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Introduction The opioid crisis is a pervasive public health threat in the U.S. Simulation modeling approaches that integrate a systems perspective are used to understand the complexity of this crisis and analyze what policy interventions can best address it. However, limitations in currently available data sources can hamper the quantification of these models. Methods To understand and discuss data needs and challenges for opioid systems modeling, a meeting of federal partners, modeling teams, and data experts was held at the U.S. Food and Drug Administration in April 2019. This paper synthesizes the meeting discussions and interprets them in the context of ongoing simulation modeling work. Results The current landscape of national-level quantitative data sources of potential use in opioid systems modeling is identified, and significant issues within data sources are discussed. Major recommendations on how to improve data sources are to: maintain close collaboration among modeling teams, enhance data collection to better fit modeling needs, focus on bridging the most crucial information gaps, engage in direct and regular interaction between modelers and data experts, and gain a clearer definition of policymakers’ research questions and policy goals. Conclusions This article provides an important step in identifying and discussing data challenges in opioid research generally and opioid systems modeling specifically. It also identifies opportunities for systems modelers and government agencies to improve opioid systems models.

The opioid crisis is a major public health challenge in the United States, killing about 70,000 people in 2020 alone. Long delays and feedbacks between policy actions and their effects on drug-use behavior create dynamic complexity, complicating policy decision-making. In 2017, the National Academies of Sciences, Engineering, and Medicine called for a quantitative systems model to help understand and address this complexity and guide policy decisions. Here, we present SOURCE (Simulation of Opioid Use, Response, Consequences, and Effects), a dynamic simulation model developed in response to that charge. SOURCE tracks the US population aged ≥12 y through the stages of prescription and illicit opioid (e.g., heroin, illicit fentanyl) misuse and use disorder, addiction treatment, remission, and overdose death. Using data spanning from 1999 to 2020, we highlight how risks of drug use initiation and overdose have evolved in response to essential endogenous feedback mechanisms, including: 1) social influence on drug use initiation and escalation among people who use opioids; 2) risk perception and response based on overdose mortality, influencing potential new initiates; and 3) capacity limits on treatment engagement; as well as other drivers, such as 4) supply-side changes in prescription opioid and heroin availability; and 5) the competing influences of illicit fentanyl and overdose death prevention efforts. Our estimates yield a more nuanced understanding of the historical trajectory of the crisis, providing a basis for projecting future scenarios and informing policy planning.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

ObjectiveTo estimate the population attributable fraction (PAF) and number of incident and fatal lung cancers in Canada from occupational exposure to diesel engine exhaust (DEE).MethodsDEE exposure prevalence and level estimates were used with Canadian Census and Labour Force Survey data to model the exposed population across the risk exposure period (REP, 1961–2001). Relative risks of lung cancer were calculated based on a meta-regression selected from the literature. PAFs were calculated using Levin’s equation and applied to the 2011 lung cancer statistics obtained from the Canadian Cancer Registry.ResultsWe estimated that 2.4% (95% CI 1.6% to 6.6%) of lung cancers in Canada are attributable to occupational DEE exposure, corresponding to approximately 560 (95% CI 380 to 1570) incident and 460 (95% CI 310 to 1270) fatal lung cancers in 2011. Overall, 1.6 million individuals alive in 2011 were occupationally exposed to DEE during the REP, 97% of whom were male. Occupations with the highest burden were underground miners, truck drivers and mechanics. Half of the attributable lung cancers occurred among workers with low exposure.ConclusionsThis is the first study to quantify the burden of lung cancer attributable to occupational DEE exposure in Canada. Our results underscore a large potential for prevention, and a large public health impact from occupational exposure to low levels of DEE.

Purpose Publication metrics are useful in evaluating academic faculty for awarding grants, recruitment, and promotion. A new metric, the relative citation ratio (RCR), was recently released by the National Institutes of Health (NIH); however, no benchmark data yet exist. We sought to create benchmark data for physician faculty in academic radiation oncology (RO) and analyze correlations associated with increased academic productivity. Methods Citation database searches were performed for all US radiation oncologists affiliated with academic RO programs. Gender, NIH funding, career duration, academic rank, RCR, and weighted RCR were collected for each faculty. RCR and weighted RCR were calculated and compared between each subgroup of interest. RCR percentiles were also created for reference. Results A total of 1,299 RO physician faculty members from 75 institutions were included in the analysis. Overall, RO physician were very productive and influential with a mean RCR of 1.57 ± 1.53 SD and median RCR (interquartile range) of 1.32 (0.87-1.94). Academic rank, career duration, and NIH funding were associated with increased mean RCR and weighted RCR. Male gender and having a PhD were associated with an increased weighted RCR but not an increased mean RCR. Conclusions Current academic radiation oncologists have a high mean RCR value relative to the benchmark NIH RCR value of 1. All subgroups analyzed had an RCR value above 1 with professor or chair and previous NIH funding having the highest RCR and weighted RCR values overall. These data may be useful for self-evaluation of ROs as well as evaluation of faculty by institutional and departmental leaders.

OBJECTIVE: To describe the normal motion pattern at the midcarpal compartment during active radial-ulnar deviation of the wrist using dynamic MRI and to determine the observer performance for measurements obtained in asymptomatic volunteers. METHODS: Dynamic MRI of 35 wrists in 19 asymptomatic volunteers (age mean 30.4 yrs, sd 8.6) was performed during active radial-ulnar deviation using a fast gradient-echo pulse sequence with 315 ms temporal resolution (acquisition time, 19 sec). Two independent readers measured the transverse translation of the trapezium at the scaphotrapezium joint (STJ) and the capitate-to-triquetrum distance (CTD). Relationships between these measurements and laterality, sex, lunate type, and wrist kinematic pattern were evaluated. RESULTS: At the STJ, the trapezium moved most in radial deviation, with an overall translation of 2.3mm between ulnar and radial deviation. Mean CTD measurements were the greatest in ulnar deviation and varied 2.4mm between ulnar and radial deviation. Mean CTD was greater in men than women in the neutral position (p=0.019), as well as in wrists with type II lunate morphology during radial and ulnar deviation (p=0.001, p=0.014). There were no significant differences in trapezium translation or CTD with wrist laterality and kinematic pattern. Intraobserver and interobserver correlation coefficients were 0.97 and 0.87 for trapezium translation and 0.84 and 0.67 for CTD. CONCLUSION: This study is the first to demonstrate the performance of dynamic MRI to quantify STJ motion and CTD. Dynamic MRI with a short acquisition time may be used as a tool to supplement static MRI in evaluation of the midcarpal compartment.

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell–mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the β2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4β7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of β2 integrin–expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non–Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10–regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

Author(s): Shaw, Calvin B; Foster, Brent H; Borgese, Marissa; Boutin, Robert D; Bateni, Cyrus; Boonsri, Pattira; Bayne, Christopher O; Szabo, Robert M; Nayak, Krishna S; Chaudhari, Abhijit J | Abstract: BackgroundCarpal instability is defined as a condition where wrist motion and/or loading creates mechanical dysfunction, resulting in weakness, pain and decreased function. When conventional methods do not identify the instability patterns, yet clinical signs of instability exist, the diagnosis of dynamic instability is often suggested to describe carpal derangement manifested only during the wrist's active motion or stress. We addressed the question: can advanced MRI techniques provide quantitative means to evaluate dynamic carpal instability and supplement standard static MRI acquisition? Our objectives were to (i) develop a real-time, three-dimensional MRI method to image the carpal joints during their active, uninterrupted motion; and (ii) demonstrate feasibility of the method for assessing metrics relevant to dynamic carpal instability, thus overcoming limitations of standard MRI.MethodsTwenty wrists (bilateral wrists of ten healthy participants) were scanned during radial-ulnar deviation and clenched-fist maneuvers. Images resulting from two real-time MRI pulse sequences, four sparse data-acquisition schemes, and three constrained image reconstruction techniques were compared. Image quality was assessed via blinded scoring by three radiologists and quantitative imaging metrics.ResultsReal-time MRI data-acquisition employing sparse radial sampling with a gradient-recalled-echo acquisition and constrained iterative reconstruction appeared to provide a practical tradeoff between imaging speed (temporal resolution up to 135 ms per slice) and image quality. The method effectively reduced streaking artifacts arising from data undersampling and enabled the derivation of quantitative measures pertinent to evaluating dynamic carpal instability.ConclusionThis study demonstrates that real-time, three-dimensional MRI of the moving wrist is feasible and may be useful for the evaluation of dynamic carpal instability.

We present a method for the statistical modeling of the displacements of wrist bones during the performance of coordinated maneuvers, such as radial-ulnar deviation (RUD). In our approach, we decompose bone displacement via a set of basis functions, identified via principal component analysis (PCA). We utilized MRI wrist scans acquired at multiple static positions for deriving these basis functions. We then utilized these basis functions to compare the displacements undergone by the bones of the left versus right wrist in the same individual, and between bones of the wrists of men and women, during the performance of the coordinated RUD maneuver. Our results show that the complex displacements of the wrist bones during RUD can be modeled with high reliability with just 5 basis functions, that captured over 91% of variation across individuals. The basis functions were able to predict intermediate wrist bone poses with an overall high accuracy (mean error of 0.26 mm). Our proposed approach found statistically significant differences between bone displacement trajectories in women versus men, however, did not find significant differences in those of the left versus right wrist in the same individual. Our proposed method has the potential to enable detailed analysis of wrist kinematics for each sex, and provide a robust framework for characterizing the normal and pathologic displacement of the wrist bones, such as in the context of wrist instability.

Homeostasis in the ileum, which is commonly disrupted in patients with Crohn’s disease, involves ongoing immune responses. To study how homeostatic processes of the ileum impact CD4(+)T cell responses, we used TCR transgenic tools to breed mice that spontaneously produced CD4(+)T cells reactive to an antigen expressed in the ileum. At an early age, the ilea of these mice exhibit crypt hyperplasia and accumulate increased numbers of T(H)17 cells bearing non-transgenic clonotypes. Half of these mice subsequently developed colitis linked to broad mucosal infiltration by T(H)17 and T(H)1 cells expressing non-transgenic clonotypes, chronic wasting disease and loss of ileal crypt hyperplasia. By contrast, adult mice with normal growth continued to exhibit T(H)17-associated ileal crypt hyperplasia and additionally accumulated ileal-reactive Treg cells. Both IL-17A and IFNγ were protective, as their deficiency precluded ileal-reactive Treg accumulation and exacerbated colitic disease. IL-23R blockade prevented progression to colitis, whereas nTreg cell transfers prevented colitic disease, ileal crypt hyperplasia and ileal-reactive Treg accumulation. Thus, our studies identify an IL-17A and IFNγ-dependent homeostatic process that mobilizes ileal-reactive Treg cells and is disrupted by IL-23.

Discussion of the relationship between human Treg potency and inhibition of TCR signaling in Teff cells.

OBJECTIVE To determine the relationship between serum levels of S100A8/A9 and S100A12 and the maintenance of clinically inactive disease during anti-tumor necrosis factor (anti-TNF) therapy and the occurrence of disease flare following withdrawal of anti-TNF therapy in patients with polyarticular forms of juvenile idiopathic arthritis (JIA). METHODS In this prospective, multicenter study, 137 patients with polyarticular-course JIA whose disease was clinically inactive while receiving anti-TNF therapy were enrolled. Patients were observed for an initial 6-month phase during which anti-TNF treatment was continued. For those patients who maintained clinically inactive disease over the 6 months, anti-TNF was withdrawn and they were followed up for 8 months to assess for the occurrence of flare. Serum S100 levels were measured at baseline and at the time of anti-TNF withdrawal. Spearman's rank correlation test, Mann-Whitney U test, Kruskal-Wallis test, receiver operating characteristic (ROC) curve, and Kaplan-Meier survival analyses were used to assess the relationship between serum S100 levels and maintenance of clinically inactive disease and occurrence of disease flare after anti-TNF withdrawal. RESULTS Over the 6-month initial phase with anti-TNF therapy, the disease state reverted from clinically inactive to clinically active in 24 (18%) of the 130 evaluable patients with polyarticular-course JIA; following anti-TNF withdrawal, 39 (37%) of the 106 evaluable patients experienced a flare. Serum levels of S100A8/A9 and S100A12 were elevated in up to 45% of patients. Results of the ROC analysis revealed that serum S100 levels did not predict maintenance of clinically inactive disease during anti-TNF therapy nor did they predict disease flare after treatment withdrawal. Elevated levels of S100A8/A9 were not predictive of the occurrence of a disease flare within 30 days, 60 days, 90 days, or 8 months following anti-TNF withdrawal, and elevated S100A12 levels had a modest predictive ability for determining the risk of flare within 30, 60, and 90 days after treatment withdrawal. Serum S100A12 levels at the time of anti-TNF withdrawal were inversely correlated with the time to disease flare (r = -0.36). CONCLUSION Serum S100 levels did not predict maintenance of clinically inactive disease or occurrence of disease flare in patients with polyarticular-course JIA, and S100A12 levels were only moderately, and inversely, correlated with the time to disease flare.

OBJECTIVE To determine the frequency, time to flare, and predictors of disease flare upon withdrawal of anti-tumor necrosis factor (anti-TNF) therapy in children with polyarticular forms of juvenile idiopathic arthritis (JIA) who demonstrated ≥6 months of continuous clinically inactive disease. METHODS In 16 centers 137 patients with clinically inactive JIA who were receiving anti-TNF therapy (42% of whom were also receiving methotrexate [MTX]) were prospectively followed up. If the disease remained clinically inactive for the initial 6 months of the study, anti-TNF was stopped and patients were assessed for flare at 1, 2, 3, 4, 6, and 8 months. Life-table analysis, t-tests, chi-square test, and Cox regression analysis were used to identify independent variables that could significantly predict flare by 8 months or time to flare. RESULTS Of 137 patients, 106 (77%) maintained clinically inactive disease while receiving anti-TNF therapy for the initial 6 months and were included in the phase of the study in which anti-TNF therapy was stopped. Stopping anti-TNF resulted in disease flare in 39 (37%) of 106 patients by 8 months. The mean/median ± SEM time to flare was 212/250 ± 9.77 days. Patients with shorter disease duration at enrollment, older age at onset and diagnosis, shorter disease duration prior to experiencing clinically inactive disease, and shorter time from onset of clinically inactive disease to enrollment were found to have significantly lower hazard ratios for likelihood of flare by 8 months (P < 0.05). CONCLUSION Over one-third of patients with polyarticular JIA with sustained clinically inactive disease will experience a flare by 8 months after discontinuation of anti-TNF therapy. Several predictors of lower likelihood of flare were identified.

Research with hazardous biologic materials (biohazards) is essential to the progress of medicine and science. The field of microbiology has rapidly advanced over the years, partially due to the development of new scientific methods such as recombinant DNA technology, synthetic biology, viral vectors, and the use of genetically modified animals. This research poses a potential risk to personnel as well as the public and the environment. Institutions must have appropriate oversight and take appropriate steps to mitigate the risks of working with these biologic hazards. This article will review responsibilities for institutional oversight of occupational health and safety for research involving biologic hazards.

Macrophages have key roles in tumor development and invasion in several human cancers, but little is known about their pathogenic role in cutaneous T-cell lymphoma (CTCL). Herein, we used PCR arrays to profile the expression of inflammatory cytokines in 12 patients with mycosis fungoides (MF), the most common variant of CTCL. Compared with normal controls, MF skin displayed increased mRNA levels of macrophage-related cytokines. Moreover, we detected CD163, a reliable marker of tumor-associated macrophages, in the tumor microenvironment of MF biopsies. To demonstrate that macrophages had a role in CTCL tumorigenesis, we xenografted human CTCL tumor cells in immunocompromised mice and compared tumor development using clodronate-containing liposomes to deplete macrophages in mice. Mice treated with clodronate-containing liposomes show markedly less tumor growth compared with mice treated with phosphate-buffered saline-containing liposomes (P

Discussion on enhancers from the Cd8 locus and their ability to drive transgenic reporter expression in different types of CD8+ cells.

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. © 2014 S. Karger AG, Basel

A short average telomere length is associated with low telomerase activity and certain degenerative diseases. Studies in animals and with human cells confirm a causal mechanism for cell or tissue dysfunction triggered by critically short telomeres, suggesting that telomerase activation may be an approach to health maintenance. Previously, we reported on positive immune remodeling in humans taking a commercial health maintenance program, PattonProtocol-1, composed of TA-65® (a natural product-derived telomerase activator) and other dietary supplements. In over a 5-year period and an estimated 7000 person-years of use, no adverse events or effects have been attributed to TA-65 by physicians licensed to sell the product. Here we report on changes in metabolic markers measured at baseline (n=97-107 subjects) and every 3-6 months (n=27-59 subjects) during the first 12 months of study. Rates of change per year from baseline determined by a multi-level model were -3.72 mg/dL for fasting glucose (p=0.02), -1.32 mIU/mL for insulin (p=0.01), -13.2 and -11.8 mg/dL for total cholesterol and low-density lipoprotein cholesterol (LDL-C) (p=0.002, p=0.002, respectively), -17.3 and -4.2 mmHg for systolic and diastolic blood pressure (p=0.007 and 0.001, respectively), and -3.6 μmole/L homocysteine (p=0.001). In a subset of individuals with bone mineral density (BMD) measured at baseline and 12 months, density increased 2.0% in the spine (p=0.003). We conclude that in addition to apparent positive immune remodeling, PattonProtocol-1 may improve markers of metabolic, bone, and cardiovascular health.

Regulatory T (Treg) cells that express the transcription factor Foxp3 are essential for maintaining tolerance at mucosal interfaces, where they act by controlling inflammation and promoting epithelial cell homeostasis. There are two major regulatory T-cell subsets, "natural" CD4(+) Treg (nTreg) cells that develop in the thymus and "induced" Treg (iTreg) cells that develop from conventional CD4(+) T (Tconv) cells in the periphery. Dysregulated Treg cell responses are associated with autoimmune diseases, including inflammatory bowel disease (IBD) and arthritis. Adoptive transfer of Treg cells can modulate innate and adaptive immune responses and cure disease in animal models, which has generated considerable interest in using Treg cells to treat human autoimmune disease, prevent rejection of transplanted organs, and to control graft-versus-host disease following hematopoietic stem cell transplantation. Herein, we describe our modifications of a treatment model of T-cell transfer colitis designed to allow mechanistic investigation of the two major Treg cell subsets and to compare their specific roles in mucosal tolerance.

To quantitatively compare diffusion metrics for human brain estimated with diffusional kurtosis imaging (DKI) at applied field strengths of 1.5 and 3T.DKI data for brain were acquired at both 1.5 and 3T from each of six healthy volunteers using a twice-refocused diffusion-weighted imaging sequence. From these data, parametric maps of mean diffusivity (MD), axial diffusivity (DDiffusion metrics measured at 1.5 and 3T were observed to be similar. Linear regression of the full datasets had coefficients of determination varying from a low of RDiffusion metrics measured with DKI at 1.5 and 3T are strongly correlated and typically differ by only a few percent. The somewhat higher discrepancy for the KFA is argued to mainly reflect the effects of signal noise. This supports the robustness DKI results with respect to field strength.3 J. Magn. Reson. Imaging 2017;45:673-680.

Introduction: Retrospective occupational exposure assessment has been challenging in case-control studies in the general population. We aimed to review (i) trends of different assessment methods used in the last 40 years and (ii) evidence of reliability for various assessment methods. Methods: Two separate literature reviews were conducted. We first reviewed all general population cancer case-control studies published from 1975 to 2016 to summarize the exposure assessment approach used. For the second review, we systematically reviewed evidence of reliability for all methods observed in the first review. Results: Among the 299 studies included in the first review, the most frequently used assessment methods were self-report/assessment (n = 143 studies), case-by-case expert assessment (n = 139), and job-exposure matrices (JEMs; n = 82). Usage trends for these methods remained relatively stable throughout the last four decades. Other approaches, such as the application of algorithms linking questionnaire responses to expert-assigned exposure estimates and modelling of exposure with historical measurement data, appeared in 21 studies that were published after 2000. The second review retrieved 34 comparison studies examining methodological reliability. Overall, we observed slightly higher median kappa agreement between exposure estimates from different expert assessors (~0.6) than between expert estimates and exposure estimates from self-reports (~0.5) or JEMs (~0.4). However, reported reliability measures were highly variable for different methods and agents. Limited evidence also indicates newer methods, such as assessment using algorithms and measurement-calibrated quantitative JEMs, may be as reliable as traditional methods. Conclusion: The majority of current research assesses exposures in the population with similar methods as studies did decades ago. Though there is evidence for the development of newer approaches, more concerted effort is needed to better adopt exposure assessment methods with more transparency, reliability, and efficiency.

In this protocol we describe a method to obtain telomere length parameters using Southern blots of terminal restriction fragments (TRFs). We use this approach primarily for epidemiological studies that examine leukocyte telomere length. However, the method can be adapted for telomere length measurements in other cells whose telomere lengths are within its detection boundaries. After extraction, DNA is inspected for integrity, digested, resolved by gel electrophoresis, transferred to a membrane, hybridized with labeled probes and exposed to X-ray film using chemiluminescence. Although precise and highly accurate, the method requires a considerable amount of DNA (3 μg per sample) and it measures both the canonical and noncanonical components of telomeres. The method also provides parameters of telomere length distribution in each DNA sample, which are useful in answering questions beyond those focusing on the mean length of telomeres in a given sample. A skilled technician can measure TRF length in ∼130 samples per week.

Cycloastragenol (CAG) is the aglycone derivative of astragaloside IV which has recently been demonstrated to activate telomerase and represents a potential drug candidate for the treatment of degenerative diseases. In the present study, intestinal absorption and metabolism of CAG were examined using the Caco-2 model and liver microsomes, respectively. The results showed that CAG rapidly passes through the Caco-2 cell monolayer by passive diffusion. Four different glucuronide conjugates and two oxidized CAG metabolites were found in the apical and basolateral sides of Caco-2 monolayer, suggesting that first-pass intestinal metabolism of CAG might occur upon passage through the intestinal epithelium. CAG underwent extensive metabolism in rat and human liver microsomes with only 17.4% and 8.2%, respectively, of the starting amount of CAG remaining after 30 min of incubation. Monohydroxylation of the parent and oxidization of the hydroxylated CAG were found in the liver samples. The present study indicates that CAG is efficiently absorbed through intestinal epithelium. However, extensive first-pass hepatic metabolism would limit the oral bioavailability of this compound.

Regulatory T cells and T helper 17 cells are two recently described lymphocyte subsets with opposing actions. In this review, we discuss the mechanisms that promote development of these cells from common precursors and the specific factors that impact their cell numbers and function. Altered regulation of this key developmental checkpoint may contribute to the pathophysiology of autoimmune diseases by tipping the balance toward inflammation. We also present recent findings that suggest how the equilibrium between regulatory T cells and proinflammatory T helper subsets might be pharmacologically restored for therapeutic benefit.

Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8+ CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8+ T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.

Telomerase is an attractive cancer target as it appears to be required in essentially all tumours for immortalization of a subset of cells, including cancer stem cells. Moreover, differences in telomerase expression, telomere length and cell kinetics between normal and tumour tissues suggest that targeting telomerase would be relatively safe. Clinical trials are ongoing with a potent and specific telomerase inhibitor, GRN163L, and with several versions of telomerase therapeutic vaccines. The prospect of adding telomerase-based therapies to the growing list of new anticancer products is promising, but what are the advantages and limitations of different approaches, and which patients are the most likely to respond?

To determine the dose of triptorelin that is sufficient to maintain complete ovarian suppression in female patients with childhood-onset systemic lupus erythematosus (SLE) who require cyclophosphamide therapy, to determine the length of time needed to achieve ovarian suppression after initiation of triptorelin treatment, and to investigate the safety of triptorelin.In this randomized, double-blind, placebo-controlled, dose-escalation study, female patients ages21 years were randomized 4:1 to receive triptorelin (n = 25) or placebo (n = 6). The starting doses of triptorelin were 25, 50, 75, and 100 μg/kg, and the dose was escalated until complete ovarian suppression was maintained. The primary outcome was the weight-adjusted dose of triptorelin that provided complete ovarian suppression in at least 90% of the patients, as determined by gonadotropin-releasing hormone agonist stimulation testing. The secondary outcome was the period of time required to achieve ovarian suppression, as measured by unstimulated follicle-stimulating hormone and luteinizing hormone levels after the initiation of triptorelin treatment.Treatment with triptorelin at a weight-adjusted dose of 120 μg/kg body weight provided sustained complete ovarian suppression in 90% of the patients. After administration of the initial dose of triptorelin, 22 days were required to achieve complete ovarian suppression. The rates of adverse events (AEs) and serious adverse events (SAEs) per 100 patient-months of followup were not higher in the triptorelin group compared with the placebo group (for AEs, 189 versus 362; for SAEs, 2.1 versus 8.5).High doses of triptorelin are needed to achieve and maintain complete ovarian suppression, but such doses appear to be well tolerated in adolescent female patients with childhood-onset SLE. Our data suggest that a lag time of 22 days after initiation of triptorelin treatment is required before cyclophosphamide therapy is started or continued.

Chronic ischemic wounds are major clinical problems, and are especially prevalent in elderly patients. Management of these wounds costs billions of dollars annually in the United States. Because of the severe impairment in tissue repair, ischemic wounds among the aged are major challenges for physicians. For example, transforming growth factor-beta1 stimulates healing of young patients' ischemic wounds, but it is totally ineffective in treating the ischemic wounds of aged patients. Therefore, our goal is to develop a better therapeutic strategy for elderly patient ischemic wounds. Because human telomerase reverse transcriptase (hTERT) has emerged as having a role in promoting cell proliferation, we hypothesized that hTERT overexpression may improve ischemic wound healing in the elderly. We successfully tested this hypothesis by demonstrating for the first time that gene delivery of hTERT by adenovirus (Ad-hTERT) dramatically improved ischemic wound healing in an aged rabbit model. Importantly, our histological data indicate that no deleterious immune response was induced in the aged rabbits. This finding has broad implications for the field of gene therapy because the foremost obstacle in the use of adenoviral vectors for gene therapy is that they provoke strong innate and adaptive immune responses in the host. Moreover, Ad-hTERT significantly improved survival of primary rabbit dermal fibroblasts that were treated with hypoxia and hydrogen peroxide (oxidative stress). This model is clinically relevant because it simulates the ischemia cycle of an ischemia-reperfusion injury, which can lead to stroke, myocardial infarction, and other tissue injuries. We conclude that Ad-hTERT is an effective and novel approach to treating the ischemic wounds of elderly patients.

Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3'-->P5' thio-phosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first- (GRN163) and second-generation (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipid-conjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Background Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes. Methods Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm. Results Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3′-P5′-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low. Conclusions Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease. © 2004 Wiley-Liss, Inc.

Human telomerase is a reverse transcriptase that is expressed in essentially all cancer cells, but not in the vast majority of normal somatic cells. Therefore, the specific inhibition of telomerase activity in tumors might have significant beneficial therapeutic effects. We have designed and evaluated oligonucleotide N3' --> P5' thio-phosphoramidates as telomerase template antagonists. In biochemical cell-free assays 11-13-mer thio-phosphoramidate oligonucleotides demonstrated sequence specific and dose dependent inhibition of telomerase with pico-molar IC50 values. Optimization of the oligonucleotide sequence and length resulted in the identification of a 13-mer-oligonucleotide thio-phosphoramidate GRN163 as a drug development candidate. In cell cultures GRN163 was able to inhibit telomerase activity in the absence of cationic lipid with approximately 1 microM IC50 values. Telomerase inhibition by GRN163 produced gradual telomere shortening, followed by cellular senescence and/or apoptosis of cancer derived cell lines.

Osteolysis syndromes are characterized by resorption of affected bones with associated swelling and pain. Various forms of multicentric osteolysis syndromes including autosomal dominant and recessive carpal-tarsal osteolysis, Torg, François, Whyte-Hemingway, Hajdu-Cheney, Winchester, and other forms have been described. Most present in pre-school years with extensive involvement and destruction of multiple bones. We present a sister-pair, both of whom presented in early teenage, i.e., 13 and 15.5 years, respectively, with bilateral ankle, knee, and later, wrist pain. Radiological examination revealed bilateral osteolysis of tali, scaphoids, and patellae, and short fourth metacarpals in both sisters. Further investigation revealed absence of renal involvement, a normal excretion of amino acids, mucopolysaccharides and oligosaccharides, and presence of chronic synovitis in both sisters. Both parents and a younger brother were without radiographic or clinical evidence of the disease and there was no history of consanguinity. Thus, our sister-pair presented with the same carpal and tarsal bone involvement at a much later age, with evidence of chronic synovitis, along with short fourth metacarpals (brachydactyly type E changes) and without renal disease, suggesting a new syndrome with probable autosomal recessive inheritance.

The inhibition of telomerase activity in actively dividing cells leads to suppression of cell growth after a time delay (inhibitory delay) required to reach a threshold telomeric DNA size. We developed a mathematical model of the dynamics of telomere size distribution and cell growth in the presence of telomere inhibitors that allowed quantification of the inhibitory delay. The model based on the solution of a system of differential equations described quantitatively recent experimental data on dynamics of cultured cells in presence of telomerase inhibitors. The analysis of the data by this model suggested the existence of at least two distinct subpopulations of cells with different proliferative activity. Size distribution of telomeres, fraction of proliferating cells, and tumor doubling times are of critical importance for the dynamics of cancer cells growth in presence of telomerase inhibitors. Rapidly growing cells with large telomeric DNA heterogeneity and small proliferating fractions as well as those with very short homogeneous telomeres would be the most sensitive to telomerase inhibitors.

What began as a study of the "end-replication problem" took on a new dimension as it became clear that telomeres are a "molecular clock" of replication in human somatic cells. Here we review the biology of telomeres in vitro and in vivo, in mice and humans. We suggest that, in humans, telomeres are involved in the biology of aging and pathobiology of disorders of aging, including cancer and cardiovascular disease. We also propose that the underlying dynamics of telomere biology is in line with broad principles of evolutionary theories.

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

The lifespan of human fibroblasts and other primary cell strains can be extended by expression of the telomerase catalytic subunit (hTERT). Since replicative senescence is accompanied by substantial alterations in gene expression, we evaluated characteristics of in vitro-aged dermal fibroblast populations before and after immortalization with telomerase. The biological behavior of these populations was assessed by incorporation into reconstituted human skin. Reminiscent of skin in the elderly, we observed increased fragility and subepidermal blistering with increased passage number of dermal fibroblasts, but the expression of telomerase in late passage populations restored the normal nonblistering phenotype. DNA microarray analysis showed that senescent fibroblasts express reduced levels of collagen I and III, as well as increased levels of a series of markers associated with the destruction of dermal matrix and inflammatory processes, and that the expression of telomerase results in mRNA expression patterns that are substantially similar to early passage cells. Thus, telomerase activity not only confers replicative immortality to skin fibroblasts, but can also prevent or reverse the loss of biological function seen in senescent cell populations.

Telomerase is one of the key enzymes responsible for the proliferative immortality of the majority of cancer cells. We recently introduced a new telomerase inhibitor, a 13-mer oligonucleotide N3' --> P5'-thio-phosphoramidate lipid conjugate, designated as GRN163L. This compound inhibits telomerase activity in various tumor cell lines with IC(50) values of 3-300 nM without any cellular uptake enhancers. GRN163L demonstrated potent and sequence specific anti-cancer activity in vivo in multiple animal models. This compound was able to significantly affect not only the growth of primary tumors, but also the spread and proliferation of metastases. GRN163L is currently in Phase I and Phase I/II clinical studies in patients with solid tumors and CLL, respectively.

Purpose: We determined the natural history of the residual native infrarenal aortic segment after conventional abdominal aortic aneurysm (AAA) repair. Methods: For the retrospective arteriographic case series, 800 hundred translumbar aortograms (TLAs) were obtained for 272 patients, before and after conventional AAA repair. The main outcome measures were changes in the aortographic diameter and the length of the infrarenal aortic segment, corrected and uncorrected for magnification by normalization to the first lumbar vertebral body height. Results: The mean follow-up time from the preoperative TLA to the most recent postoperative TLA was 42 months (range, 1 to 257 months). Vertebral body height did not change p = 0.35). The length of the native infrarenal aorta cephalad to the proximal anastomosis increased a mean of 3 mm, from 23 to 26 mm p = 0.001). However, in 115 patients (43%), this aortic segment elongated more than 5 mm, and in 63 patients (24%), it elongated more than 10 mm. The native residual infrarenal aorta above the proximal anastomosis dilated a mean of 1 mm, from 23 to 24 mm p = 0.001), but in 21 patients (8%), it dilated more than 5 mm. There was a weak positive correlation between the increase in residual native aortic diameter and duration of follow-up. There was a negative correlation between this increase and the initial size. The diameters of the proximal anastomosis and proximal graft did not change. Marked variability in the changes in aortic dimensions was observed. Conclusions: A mean period of 42 months after conventional AAA repair, the native infrarenal aortic segment elongates and dilates. Although such enlargement is statistically significant, the average increase appears to be small. However, residual aortic cuff diameter increased more than 5 mm and neck length more than 10 mm in a significant number of patients, with potentially serious implications for endovascular treatment of AAA. (J Vasc Surg 1998;27:805-12.)

Purpose: To characterize the use and utility of lower extremity noninvasive venous testing (NIVT) in the diagnosis of pulmonary embolism (PE). Methods: The study is a retrospective case series of consecutive patients in whom PE was suspected who were referred to a large, urban tertiary care center for NIVT. The main outcome measures of the study were the rate of positive results of NIVT, the amount of new information provided by NIVT, and the frequency of management changes that were attributable to NIVT. Results: Forty-one of 450 patients (9%) had deep venous thrombosis (DVT) by NIVT. The prevalence of DVT by NIVT among patients not evaluated by ventilation/perfusion (V/Q) scanning was 8%. The prevalence of DVT by NIVT among patients with a high-probability V/Q scan result before NIVT was 39%, but no management decisions in this group were based on a positive NIVT result and only two decisions were based on negative NIVT results. The prevalence of DVT according to NIVT among patients who had a negative "diagnostic" (low, or very low probability, or normal) result of V/Q scan before NIVT was 2%. The overall frequency of management changes attributed to NIVT was only 2.5%. In the remaining 97% of patients, management was determined by the result of V/Q scanning or of subsequent pulmonary arteriography. Conclusions: In patients in whom PE is suspected, results of NIVT are usually negative for acute DVT. Management decisions are almost always based on V/Q scan or results of pulmonary arteriography and not on NIVT. The utility of NIVT to identify DVT in these patients appears limited, and a more selective approach to its application for the diagnosis of PE should be considered. (J Vasc Surg 1997 26:757-63.)

Telomere shortening is correlated with cell senescence in vitro and cell aging in vivo. The telomere hypothesis suggests that telomere length serves as a mitotic clock for timing cellular replicative life span. Expression of telomerase stabilizes telomere length and allows for continual replication, or cell immortality. This article reviews recent evidences for the role of telomere length and telomerase in the regulation of cellular replicative life span. The therapeutic potential of manipulating telomerase expression and telomere length is also discussed.

To study regulatory T (Treg) cell control of chronic autoimmunity in a lymphoreplete host, we created and characterized a new model of autoimmune lung inflammation that targets the medium and small airways. We generated transgenic mice that express a chimeric membrane protein consisting of hen egg lysozyme and a hemoglobin epitope tag under the control of the Clara cell secretory protein promoter, which largely limited transgene expression to the respiratory bronchioles. When Clara cell secretory protein–membrane hen egg lysozyme/hemoglobin transgenic mice were crossed to N3.L2 TCR transgenic mice that recognize the hemoglobin epitope, the bigenic progeny developed dense, pseudo-follicular lymphocytic peribronchiolar infiltrates that resembled the histological pattern of follicular bronchiolitis. Aggregates of activated IFN-γ– and IL-17A–secreting CD4+ T cells as well as B cells surrounded the airways. Lung pathology was similar in Ifng−/− and Il17a−/− mice, indicating that either cytokine is sufficient to establish chronic disease. A large number of Ag-specific Treg cells accumulated in the lesions, and Treg cell depletion in the affected mice led to an interstitial spread of the disease that ultimately proved fatal. Thus, Treg cells act to restrain autoimmune responses, resulting in an organized and controlled chronic pathological process rather than a progressive disease.

A computationally efficient approach that computes the optimal regularization parameter for the Tikhonov-minimization scheme is developed for photoacoustic imaging. This approach is based on the least squares-QR decomposition which is a well-known dimensionality reduction technique for a large system of equations. It is shown that the proposed framework is effective in terms of quantitative and qualitative reconstructions of initial pressure distribution enabled via finding an optimal regularization parameter. The computational efficiency and performance of the proposed method are shown using a test case of numerical blood vessel phantom, where the initial pressure is exactly known for quantitative comparison. (C) 2013 Society of Photo-Optical Instrumentation Engineers (SPIE)

Objective : To test the hypothesis that the expanded population of non-proliferative CD28- CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. Design : CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28- CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28- CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. Methods : CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. Results : The TRF lengths of sorted CD28- CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28- CD8+ cells in uninfected subjects (P= 0.003). TheTRF length in CD28- CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. Conclusions : The shortened telomeres in the CD28- CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

Purpose : The purpose of this study was to review the complications of transaxillary arteriography (TRAX), determine clinical factors associated with their occurrence, and define optimal treatment methods. Methods : A retrospective review of 842 consecutive TRAX studies performed in a large, urban, tertiary care, academic medical center was undertaken. Patients with complications were compared with a concurrent randomized control group without complications with the use of a multivariate analysis model. Results of operative therapy for nerve injury were compared with those of nonoperative therapy. Results : Nineteen (2.3%) complications were identified including 14 nerve injuries, four expanding hematomas/pseudoaneurysms without neurologic deficit, and one puncture site thrombosis. Several statistically significant or suggestive findings associated with the occurrence of complications were identified: female sex (odds ratio [OR[ = 4.7), systolic blood pressure ≥150 mm Hg at the conclusion of TRAX (OR = 9.5), periprocedural systemic heparin anticoagulation (OR = 7.9), concomitant use of intraarterial thrombolysis or percutaneous angioplasty (OR = 12.0), and duration of procedure ≥90 minutes (OR = 4.0). Patients who underwent prompt exploration (≤4 hours from symptom onset) for nerve injuries were more likely to have complete resolution of their neurologic deficits (five of six patients) than those who were observed or underwent delayed operation (three of eight patients) (OR = 8.3). Conclusions : Aggressive treatment of post-TRAX hypertension, limitation of TRAX duration, delay of postprocedure anticoagulation, and use of alternative sites for arterial puncture in female patients or patients undergoing catheter-based intervention may reduce the incidence of TRAX-related complications. In patients who have neurologic deficits prompt surgical exploration of the puncture site with decompression of the involved nerve(s) may reduce the incidence of prolonged deficits. (J Vasc Surg 1996;23:844-50.)