Characterization of Teicoplanin-Specific T-Cells from Drug Naïve Donors Expressing HLA-A*32:01.

Teicoplanin is a glycopeptide antibiotic deployed to combat Gram-positive bacterial infection and has recently been associated with development of adverse drug reactions, particularly following previous exposure to vancomycin. In this study, we generated teicoplanin-specific monoclonal T-cell populations from healthy volunteers expressing HLA-A*32:01 and defined pathways of T-cell activation and HLA allele restriction. Teicoplanin-responsive T-cells were CD8+, HLA class I-restricted, and cross-reacted with the lipoglycopeptide daptomycin in proliferation and cytokine/cytolytic molecule (granzyme B, Perforin, and FasL) release assays. These data show that teicoplanin activates T-cells, which may play a role in the pathogenesis of teicoplanin-induced adverse events, in HLA-A*32:01 positive donors.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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-ß. 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.

New Approaches to Investigate Drug-Induced Hypersensitivity.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.