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The success of preschool programs is largely dependent upon a high-quality teaching workforce. Preschool educators are critical in supporting a child’s socio-emotional development, motivation, school readiness, achievement, and overall learning. While expected to be professionally prepared similarly to their colleagues from elementary and secondary education, public preschool teachers face substantial underpayment and a higher workload. This study, which was part of a larger study focused on the Kentucky early childhood workforce, presents findings on compensation inequity for teachers and teaching assistants working with children aged three to five years in public preschool programs. The average public preschool teacher reported having a college degree with 11 years of experience in their current position and 12 years of experience in public preschool. The average teaching assistant reported having some college credits or a Child Development Associate degree with six years of experience in their current position and nearly nine years of preschool-based experience. Teachers and teaching assistants reported low wages in comparison with similarly qualified teaching staff at kindergarten through high school levels, with some having to work secondary jobs. Despite the wage gap, teaching preschool was a career choice for most respondents. To keep high-quality teachers and teaching assistants in public preschool classrooms, it is necessary to pay them a higher wage and provide support for their ongoing professional development.

The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein–protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1–POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein–protein interaction intact likely affect some other essential function of POPDC proteins.

BackgroundThe Popeye domain containing (POPDC) genes encode sarcolemma-localised cAMP effector proteins. Mutations inBVES (POPDC1)andPOPDC2have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms.MethodsBiopsy material of patients carrying mutations inBVESwere immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein interaction.FindingsPatients carrying a novel homozygous variant,BVES(c.547G>T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. This is in contrast to variants such asBVESp.Q153X orPOPDC2p.W188X, which were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex.InterpretationMutations in POPDC proteins that cause an impairment in membrane localisation affect POPDC complex formation while mutations which leave the protein interaction intact likely affect some other essential function of POPDC proteins.FundingThis study was funded by an EPSRC/British Heart Foundation co-funded Imperial Institute of Chemical Biology (ICB) Centre for Doctoral Training (CDT) PhD studentship (EP/S023518/1), a project grant of the British Heart Foundation (PG19/13/34247) and the Deutsche Forschungsgemeinschaft (DE1482/9-1).Research in ContextEvidence before this studySeveral biallelic missense and nonsense variants inBVES (POPDC1)have been described and are associated with heart and skeletal muscle disease. Skeletal muscle biopsies of homozygous carriers of these variants display a loss of sarcolemmal localisation of POPDC1 and POPDC2.Added value of this studyWe demonstrate that POPDC1 and POPDC2 form a heteromeric complex and that complex formation is required for plasma membrane trafficking of POPDC proteins. Transfection of different disease variants in HEK293 cells replicates their defective membrane targeting observed in biopsy material. Structural modelling and site-directed mutagenesis identifies an interface of strongly conserved hydrophobic residues in POPDC proteins, which likely mediate the interaction of POPDC proteins.Implications of all the available evidenceThese data provide novel insight into the membrane targeting requirements of POPDC proteins. We recommend testing the membrane targeting properties of any novel variant in POPDC isoforms using a newly developed co-transfection assay in HEK293 cells to characterise its pathogenicity. Our novel insight into the requirement of heterodimerization for proper membrane targeting may also offer novel opportunities to treat patients carrying mutations in POPDC proteins.

Reducing infarct size (IS) by interfering with mechanisms for cardiomyocyte death remains an elusive goal. DMX-5804, a selective inhibitor of the stress-activated kinase MAP4K4, suppresses cell death in mouse myocardial infarction (MI), human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), and 3D human engineered heart tissue, whose fidelity to human biology is hoped to strengthen the route to clinical success. Here, DMX-10001, a soluble, rapidly cleaved pro-drug of DMX-5804, was developed for i.v. testing in large-mammal MI. Following pharmacodynamic studies, a randomized, blinded efficacy study was performed in swine subjected to LAD balloon occlusion (60 min) and reperfusion (24 h). Thirty-six animals were enrolled; 12 were excluded by pre-defined criteria, death before infusion, or technical issues. DMX-10001 was begun 20 min before reperfusion (30 min, 60 mg/kg/h; 23.5 h, 17 mg/kg/h). At all times tested, beginning 30 min after the start of infusion, DMX-5804 concentrations exceeded > fivefold the levels that rescued hPSC-CMs and reduced IS in mice after oral dosing with DMX-5804 itself. No significant reduction occurred in IS or no-reflow corrected for the area at ischemic risk, even though DMX-10001 reduced IS, expressed in grams or % of LV mass, by 27%. In summary, a rapidly cleaved pro-drug of DMX-5804 failed to reduce IS in large-mammal MI, despite exceeding the concentrations for proven success in both mice and hPSC-CMs.

Graphical abstract, SARS-CoV-2, which causes COVID-19, was first identified in humans in late 2019 and is a coronavirus which is zoonotic in origin. As it spread around the world there has been an unprecedented effort in developing effective vaccines. Computational methods can be used to speed up the long and costly process of vaccine development. Antigen selection, epitope prediction, and toxicity and allergenicity prediction are areas in which computational tools have already been applied as part of reverse vaccinology for SARS-CoV-2 vaccine development. However, there is potential for computational methods to assist further. We review approaches which have been used and highlight additional bioinformatic approaches and PK modelling as in silico approaches which may be useful for SARS-CoV-2 vaccine design but remain currently unexplored. As more novel viruses with pandemic potential are expected to arise in future, these techniques are not limited to application to SARS-CoV-2 but also useful to rapidly respond to novel emerging viruses.

SUMMARYThe global outbreak of SARS-CoV-2 necessitates the rapid development of new therapies against COVID-19 infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2-induced protein (SIP) network, based on disease signatures defined by COVID-19 multi-omic datasets(Bojkova et al., 2020; Gordon et al., 2020), and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2-induced pathways, 40 of which are already in COVID-19 clinical trials(Clinicaltrials.gov, 2020) testifying to the validity of the approach. Using artificial neural network analysis we classified these 200 drugs into 9 distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (130) and immune response (70). A subset of drugs implicated in viral replication were tested in cellular assays and two (proguanil and sulfasalazine) were shown to inhibit replication. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.

G-protein-coupled receptor (GPCR) drug research is presently hindered by the technical challenges associated with generating purified receptors. Consequently, the application of critical modern discovery technologies has been limited, and the vast untapped opportunity for new GPCR-directed medicines is not being realised. A simple but transformative solution is to purify receptors without removing them from their native phospholipid environment by using polymer lipid particle (PoLiPa) technology, with reagents such as styrene-maleic acid co-polymer (SMA). Compared with contemporary detergent-based and stabilising mutagenesis methods, the PoLiPa approach is simple and generic and, therefore, offers huge advantages, with the potential to revolutionise GPCR research by facilitating the availability of the purified receptors that are required for structural biology, biophysical, and panning technologies.

The IKK‐related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll‐like receptors to regulate NF‐κB signalling. We investigated the expression and function of IKKε and TBK1, in diffuse large B‐cell lymphoma (DLBCL). DLBCL cell lines and patient‐derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non‐germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL‐10 production from Ly10 and repressed NF‐κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF‐κB signalling in lymphoma.

Background The ActualHCA™ system continuously monitors the activity, temperature and behavior of group-housed rats without invasive surgery. The system was validated to detect the contrasting effects of sedative and stimulant test agents (chlorpromazine, clonidine and amphetamine), and compared with the modified Irwin test (mIT) with rectal temperature measurements. Methods Six male Han Wistar rats per group were used to assess each test agent and vehicle controls in separate ActualHCA™ recordings and mIT. The mIT was undertaken at 15, 30 mins, 1, 2, 4 and 24 h post-dose. ActualHCA™ recorded continuously for 24 h post-dose under 3 experimental conditions: dosed during light phase, dark phase, and light phase with a scheduled cage change at the time of peak effects determined by mIT. Results ActualHCA™ detected an increase stimulated activity from the cage change at 1-2 h post-dose which was obliterated by chlorpromazine and clonidine. Amphetamine increased activity up to 4 h post-dose in all conditions. Temperature from ActualHCA™ was affected by all test agents in all conditions. The mIT showed effects on all 3 test agents up to 4 h post-dose, with maximal effects at 1-2 h post-dose. The maximal effects on temperature from ActualHCA™ differed from mIT. Delayed effects on activity were detected by ActualHCA™, but not on mIT. Conclusions Continuous monitoring has the advantage of capturing effects over time that may be missed with manual tests using pre-determined time points. This automated behavioural system does not replace the need for conventional methods but could be implemented simultaneously to improve our understanding of behavioural pharmacology.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Central Nervous System (CNS)-related safety concerns are major contributors to delays and failure during the development of new candidate drugs (CDs). CNS-related safety data on 141 small molecule CDs from five pharmaceutical companies were analyzed to identify the concordance between rodent multi-parameter neurofunctional assessments (Functional Observational Battery: FOB, or Irwin test: IT) and the five most common adverse events (AEs) in Phase I clinical trials, namely headache, nausea, dizziness, fatigue/somnolence and pain. In the context of this analysis, the FOB/IT did not predict the occurrence of these particular AEs in man. For AEs such as headache, nausea, dizziness and pain the results are perhaps unsurprising, as the FOB/IT were not originally designed to predict these AEs. More unexpected was that the FOB/IT are not adequate for predicting ‘somnolence/fatigue’ nonclinically. In drug development, these five most prevalent AEs are rarely responsible for delaying or stopping further progression of CDs. More serious AEs that might stop CD development occurred at too low an incidence rate in our clinical dataset to enable translational analysis.

Introduction The Safety Pharmacology Society (SPS) and National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) conducted a survey and workshop in 2015 to define current industry practices relating to housing of non-rodents during telemetry recordings in safety pharmacology and toxicology studies. The aim was to share experiences, canvas opinion on the study procedures/designs that could be used and explore the barriers to social housing. Methods Thirty-nine sites, either running studies (Sponsors or Contract Research Organisations, CROs) and/or outsourcing work responded to the survey (51% from Europe; 41% from USA). Results During safety pharmacology studies, 84, 67 and 100% of respondents socially house dogs, minipigs and non-human primates (NHPs) respectively on non-recording days. However, on recording days 20, 20 and 33% of respondents socially house the animals, respectively. The main barriers for social housing were limitations in the recording equipment used, study design and animal temperament/activity. During toxicology studies, 94, 100 and 100% of respondents socially house dogs, minipigs and NHPs respectively on non-recording days. However, on recording days 31, 25 and 50% of respondents socially house the animals, respectively. The main barriers for social housing were risk of damage to and limitations in the recording equipment used, food consumption recording and temperament/activity of the animals. Conclusions Although the majority of the industry does not yet socially house animals during telemetry recordings in safety pharmacology and toxicology studies, there is support to implement this refinement. Continued discussions, sharing of best practice and data from companies already socially housing, combined with technology improvements and investments in infrastructure are required to maintain the forward momentum of this refinement across the industry.

Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio™, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory. We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors have made recommendations for a data-driven approach to the toxicological assessment of mAb biosimilars that minimises unnecessary use of animals and can be used across all regions of the world.

PURPOSE/OBJECTIVES To explore differences in cancer risk knowledge and colorectal cancer (CRC) knowledge among adults in Alabama. . RESEARCH APPROACH Telephone interviews were conducted using an 80-item questionnaire that included 14 demographic questions and 26 general questions regarding healthcare quality, sources of health-related information, and cancer fears and risk factors. Also included were 40 questions dealing with CRC perceptions and experiences (asked only of respondents aged 50 years or older). . SETTING A standard random-digit-dialed statewide telephone survey, targeting adult residents of Alabama. Interviews were conducted during about seven weeks in 2012. . PARTICIPANTS 1,024 participants, including 615 who were aged 50 years or older. Most of the participants identified as Caucasian. . METHODOLOGIC APPROACH A secondary data analysis using data from a CRC screening survey of Alabama residents conducted in 2012 by the University of South Alabama polling group via telephone. . FINDINGS Knowledge of general cancer risk factors predicted disagreement with the statement, "There is nothing you can do to avoid getting cancer," and disagreement with the statement, "Only people with signs or symptoms should be screened for CRC." Binary logistic regression showed that those higher in CRC risk knowledge were more likely to have been screened for CRC. . CONCLUSIONS Knowledge of general cancer risk factors may increase self-efficacy. In addition, individuals with increased knowledge of colorectal cancer risk factors were more likely to participate in CRC screening. . INTERPRETATION Nurses play an instrumental role in addressing CRC, a preventable and treatable cancer. An opportunity exists for nurses and other healthcare providers to develop culturally appropriate educational interventions to increase knowledge related to CRC, risk factors, and screening, particularly among those who are at increased risk. This education needs to occur in clinical practice and within the community.

Measuring the activity and temperature of rats is commonly required in biomedical research. Conventional approaches necessitate single housing, which affects their behavior and wellbeing. We have used a subcutaneous radiofrequency identification (RFID) transponder to measure ambulatory activity and temperature of individual rats when group-housed in conventional, rack-mounted home cages. The transponder location and temperature is detected by a matrix of antennae in a baseplate under the cage. An infrared high-definition camera acquires side-view video of the cage and also enables automated detection of vertical activity. Validation studies showed that baseplate-derived ambulatory activity correlated well with manual tracking and with side-view whole-cage video pixel movement. This technology enables individual behavioral and temperature data to be acquired continuously from group-housed rats in their familiar, home cage environment. We demonstrate its ability to reliably detect naturally occurring behavioral effects, extending beyond the capabilities of routine observational tests and conventional monitoring equipment. It has numerous potential applications including safety pharmacology, toxicology, circadian biology, disease models and drug discovery.

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use.Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends.The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Availability, affordability, and acceptability are critical factors in relation to low F&V consumption. This thesis aims to understand the cost environment for F&V, their availability, and quality, in retail outlets; and consumer perceptions and behaviours, to understand the impact on consumption. Both supply and demand sides of the F&V equation are examined. The thesis is underpinned by a social-ecological framework examining both individual and contextual systems. The first phase involved three Food Basket surveys in 2006, 2008 and 2009 across NSW. Trends and differences in the costs, availability and quality of F&V were analysed by ARIA score (measure of postcode rurality), SEIFA score (postcode socioeconomic status), store type, and effects of store competition. Phase 1 included a comparison of the cost and nutritional quality of generic versus branded products. Phase two involved an online community survey of 2474 adults in NSW. Participants were asked about their F&V consumption and knowledge of recommended servings; barriers to consumption; stages of change in relation to F&V recommendations; spending on F&V; and beliefs about food costs and affordability. Food prices varied across NSW, with total food and F&V more expensive in remote areas. Significant savings could be achieved by purchasing generic products without compromising nutritional quality in Sydney supermarkets. The community survey confirmed the ongoing trend of low F&V consumption, and poor knowledge of recommended servings. Those younger and less educated had lower intakes of F&V. Approximately a third of food budget was spent on F&V. This thesis increases knowledge of the cost environment for F&V, and overlays this with information on consumer perceptions, beliefs and behaviours, in order to understand their impact on consumption. The findings will be useful in designing effective interventions. The lack of social marketing may be contributing to poor knowledge and low consumption.

The Alabama Comprehensive Cancer Control Coalition (ACCCC) has developed an integrated and coordinated approach to reducing cancer incidence, morbidity, and mortality, and to improving the quality of life for cancer survivors, their families, and their caregivers. The ACCCC is currently in a maintenance phase and a formal plan for sustainability of the coalition was needed to keep the members engaged and productive. A training session in coalition sustainability conducted in 2013 identified the following elements as essential to success: (1) increased marketing of the coalition by simplifying its mission; (2) improved networking including flexibility in coalition meeting location and attendance; (3) increased membership satisfaction through transformational leadership; (4) revision of the working structure of committees and improved accountability; and (5) enhancement of partner satisfaction with coalition activities designed to recruit and retain new partners. A self-administered membership satisfaction survey was given to assess coalition mission, meeting logistics, organization, capacity building, and coalition goals. Results indicated that the subcategories of communication, mission, and meeting logistics were rated satisfied to very satisfied on a five-point scale. Although the ACCCC had clearly written goals, improvement could be made in leadership participation and new member orientation could be improved. Most members rated their parent organization as highly involved with the ACCCC and many offered suggestions on capacity building. Results of the sustainability training have clarified the ACCCC's plans to ensure coalition viability and improve strategies to inform stakeholders of the benefits of participation in the coalition.

Short term toxicity studies are conducted in animals to provide information on major adverse effects typically at the maximum tolerated dose (MTD). Such studies are important from a scientific and ethical perspective as they are used to make decisions on progression of potential candidate drugs, and to set dose levels for subsequent regulatory studies. The MTD is usually determined by parameters such as clinical signs, reductions in body weight and food consumption. However, these assessments are often subjective and there are no published criteria to guide the selection of an appropriate MTD. Even where an objective measurement exists, such as body weight loss (BWL), there is no agreement on what level constitutes an MTD. A global initiative including 15 companies, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), has shared data on BWL in toxicity studies to assess the impact on the animal and the study outcome. Information on 151 studies has been used to develop an alert/warning system for BWL in short term toxicity studies. The data analysis supports BWL limits for short term dosing (up to 7days) of 10% for rat and dog and 6% for non-human primates (NHPs).

This Alabama statewide cancer control plan for 2011-2015 seeks to build on the successes of two previous 5-year plans while developing new objectives that address cancer disparities and cancer prevention over the entire lifespan. The approach to defining objectives for this Plan was systematic and sought input from all members of the Alabama Comprehensive Cancer Control Coalition (ACCCC). The Plan that was fashioned is based on input from academic medical centers, private physicians, government agencies, regulatory agencies, health societies, private citizens, and cancer survivors, all of whom are active Coalition members who exchange information, opinions, and knowledge from their respective points of view. The Plan could not have taken shape without the full input of health professionals, statisticians, graduate students, former patients, and concerned citizens; it is truly an example of the synergy of professional, public, and patient education.

TRPM2 Ca2+-permeable cation channel is widely expressed and activated by markers of cellular stress. Since inflammation and stress play a major role in insulin resistance, we examined the role of TRPM2 Ca2+ channel in glucose metabolism. A 2-h hyperinsulinemic euglycemic clamp was performed in TRPM2-deficient (KO) and wild-type mice to assess insulin sensitivity. To examine the effects of diet-induced obesity, mice were fed a high-fat diet for 4–10 mo, and metabolic cage and clamp studies were conducted in conscious mice. TRPM2-KO mice were more insulin sensitive partly because of increased glucose metabolism in peripheral organs. After 4 mo of high-fat feeding, TRPM2-KO mice were resistant to diet-induced obesity, and this was associated with increased energy expenditure and elevated expressions of PGC-1α, PGC-1β, PPARα, ERRα, TFAM, and MCAD in white adipose tissue. Hyperinsulinemic euglycemic clamps showed that TRPM2-KO mice were more insulin sensitive, with increased Akt and GSK-3β phosphorylation in heart. Obesity-mediated inflammation in adipose tissue and liver was attenuated in TRPM2-KO mice. Overall, TRPM2 deletion protected mice from developing diet-induced obesity and insulin resistance. Our findings identify a novel role of TRPM2 Ca2+ channel in the regulation of energy expenditure, inflammation, and insulin resistance.

The changing environment of monoclonal antibody (mAb) development is impacting on the cost of drug development and the use of experimental animals, particularly non-human primates (NHPs). The drive to reduce these costs is huge and involves rethinking and improving nonclinical studies to make them more efficient and more predictive of man. While NHP use might be unavoidable in many cases because of the exquisite specificity and consequent species selectivity of mAbs, our increasing knowledge base can be used to improve drug development and maximise the output of experimental data. Data on GLP regulatory toxicology studies for 58mAbs were obtained from 10 companies across a wide range of therapeutic indications. These data have been used to investigate current practice and identify study designs that minimise NHP use. Our analysis shows that there is variation in the number of animals used for similar studies. This information has been used to develop practical guidance and make recommendations on the use of science-based rationale to design studies using fewer animals taking into account the current regulatory guidance. There are eight recommendations intended to highlight areas for consideration. They include guidance on the main group size, the inclusion of recovery groups and the number of dose groups used in short and long term chronic toxicology studies.

Toxicity studies in animals are carried out to identify the intrinsic hazard of a substance to support risk assessment for humans. In order to identify opportunities to minimise animal use in regulatory toxicology studies, a review of current study designs was carried out. Pharmaceutical companies and contract research organisations in the UK shared data and experience of standard toxicology studies (ranging from one to nine months duration) in rodents and non-rodents; and carcinogenicity studies in the rat and mouse. The data show that variation in study designs was primarily due to (i) the number of animals used in the main study groups, (ii) the use of animals in toxicokinetic (TK) satellite groups, and (iii) the use of animals in off-treatment recovery groups. The information has been used to propose a series of experimental designs where small adjustments could reduce animal use in practice, while maintaining the scientific objectives.

Selection of a pharmacologically responsive species can represent a major challenge in designing nonclinical safety assessment programs for many biopharmaceuticals (eg, monoclonal antibodies (mAbs)). Frequently, the only relevant species for nonclinical testing of mAbs is the non-human primate (NHP). This situation, coupled with a rapidly increasing number of mAb drugs in development, has resulted in a significant increase in the number of NHPs used in nonclinical safety assessment. Apart from ethical considerations related to responsible animal use, there is a clear need for more efficient and innovative approaches to drug discovery and development; these factors drive the need to investigate alternative approaches and strategies for the safety assessment. This review summarizes important scientific and regulatory perspectives derived from presentations and audience discussions in an educational forum at the 2010 annual American College of Toxicology meeting regarding opportunities for employing alternative approaches to minimize NHP use in mAb drug development.

Gathering quality data on physician practices and patterns of care is crucial to enhancing efficiency in health care delivery while continuing to improve patient outcomes. One of the more common approaches to gathering such data has been survey research in the form of mail questionnaires. This approach has significant advantages over other modes of data collection: reasonably accurate contact information is readily available; survey delivery and return are relatively inexpensive; and no technologically advanced skills or capabilities are required for the researchers or the respondents. The only significant disadvantage to mail questionnaires involves the unwillingness of some physicians to participate, resulting in low response rates, which in turn raise issues of nonresponse bias and problems of generalizability. This article describes an attempt to boost response rates in a survey of primary care physicians in Alabama by offering a range of electronic options for survey completion.

Context The literature predominately describes hospice utilization among Medicare recipients, with a limited number of reports describing use among all age groups. Objectives This study aimed to describe and compare patterns of hospice use among decedents of all ages in Alabama using a population-based approach. Methods We obtained death certificates for Alabama residents who died from January 1, 2002 to December 31, 2005 (n=178,420). To ascertain hospice use, we linked death certificates to the hospice administering care using state-mandated listings of deaths reported by hospices. Additionally, each decedent's residence at death was geocoded and area-level socioeconomic status (SES) measures were added. Results From 2002 to 2005, a total of 43,638 Alabamians died while under hospice care, representing a quarter (24.5%) of all deaths in the state. During this four-year span, the rate of hospice use increased by nearly 15% (22.2%–25.6%). As expected, rates of hospice use increased with age at death. For the SES indicators for poverty, education, and income, rates of hospice use increased as SES improved. However, this pattern was found to vary by race and metro/nonmetro status. Conclusions In addition to revealing racial, geographic, and other disparities in hospice care across Alabama, our results indicate usage rates in Alabama trail behind those observed nationally. We also identified previously unreported interactions between race, urbanization level, and poverty classification. Future studies should explore whether such relationships exist elsewhere and the rationale for their occurrence.

Few studies have evaluated barriers to hospice from a hospice provider perspective. We assessed such views via a postal survey to all licensed hospices in Alabama (N = 193)—response = 55.4%. Most providers considered physicians and health care professionals to be somewhat knowledgeable of hospice, but also indicated a lack of knowledge constituted the barrier with the most impact in their communities. Respondents also cited physician difficulties with discussing end of life with patients and prognosticating death within 6 months as leading barriers. Providers also described Medicare reimbursement cap issues that have resulted in barriers to hospice. Our findings were similar to previous investigations assessing provider perceptions. Future studies should explore how reimbursement cap issues affect the receipt and delivery of hospice care.

Compensatory release of immunosuppressive cytokines, such as IL-10, by macrophages present in the tumor microenvironment has been implicated as a mechanism for adaptive resistance to a number of immunotherapies. Our drug discovery effort utilises Esterase Motif TechnologyTM (ESMTM) which selectively targets myelomonocytic cells sparing the concomitant lymphocyte anti-tumor immune response. TLR agonists are known to be stimulators of the immune response, a key component of which is the production of myeloid cell IL-12p70. However, their therapeutic potential has been limited by their accompanying induction of IL-10 and other factors. MPL-5821 is an ESMTM p38 MAPK inhibitor which not only inhibits IL-10 but also enhances LPS stimulated IL-12p70 and in contrast to conventional p38 MAPK inhibitors provides enhancement of lymphocyte IFNγ production. The present studies contrast MPL-5821 with multiple non-targeted agents, including inhibitors of HDAC, JAK, PI3K, MEK and CSF-1R, in human PBMC assays. These demonstrated the benefit of ESMTM-targeting as applied to p38 MAP kinase inhibition to not only inhibit TLR agonist induced immunosuppression but also enhance IFNγ due to its sparing of the myeloid-lymphocyte axis. None of the other modalities were able to achieve the effects observed for MPL-5821. Having established the unique ability of MPL-5281 to inhibit LPS induced IL-10 production whilst still maintaining lymphocyte IFNγ production, we extended our studies to human cancer ex vivo models. We chose to evaluate MPL-5821 in combination with TLR agonists in ex vivo assays using tissue and ascites derived from ovarian and cervical cancer patients. A single cell suspension was prepared from cervical cancer tumor draining lymph nodes and incubated with MPL-5821 +/- the TLR 7/8 agonist R848 for 24 and 48 hours. MPL-5821 potently inhibited the R848 induced IL-10 production as measured by Cytometric Bead Array and in contrast to a conventional p38 MAP inhibitor LY2228820 also enhanced IFNγ production. We studied MPL-5821 in cell suspensions prepared from human ovarian tumor and ascites. For the tumor sample, a single cell homogenate was prepared by mechanical and enzymatic digestion and for the ascites the cells were isolated by centrifugation. The cell populations were then analysed by flow cytometry and the cell preparation cultured for 72 hours with anti-CD3 or TLR agonist in the presence of test compound. Cytokine production was measured after 72 hours by ELISA or Luminex bead array. MPL-5821 again showed potent inhibition of TLR agonist induced IL-10 with concomitant enhancement of IFNγ production. We conclude that application of ESMTM technology to macrophage selective delivery of p38 MAPK inhibitors has the potential to inhibit TLR agonist induction of IL-10, which is implicated in limiting the performance of TLR agonists in the clinic. Citation Format: David Moffat, Martin Perry, A. Marijne Heeren, Tanja D. de Gruijl, Justyna Rzepecka, Lucia Janicova, Anastasia Nika, Darryl Turner, Clare Doris, Claire Tebbutt, Kathryn Chapman, Gary Newton, Stephen Anderton. MPL-5821, a macrophage targeted ESMTM p38 MAPK inhibitor, inhibits the production of TLR agonist induced IL-10 whilst sparing T-cell functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3877.

It has been predicted that the use of non-human primates (NHPs) is going to increase considerably in the development of monoclonal antibodies (mAbs). Opportunities exist to focus on a rigorous, science-based approach to drug development, however, which will minimize this increase. In this article, the authors review current and future NHP use in mAb development based on surveys, experience and expert opinion and propose a framework that will minimize future NHP use and continue to support science and innovation.

Toxicological assessment of pharmaceutical and non-pharmaceutical chemicals is a regulatory requirement to ensure all compounds likely to be exposed to humans or the environment are safe. These studies rely on the use of large numbers of animals and involve a number of assumptions and extrapolations that remain controversial in assuring consumer safety. The UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has taken a collaborative approach to identify opportunities for implementation of the 3Rs principles (Replacement, Reduction and Refinement) to drive innovation and support animal welfare in toxicity testing. This review highlights the mechanisms by which the NC3Rs is working with the pharmaceutical and chemical industries and regulatory authorities to achieve these goals.

Regulatory guidelines indicate acute toxicity studies in animals are considered necessary for pharmaceuticals intended for human use. This is the only study type where lethality is mentioned as an endpoint. The studies are carried out, usually in rodents, to support marketing of new drugs and to identify the minimum lethal dose. A European initiative including 18 companies has undertaken an evidence-based review of acute toxicity studies and assessed the value of the data generated. Preclinical and clinical information was shared on 74 compounds. The analysis indicated acute toxicity data was not used to (i) terminate drugs from development (ii) support dose selection for repeat dose studies in animals or (iii) to set doses in the first clinical trials in humans. The conclusion of the working group is that acute toxicity studies are not needed prior to first clinical trials in humans. Instead, information can be obtained from other studies, which are performed at more relevant doses for humans and are already an integral part of drug development. The conclusions have been discussed and agreed with representatives of regulatory bodies from the US, Japan and Europe.

Selecting a pharmacologically relevant animal species for testing the safety and toxicity of novel monoclonal antibody (mAb) therapies to support clinical testing can be challenging. Frequently, the species of choice is the primate. With the increased number of mAbs in the pharmaceutical pipeline, this has significant implications for primate use, and so raises several important scientific, ethical and economic issues. Here, following a recent international workshop held to debate this topic, we discuss issues in the preclinical testing of mAbs, with a particular focus on species relevance and primate use, and provide suggestions for how these issues might be addressed.

The National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) is an independent scientific organization that is based in the United Kingdom, which was set up by the government to lead the discovery and application of new technologies and approaches that minimize the use of animals in research and improve animal welfare. The NC3Rs uses a range of strategies to improve and advance science through application of the 3Rs. These include funding basic research, open innovation (CRACK IT), and programs run by inhouse scientists. We present several case studies from the NC3Rs portfolio, featuring asthma research, the use of nonhuman primates in monoclonal antibody development, and CRACK IT. Finally, we anticipate the future, as we use our experience to move into new research fields and expand toward international collaboration. Here we highlight how equipping scientists with relevant and emerging 3Rs tools can help overcome the challenges and limitations of the use of animals in research to the benefit of the whole bioscience community.

There are many opportunities to benefit science and business through application of the “3 Rs” (replacement, reduction, and refinement) to nonhuman primate use in drug development and safety assessment studies. Scientifically driven approaches that consider individual studies on a case-by-case basis and how these studies fit into the drug development program as a whole can reduce the number of animals used without affecting the development outcome. Some examples include minimizing the number of dose groups, the number of animals per group, and the inclusion of recovery animals. In addition, studies that involve nonhuman primates can be refined through spacious and well-designed accommodation, effective environmental enrichment, housing in stable social groups, well-trained staff, use of the best humane technical practices, and positive reinforcement training of the animals to cooperate with procedures. All the elements within this chapter are interlinked and support improved animal welfare to generate more robust and reliable data to predict human safety.