNA binding activity of these proteins indicates that the biochemical function of several RNA binding proteins is compromised during cellular senescence, raising an intriguing possibility that a change in pre-mRNA metabolism may contribute to the multitude of changes in gene expression observed in cellular senescence.

The product of the c-myc proto-oncogene is an important positive regulator of cell growth and proliferation. Recently, c-Myc has also been demonstrated to be a potent inducer of apoptosis when expressed in the absence of serum or growth factors. To further examine Myc-induced apoptosis, we coexpressed the proto-oncogene bcl2, which has been shown to block apoptosis in other systems, with c-myc in serum-deprived Rat 1a fibroblasts. Here we report that ectopic expression of bcl2 specifically blocks apoptosis induced by constitutive c-myc expression. Constitutive c-myc expression in serum-deprived Rat 1a cells caused a > 15-fold increase in the number of dead cells, accompanied by DNA fragmentation. However, coexpression of bcl2 with c-myc in these cells led to a 10-fold increase in the number of live cells and a significant decrease in DNA fragmentation. Thus, Bcl-2 effectively inhibits Myc-induced apoptosis in serum-deprived Rat 1a fibroblasts without blocking entry into the cell cycle. These results imply that apoptosis serves as a protective mechanism to prevent tumorigenicity elicited by deregulated Myc expression. This protective mechanism is abrogated, however, by Bcl-2 and therefore may explain the synergism between Myc and Bcl-2 observed in certain tumor cells.

Apoptosis, or programmed cell death, plays an essential role in specific cell deletion during normal embryonic and adult development in vertebrate and invertebrate species. Recent evidence suggests that signal transduction pathways governing cellular proliferation and cell cycle progression also mediate the physiological response to changes in the extracellular environment that trigger the anti-proliferative state characteristic of apoptosis.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.