Publications by authors named "Catherine J Betts"

27 Publications

  • Page 1 of 1

Precision cut lung slices: an ex vivo model for assessing the impact of immunomodulatory therapeutics on lung immune responses.

Arch Toxicol 2021 08 30;95(8):2871-2877. Epub 2021 Jun 30.

Clinical Testing, Global Procurement, Operations, AstraZeneca, Gothenburg, Sweden.

Chronic inflammatory diseases of the respiratory tract, such as chronic obstructive pulmonary disease (COPD) and asthma, are severe lung diseases that require effective treatments. In search for new medicines for these diseases, there is an unmet need for predictive and translatable disease-relevant in vitro/ex vivo models to determine the safety and efficacy of novel drug candidates. Here, we report the use of precision cut lung slices (PCLS) as a potential ex vivo platform to study compound effects in a physiologically relevant environment. PCLS derived from an elastase-challenged mouse model display key characteristics of increased inflammation ex vivo, which is exacerbated further upon challenge with LPS, mimicking the immune insult of a pathogen triggering disease exacerbation. Such LPS-induced inflammatory conditions are significantly abrogated by immunomodulatory agents targeting specific inflammatory signaling pathways in the absence of cytotoxic effects in lung slices. Thus, an ex vivo model of PCLS with a simulated pathogenic insult can replicate proposed in vivo pharmacological effects and thus could potentially act as a valuable tool to investigate the underlying mechanisms associated with lung safety, therapeutic efficacy and exacerbations with infection.
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http://dx.doi.org/10.1007/s00204-021-03096-yDOI Listing
August 2021

The Impact of Pre-existing Comorbidities and Therapeutic Interventions on COVID-19.

Front Immunol 2020 11;11:1991. Epub 2020 Aug 11.

Immunotoxicology, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom.

Evidence from the global outbreak of SARS-CoV-2 has clearly demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from COVID-19. This is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. Not all comorbidities confer the same risk, however, many affect the function of the immune system, which in turn directly impacts the response to COVID-19. Furthermore, the myriad of drugs prescribed for these comorbidities can also influence the progression of COVID-19 and limit additional treatment options available for COVID-19. Here, we review immune dysfunction in response to SARS-CoV-2 infection and the impact of pre-existing comorbidities on the development of COVID-19. We explore how underlying disease etiologies and common therapies used to treat these conditions exacerbate COVID-19 progression. Moreover, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of COVID-19 in patients with pre-existing comorbidities.
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http://dx.doi.org/10.3389/fimmu.2020.01991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437504PMC
September 2020

Recent Advancements and Applications of Human Immune System Mice in Preclinical Immuno-Oncology.

Toxicol Pathol 2020 02 18;48(2):302-316. Epub 2019 Dec 18.

Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Campus, Cambridge, United Kingdom.

Significant advances in immunotherapies have resulted in the increasing need of predictive preclinical models to improve immunotherapeutic drug development, treatment combination, and to prevent or minimize toxicity in clinical trials. Immunodeficient mice reconstituted with human immune system (HIS), termed humanized mice or HIS mice, permit detailed analysis of human immune biology, development, and function. Although this model constitutes a great translational model, some aspects need to be improved as the incomplete engraftment of immune cells, graft versus host disease and the lack of human cytokines and growth factors. In this review, we discuss current HIS platforms, their pathology, and recent advances in their development to improve the quality of human immune cell reconstitution. We also highlight new technologies that can be used to better understand these models and how improved characterization is needed for their application in immuno-oncology safety, efficacy, and new modalities therapy development.
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http://dx.doi.org/10.1177/0192623319886304DOI Listing
February 2020

Drug-induced skin toxicity: gaps in preclinical testing cascade as opportunities for complex in vitro models and assays.

Lab Chip 2020 01 10;20(2):199-214. Epub 2019 Oct 10.

MPS Center of Excellence, Drug Safety & Metabolism, IMED Biotech Unit, AstraZeneca, Waltham, MA, USA.

Skin is the largest organ of the body and serves as the principle barrier to the environment. Composed of multiple cell types arranged in stratified layers with highly specialized appendages, it serves sensory and immune surveillance roles in addition to its primary mechanical function. Several complex in vitro models of skin (i.e. microphysiological systems (MPS) including but not limited to 3D tissues, organ-on-a-chip, organoids), have been developed and assays validated for regulatory purposes. As such, skin is arguably the most advanced organ with respect to model development and adoption across industries including chemical, cosmetic, and to a somewhat lesser extent, pharmaceutical. Early adoption of complex skin models and associated assays for assessment of irritation and corrosion spurred research into other areas such as sensitization, absorption, phototoxicity, and genotoxicity. Despite such considerable advancements, opportunities remain for immune capabilities, inclusion of appendages such as hair follicles, fluidics, and innervation, among others. Herein, we provide an overview of current complex skin model capabilities and limitations within the drug development scheme, and recommendations for future model development and assay qualification and/or validation with the intent to facilitate wider adoption of use within the pharmaceutical industry.
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http://dx.doi.org/10.1039/c9lc00519fDOI Listing
January 2020

Investigating the Value of Urine Volume, Creatinine, and Cystatin C for Urinary Biomarkers Normalization for Drug Development Studies.

Int J Toxicol 2019 Jan/Feb;38(1):12-22. Epub 2019 Jan 23.

2 Janssen Research & Development, LLC, San Diego, CA, USA.

Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there seems to be no standardized normalization method for analyzing these urinary biomarkers, as some users normalize with urinary creatinine (uCr), urine volume (uVol), or leave biomarker un-normalized. More recently, urinary cystatin C is also emerging as a urinary biomarker normalizer, given some of its characteristics as a glomerular filtration marker. The purpose of this study was to identify an optimal drug-induced kidney injury biomarker normalization method that can be adopted more uniformly in the field. To this end, we compared the variability of uVol, urinary cystatin C, and Cr in healthy rats; we evaluated the sensitivity of the renal biomarkers to renal injury after normalization with uVol, uCr, and cystatin C in rats with cisplatin-induced renal injury. We showed that, over time, uCr was less variable than urinary cystatin C and uVol. When the renal biomarkers were normalized with the 3 normalizing end points, the biomarkers showed (1) least variability following normalization with Cr in healthy animals and (2) poor sensitivity when normalized with urinary cystatin C in animals with renal injury. Overall, the results suggested that uCr is better than urinary cystatin C and uVol for normalizing renal biomarkers in rats under controlled preclinical conditions. To our knowledge, this is the first report that compared the variability of uVol, cystatin C, and Cr in the context of renal biomarkers' normalization.
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http://dx.doi.org/10.1177/1091581818819791DOI Listing
December 2019

A 3D Human Airway Model Enables Prediction of Respiratory Toxicity of Inhaled Drugs In Vitro.

Toxicol Sci 2018 03;162(1):301-308

Regulatory Safety, Drug Safety and Metabolism.

Respiratory tract toxicity represents a significant cause of attrition of inhaled drug candidates targeting respiratory diseases. One of the key issues to allow early detection of respiratory toxicities is the lack of reliable and predictive in vitro systems. Here, the relevance and value of a physiologically relevant 3D human airway in vitro model (MucilAir) were explored by repeated administration of a set of compounds with (n = 8) or without (n = 7) respiratory toxicity following inhalation in vivo. Predictability for respiratory toxicity was evaluated by readout of cytotoxicity, barrier integrity, viability, morphology, ciliary beating frequency, mucociliary clearance and cytokine release. Interestingly, the data show that in vivo toxicity can be predicted in vitro by studying cell barrier integrity by transepithelial electrical resistance (TEER), and cell viability determined by the Resazurin method. Both read-outs had 88% sensitivity and 100% specificity, respectively, while the former was more accurate with receiver operating characteristic (ROC) AUC of 0.98 (p = .0018) compared with ROC AUC of 0.90 (p = .0092). The loss of cell barrier integrity could mainly, but not fully, be attributed to a loss of cell coverage in 6 out of 7 compounds with reduced TEER. Notably, these effects occurred only at 400 µM, at concentration levels significantly above primary target cell potency, suggesting that greater attention to high local lung concentrations should be taken into account in safety assessment of inhaled drugs. Thus, prediction of respiratory toxicity in 3D human airway in vitro models may result in improved animal welfare and reduced attrition in inhaled drug discovery projects.
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http://dx.doi.org/10.1093/toxsci/kfx255DOI Listing
March 2018

Characterization of Drug-Specific Signaling Between Primary Human Hepatocytes and Immune Cells.

Toxicol Sci 2017 07;158(1):76-89

Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool L69 3GE, UK.

It is now apparent that antigen-specific T-cells are activated in certain patients with drug-induced liver injury (DILI). Since cross-talk between hepatocytes and immune cells is likely to be critical in determining the outcome of drug exposure, the aim of this study was to profile the signals released by drug-treated hepatocytes and to characterize the impact of these molecules on dendritic cells. Human hepatocytes were exposed to 3 drugs (flucloxacillin, amoxicillin, and isoniazid) associated with DILI potentially mediated by the adaptive immune system as drug-specific T-cells have been isolated from DILI patients, and the metabolite nitroso-sulfamethoxazole (SMX-NO). Hepatocyte toxicity, cytokine release and activation of oxidative stress pathways were measured. Supernatants were transferred to monocyte-derived dendritic cells and cell phenotype and function were assessed. High-mobility group box 1 protein (HMGB1) and lactate dehydrogenase release as well as adenosine triphosphate depletion occurred in a drug-, time-, and concentration-dependent manner with SMX-NO and flucloxacillin, whereas isoniazid and amoxicillin were nontoxic. Furthermore, drug-induced activation of nuclear factor (erythroid-derived 2)-like 2 marker genes was observed when hepatocytes were exposed to test drugs. The disulfide isoform of HMGB1 stimulated dendritic cell cytokine release and enhanced the priming of naive T-cells. Incubation of dendritic cells with supernatant from drug-treated hepatocytes resulted in 2 distinct cytokine profiles. SMX-NO/flucloxacillin stimulated secretion of TNF-α, IL-6, IL-1α, and IL-1-β. Isoniazid which did not induce significant hepatocyte toxicity, compared with SMX-NO and flucloxacillin, stimulated the release of a panel of cytokines including the above and IFN-γ, IL-12, IL-17A, IP-10, and IL-10. Collectively, our study identifies drug-specific signaling pathways between hepatocytes and immune cells that could influence whether drug exposure will result in an immune response and tissue injury.
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http://dx.doi.org/10.1093/toxsci/kfx069DOI Listing
July 2017

New Approaches to Investigate Drug-Induced Hypersensitivity.

Chem Res Toxicol 2017 01 15;30(1):239-259. Epub 2016 Nov 15.

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool , Ashton Street, Liverpool L69 3GE, U.K.

The workshop on "New Approaches to Investigate Drug-Induced Hypersensitivity" was held on June 5, 2014 at the Foresight Center, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical, and chemical basis of small molecule drug hypersensitivity, (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, and (3) identify the limitations, knowledge gaps, and challenges that limit the use of these assays and utilize the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective reviews the clinical and immunological bases of drug hypersensitivity and summarizes various experts' views on the different topics covered during the meeting.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00333DOI Listing
January 2017

An integrated characterization of serological, pathological, and functional events in doxorubicin-induced cardiotoxicity.

Toxicol Sci 2014 Jul 27;140(1):3-15. Epub 2014 Mar 27.

Drug Safety & Metabolism, Innovative Medicines, AstraZeneca R&D, Alderley Park Macclesfield, Cheshire, SK10 4TF, UK

Many efficacious cancer treatments cause significant cardiac morbidity, yet biomarkers or functional indices of early damage, which would allow monitoring and intervention, are lacking. In this study, we have utilized a rat model of progressive doxorubicin (DOX)-induced cardiomyopathy, applying multiple approaches, including cardiac magnetic resonance imaging (MRI), to provide the most comprehensive characterization to date of the timecourse of serological, pathological, and functional events underlying this toxicity. Hannover Wistar rats were dosed with 1.25 mg/kg DOX weekly for 8 weeks followed by a 4 week off-dosing "recovery" period. Electron microscopy of the myocardium revealed subcellular degeneration and marked mitochondrial changes after a single dose. Histopathological analysis revealed progressive cardiomyocyte degeneration, hypertrophy/cytomegaly, and extensive vacuolation after two doses. Extensive replacement fibrosis (quantified by Sirius red staining) developed during the off-dosing period. Functional indices assessed by cardiac MRI (including left ventricular ejection fraction (LVEF), cardiac output, and E/A ratio) declined progressively, reaching statistical significance after two doses and culminating in "clinical" LV dysfunction by 12 weeks. Significant increases in peak myocardial contrast enhancement and serological cardiac troponin I (cTnI) emerged after eight doses, importantly preceding the LVEF decline to <50%. Troponin I levels positively correlated with delayed and peak gadolinium contrast enhancement, histopathological grading, and diastolic dysfunction. In summary, subcellular cardiomyocyte degeneration was the earliest marker, followed by progressive functional decline and histopathological manifestations. Myocardial contrast enhancement and elevations in cTnI occurred later. However, all indices predated "clinical" LV dysfunction and thus warrant further evaluation as predictive biomarkers.
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http://dx.doi.org/10.1093/toxsci/kfu057DOI Listing
July 2014

Islets of Langerhans from prohormone convertase-2 knockout mice show α-cell hyperplasia and tumorigenesis with elevated α-cell neogenesis.

Int J Exp Pathol 2014 Feb;95(1):29-48

Department of Pathological Sciences, AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK.

Antagonism of the effects of glucagon as an adjunct therapy with other glucose-lowering drugs in the chronic treatment of diabetes has been suggested to aggressively control blood glucose levels. Antagonism of glucagon effects, by targeting glucagon secretion or disabling the glucagon receptor, is associated with α-cell hyperplasia. We evaluated the influence of total glucagon withdrawal on islets of Langerhans using prohormone convertase-2 knockout mice (PC2-ko), in which α-cell hyperplasia is present from a young age and persists throughout life, in order to understand whether or not sustained glucagon deficit would lead to islet tumorigenesis. PC2-ko and wild-type (WT) mice were maintained drug-free, and cohorts of these groups sampled at 3, 12 and 18 months for plasma biochemical and morphological (histological, immunohistochemical, electron microscopical and image analytical) assessments. WT mice showed no islet tumours up to termination of the study, but PC2-ko animals displayed marked changes in islet morphology from α-cell hypertrophy/hyperplasia/atypical hyperplasia, to adenomas and carcinomas, these latter being first encountered at 6-8 months. Islet hyperplasias and tumours primarily consisted of α-cells associated to varying degrees with other islet endocrine cell types. In addition to substantial increases in islet neoplasia, increased α-cell neogenesis associated primarily with pancreatic duct(ule)s was present. We conclude that absolute blockade of the glucagon signal results in tumorigenesis and that the PC2-ko mouse represents a valuable model for investigation of islet tumours and pancreatic ductal neogenesis.
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http://dx.doi.org/10.1111/iep.12066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919648PMC
February 2014

Inhibin B in plasma samples from male volunteer panel selected for health but not fertility: sources of variability.

Birth Defects Res B Dev Reprod Toxicol 2013 Feb 24;98(1):104-9. Epub 2013 Jan 24.

Global Safety Assessment, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.

Background: Inhibin B was measured in plasma samples obtained from 34 healthy male subjects selected on criteria typical for a phase I clinical trial across a wide age range (19-70 years).

Methods: Mutiple samples (up to seven per subject) were obtained as a set consisting of one baseline sample then three pairs of morning and evening samples. This allowed assessment of the fed/fasted state and diurnal effects. Samples were analyzed using a commercially available inhibin B ELISA assay. Across all time points, the mean plasma inhibin B was 197 pg/ml ± 67pg/ml.

Results: The results confirmed a diurnal effect where inhibin B concentration is on average about 40 pg/ml greater in the morning and showed a negative influence of age on inhibin B concentrations. There was no overt influence of body mass index on inhibin B. A variance components analysis revealed that more than 80% of the total variability was due to the variability observed between individuals. Within the fed-fasted sampling schedule of this study, inhibin B levels were slightly lower when volunteers had eaten but the magnitude of this effect was within the variance encountered between occasions.

Conclusion: These results illustrate that when undertaking longitudinal monitoring of inhibin B in clinical trials as means of monitoring testicular function, it is important to obtain samples from an individual at the same time of day and to use statistical methods which analyze the magnitude of deviation of an individual from their personal baseline as well as looking at group means and influence of study duration.
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http://dx.doi.org/10.1002/bdrb.21049DOI Listing
February 2013

Analytic evaluation of a human ELISA kit for measurement of inhibin B in rat samples.

Birth Defects Res B Dev Reprod Toxicol 2013 Feb 24;98(1):4-16. Epub 2013 Jan 24.

AstraZeneca Research & Development, Global Safety Assessment, Alderley Park, Macclesfield, United Kingdom.

Background: A cross-laboratory analytic evaluation of a commercially available human inhibin B ELISA for measuring inhibin B in rat serum and plasma has been undertaken.

Methods: Dilution linearity, spiked recovery, intra- and inter-assay precision, functional sensitivity, matrix effects, and frozen stability were assessed across five laboratories. Reference ranges were generated for male Sprague Dawley and Han Wistar rats.

Results: Acceptable performance was defined as an overall assay coefficient of variation ≤ 20% with an intraday LLOQ ≤ 20 pg/ml. Intra- and inter-assay precision and functional sensitivity (≤6.4 pg/ml) generally met these criteria, but with occasional evidence of greater variability, particularly at lower concentrations. Dilution linearity was acceptable with occasional low recovery. Acceptable recovery of kit calibrators from rat serum confirmed the absence of matrix effects. Matched serum and plasma samples gave comparable results. The signal increased on freezing, remained constant for ≥3 freeze-thaw cycles and was generally stable for at least 8 weeks. Mean inhibin B ranged from 33.5 to 140.6 pg/ml in adult rats across laboratories, with some evidence for a decline from 6 to 9 weeks of age. Power calculations using preliminary reference range data indicated 10 animals/group would generally detect a 40% decrease in inhibin B at AstraZeneca, but laboratories with lower control values would require larger groups.

Conclusions: The assay meets the analytical performance criteria; however, precision at the low end of the standard curve, biological variability, and low control values observed in some laboratories indicate that the utility of the assay may be limited in some laboratories.
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http://dx.doi.org/10.1002/bdrb.21047DOI Listing
February 2013

Evaluation of novel urinary renal biomarkers: biological variation and reference change values.

Toxicol Pathol 2012 Apr 6;40(3):541-9. Epub 2012 Mar 6.

Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, Cheshire, UK.

A number of novel urinary biomarkers have been identified and partially qualified for use as markers for renal injury in rats. We used two multiplex assays for these novel biomarkers to quantify biomarker concentration in serial urine collections from rats of both sexes administered varying concentrations of cisplatin. From these data, we calculate inter-individual variation and reference ranges from predose animals and intra-individual variation and reference change values from undosed control animals. The biomarkers evaluated are albumin, α glutathione s-transferase, glutathione S-transferase-yb1, lipocalin-2, kidney injury molecule-1, osteopontin, and renal papillary antigen 1. For any creatinine-corrected novel biomarkers, we found intra-individual variation to be no greater than 44% and inter-individual variation to be no greater than 46%. Reference change values for most corrected analytes (except osteopontin) were 50-100%, indicating that a >100% increase in analyte concentration between serial samples would be unlikely to be associated with inherent analytical or biological variation.
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http://dx.doi.org/10.1177/0192623311432436DOI Listing
April 2012

Evaluation of novel urinary renal biomarkers with a cisplatin model of kidney injury: effects of collection period.

Toxicol Pathol 2012 Apr 13;40(3):534-40. Epub 2012 Jan 13.

Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, Cheshire, UK.

A number of novel urinary biomarkers have been identified and partially qualified for use as markers for renal injury in rats. To date, all evaluation studies have been made using 18 to 24 hour collection periods. However, shorter, more welfare friendly, urine collection periods are also used in industry. In this article, we quantify urinary biomarker concentration in serial paired sequential short and long urine collections from male rats administered varying concentrations of cisplatin. We calculate the rate of biomarker excretion in normal animals for both collection periods and the bias and correlation in urinary biomarker concentration between collection periods in dosed and control animals, and we estimate the level of agreement in biomarker concentration between both collection periods. We conclude that although there are minor differences in the concentration of some urinary biomarkers that are dependent upon the time and duration of collection, shorter collection protocols do not influence subsequent interpretation of normalized urinary biomarker data for most biomarkers.
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http://dx.doi.org/10.1177/0192623311432437DOI Listing
April 2012

The reduced local lymph node assay: the impact of group size.

J Appl Toxicol 2008 May;28(4):518-23

The Procter & Gamble Company, Cincinnati, OH 45253-8707, USA.

The local lymph node assay (LLNA) is a skin sensitization test that provides animal welfare benefits. To reduce animal usage further, a modified version (rLLNA) was proposed. Conducting the rLLNA as a screening test with a single high dose group and vehicle control differentiated accurately between skin sensitizers and non-sensitizers. This study examined whether a reduction in animal number/group is feasible. Historical data were utilized to examine the impact of conducting the rLLNA with two mice/group. To assess the effect on the stimulation index (SI) 41 datasets with individual animal data derived using five mice/group were analysed. SIs were calculated on all possible combinations of two control and two high dose group disintegrations per minute (dpm) values. For 25 of 33 sensitizer datasets, > 96% of possible dpm combinations resulted in a calculated SI > 3. The lowest percentages of positive SIs were observed with weak allergens when, in the standard LLNA, the mean SIs would have been nearer to the threshold value of 3. The results indicate that moderate, strong and extreme allergens are more likely than weak allergens to be identified as sensitizers when group sizes of two mice are used within the rLLNA. It is concluded that a rLLNA with two mice/group would display decreased sensitivity and is inappropriate for use in hazard identification.
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http://dx.doi.org/10.1002/jat.1303DOI Listing
May 2008

Comparative analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl acrylate, and ethylhexyl acrylate) using the local lymph node assay.

Contact Dermatitis 2007 Oct;57(4):242-7

Immunology, Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK1 4TJ, UK.

There are currently available no systematic experimental data on the skin sensitizing properties of acrylates that are of relevance in occupational settings. Limited information from previous guinea-pig tests or from the local lymph node assay (LLNA) is available; however, these data are incomplete and somewhat contradictory. For those reasons, we have examined in the LLNA 4 acrylates: butyl acrylate (BA), ethyl acrylate (EA), methyl acrylate (MA), and ethylhexyl acrylate (EHA). The LLNA data indicated that all 4 compounds have some potential to cause skin sensitization. In addition, the relative potencies of these acrylates were measured by derivation from LLNA dose-response analyses of EC3 values (the effective concentration of chemical required to induce a threefold increase in proliferation of draining lymph node cells compared with control values). On the basis of 1 scheme for the categorization of skin sensitization potency, BA, EA, and MA were each classified as weak sensitizers. Using the same scheme, EHA was considered a moderate sensitizer. However, it must be emphasized that the EC3 value for this chemical of 9.7% is on the borderline between moderate (<10%) and weak (>10%) categories. Thus, the judicious view is that all 4 chemicals possess relatively weak skin sensitizing potential.
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http://dx.doi.org/10.1111/j.1600-0536.2007.01215.xDOI Listing
October 2007

B220 analysis with the local lymph node assay: proposal for a more flexible prediction model.

J Appl Toxicol 2007 Sep-Oct;27(5):506-10

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK.

The mouse local lymph node assay (LLNA) has been developed and validated for the identification of chemicals that have the potential to induce skin sensitisation. In common with other predictive test methods the accuracy of the LLNA is not absolute and experience has revealed that a few chemicals, including for instance a minority of skin irritants, may elicit false-positive reactions in the assay. To improve further the performance of the LLNA, and to eliminate or reduce false-positives, there has been interest in an adjunct method in which the ability of chemicals to cause increases in the frequency of B220(+) lymphocytes in skin-draining lymph nodes is measured. Previous studies suggest that the use of B220 analyses aligned with the standard LLNA may serve to distinguish further between contact allergens and skin irritants. In the original predictive model, chemicals were regarded as being skin sensitisers if they were able to induce a 1.25-fold or greater increase in the percentage of B220(+) cells within lymph nodes compared with concurrent vehicle controls. Although this first prediction model has proven useful, in the light of more recent experience, and specifically as a consequence of some variability observed in the frequency of B220(+) lymphocytes in nodes taken from vehicle control-treated animals, it is timely now to reconsider and refine the model. As a result a new prediction model is proposed in which reliance on the use of absolute thresholds is reduced, and in which small changes in control values can be better accommodated.
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http://dx.doi.org/10.1002/jat.1269DOI Listing
October 2007

The use of ethanol:diethylphthalate as a vehicle for the local lymph node assay.

Contact Dermatitis 2007 Feb;56(2):70-5

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK.

The murine local lymph node assay (LLNA) is used for the prospective identification of contact allergens. This method is not only accepted as a stand-alone test for the identification of contact allergenic hazard but also used increasingly for the measurement of the relative potency of skin-sensitizing chemicals as an integral component of the risk assessment process. During the development and validation phases of the method, a list of standard vehicles for use in the LLNA was identified, among them being the vehicle most commonly used acetone/olive oil (4:1, AOO). We have now explored the performance in the LLNA of a non-standard vehicle, ethanol:diethyl phthalate (1:3, EtOH:DEP), that is used frequently to evaluate dermal effects of fragrance materials in both human and experimental studies. Current investigations have demonstrated that EtOH:DEP induces similar levels of background proliferative responses in lymph nodes comparable with the standard vehicle AOO. Moreover, expected levels of activity are observed when EtOH:DEP is used to deliver a known contact allergen in the LLNA. The conclusion drawn is that EtOH:DEP provides a suitable vehicle for use in the LLNA and that the approach described provides a basis for future evaluation of novel vehicles.
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http://dx.doi.org/10.1111/j.1600-0536.2007.01036.xDOI Listing
February 2007

Skin sensitization potency of methyl methacrylate in the local lymph node assay: comparisons with guinea-pig data and human experience.

Contact Dermatitis 2006 Sep;55(3):140-7

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK1 4TJ, UK.

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.
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http://dx.doi.org/10.1111/j.1600-0536.2006.00898.xDOI Listing
September 2006

The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?

Contact Dermatitis 2006 Apr;54(4):181-5

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK.

The local lymph node assay (LLNA), an alternative approach to skin-sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut-down assay comprising a single high-dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.
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http://dx.doi.org/10.1111/j.0105-1873.2006.00827.xDOI Listing
April 2006

Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment.

Toxicol Sci 2005 Nov 13;88(1):4-11. Epub 2005 Jul 13.

Miami Valley Innovation Center, Central Product Safety Department, The Procter & Gamble Company, Cincinnati, Ohio 45253-8707, USA.

The identification of potential skin sensitizing chemicals is a key step in the overall skin safety risk assessment process. Traditionally, predictive testing has been conducted in guinea pigs. More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential. However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop non-animal test methods for skin sensitization. Mechanistic understanding of allergic contact dermatitis has increased substantially in recent years. For example, a number of changes are known to occur in epidermal Langerhans cells, the principal antigen-presenting dendritic cell in the skin, as a result of exposure to chemical allergens, including the internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis, the induction of tyrosine phosphorylation, the modulation of cell surface markers, and cytokine expression. The application of this knowledge to the design of predictive in vitro alternative tests provides both unique opportunities and challenges. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells and the potential use of that information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.
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http://dx.doi.org/10.1093/toxsci/kfi245DOI Listing
November 2005

Potency and risk assessment of a skin-sensitizing disperse dye using the local lymph node assay.

Contact Dermatitis 2005 May;52(5):268-72

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK1 4TJ, UK.

Disperse Blue 106 is an acknowledged skin-sensitization hazard. However, information about the relative sensitization potency of this chemical is lacking, and to provide this information was the purpose of the investigations described here. The approach taken was to measure dose-response relationships for C.I. Disperse Blue in the local lymph node assay, a method for the assessment of skin-sensitization potential in which activity is measured as a function of lymphocyte proliferative responses induced in draining lymph nodes. From these data, it was possible to derive EC3 values (such being the estimated concentration of chemical required to elicit a 3-fold increase in proliferation) that have been shown previously to reflect the relative sensitizing potency of contact allergens. These analyses revealed that Disperse Blue 106 had a relatively low EC3 value (0.01%), comparable to that measured concurrently for 2,4-dinitrochlorobenzene, a potent contact allergen. Collectively, these data reveal that Disperse Blue 106 represents a significant skin-sensitization hazard, and, in combination with information on dye migration and percutaneous penetration from various types of fabric and use conditions provide a basis for the development of effective and accurate risk assessments.
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http://dx.doi.org/10.1111/j.0105-1873.2005.00578.xDOI Listing
May 2005

Intradermal exposure of BALB/c strain mice to peanut protein elicits a type 2 cytokine response.

Food Chem Toxicol 2004 Oct;42(10):1589-99

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK.

There is a growing need for the development of methods to characterize the allergenic properties of novel proteins, particularly those expressed by transgenic crop plants. Hence, there is considerable interest in the development of suitable animal models for this purpose. The production of specific IgE antibody has been reported following sensitization with food allergen via oral or systemic (intraperitoneal) routes of exposure. We have characterized cytokine profiles induced by intradermal treatment of BALB/c strain mice with a purified peanut allergen, Arachis hypogea lectin. Mice were exposed to peanut lectin by intradermal administration and the cytokine responses in the lymph node draining the site of exposure analyzed at the secreted protein level by enyzme-linked immunosorbent assay (ELISA) and cytokine mRNA level by ribonuclease protection assay (RPA). Exposure to peanut lectin, under conditions that induced robust IgE antibody titers, was found to be associated with a T helper 2 (Th2)-type cytokine expression profile at both the mRNA and secreted protein levels. Culture of naïve lymph node cells with peanut lectin failed to stimulate marked proliferation or cytokine production, confirming this protein is not mitogenic for mouse lymphocytes. Furthermore, the expression of Th2 cytokines was associated with the effector/memory CD62L- cell population. Similar treatment with a non-allergenic protein, potato acid phosphatase, failed to induce Th2 cytokine expression. These data demonstrate that exposure of mice to peanut allergen results in the selective stimulation of a Th2-type response.
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http://dx.doi.org/10.1016/j.fct.2004.05.004DOI Listing
October 2004

Assessment of the allergenic potential of proteins.

Toxicol Lett 2003 Apr;140-141:297-302

Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire SK10 4TJ, Macclesfield, UK.

The development of novel foods, including foods derived from genetically modified plants, has generated considerable interest in the design and application of appropriate safety assurance measures. A specific focus of attention has been on allergenicity, and in particular the need to determine whether the products of novel genes introduced into food plants have the potential to cause allergic sensitisation. Among the approaches applied currently are considerations of whether a new protein has structural, sequence and/or antigenic similarities with known food allergens, and whether or not it displays resistance to digestion within a simulated gastric fluid, or by pepsin. Although such data are useful in an overall hazard assessment, they are neither individually, nor collectively, able to provide a direct evaluation of inherent sensitising potential. For this reason there is a need to develop and apply appropriate animal models that will offer a more holistic view of sensitising activity. Several methods have been suggested, but as yet none has been evaluated fully or validated. Nevertheless, significant progress has been made and in this article an experimental approach using BALB/c strain mice in which animals are exposed to the test protein via systemic (intraperitoneal, or in certain circumstances, intradermal) administration is described. Inherent sensitising potential is measured as a function of induced IgE antibody responses. Experience to date is encouraging and the data available reveal that this method is able to distinguish between proteins of different allergenic potential.
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http://dx.doi.org/10.1016/s0378-4274(03)00025-0DOI Listing
April 2003

Chemical allergy: considerations for the practical application of cytokine profiling.

Toxicol Sci 2003 Feb;71(2):137-45

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, United Kingdom.

Chemical respiratory allergy is an important occupational health problem, but there are currently available no validated methods for hazard identification. This is due in part to the fact that the relevant cellular and molecular mechanisms of sensitization of the respiratory tract have been unclear, with particular controversy regarding the role of IgE. There is now increasing evidence that respiratory sensitization is associated with the preferential activation of type 2 T lymphocytes and the expression of type 2 cytokines interleukin (IL)-4, IL-5, IL-10, and IL-13. Type 2 cell products favor immediate type hypersensitivity reactions, serving as growth and differentiation factors for mast cells and eosinophils, the cellular effectors of the clinical manifestations of the allergic responses, and promoting IgE antibody production. There has been considerable interest in the application of cytokine profiling for the characterization of chemical allergens, with cytokine phenotypes analyzed in freshly isolated tissue, or following culture in the presence or absence of mitogen at the level of protein secretion or mRNA expression. Experience to date suggests that the measurement of induced cytokine secretion profiles shows promise for the hazard identification and characterization of chemical respiratory allergens. The purpose of this brief review article is to consider the approaches available and to highlight key procedural issues.
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http://dx.doi.org/10.1093/toxsci/71.2.137DOI Listing
February 2003

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation.

Toxicology 2003 Mar;185(1-2):103-17

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK.

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.
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http://dx.doi.org/10.1016/s0300-483x(02)00598-xDOI Listing
March 2003

Temporal changes in cytokine gene expression profiles induced in mice by trimellitic anhydride.

Toxicol Lett 2002 Dec;136(2):121-32

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK.

Prolonged (13 day) topical exposure of BALB/c strain mice to the chemical respiratory allergen trimellitic anhydride (TMA) induces a selective T helper (Th) 2 profile of cytokine secretion in cells isolated from the draining lymph node. The ability of chemical respiratory allergens to elicit preferential type 2 immune responses is a distinguishing characteristic and provides the theoretical basis for cytokine fingerprinting, a novel approach to hazard identification. This study aimed to further characterize the cytokine expression profile induced by TMA, and to investigate the kinetics of cytokine production at both the protein and mRNA level by comparison of acute (3 day) and chronic (13 day) exposure regimes. Acute exposure resulted in the expression of high levels of mRNA for both Th1- and Th2-type cytokines, including interleukins 4, 10, 15 (IL-4, IL-10, IL-15) and interferon gamma (IFN-gamma), and the inflammatory cytokine IL-6, as determined by ribonuclease protection assay (RPA). However, following chronic exposure marked down-regulation of message for IL-6 and IFN-gamma was observed along with concomitant up-regulation of IL-4 and IL-10 expression. These cytokine mRNA profiles were broadly paralleled at the protein level. There was also a marked increase with time of mRNA for the Th2 cytokine IL-9, a cytokine not associated previously with chemical allergy. These data show that as the immune response to TMA develops, the cytokine gene expression profile of allergen-activated lymph node cells evolves from a mixed Th1/Th2 phenotype to a more polarized Th2 profile.
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http://dx.doi.org/10.1016/s0378-4274(02)00289-8DOI Listing
December 2002
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