Poor Translatability of Biomedical Research Using Animals - A Narrative Review.

The failure rate for the translation of drugs from animal testing to human treatments remains at over 92%, where it has been for the past few decades. The majority of these failures are due to unexpected toxicity - that is, safety issues revealed in human trials that were not apparent in animal tests - or lack of efficacy. However, the use of more innovative tools, such as organs-on-chips, in the preclinical pipeline for drug testing, has revealed that these tools are more able to predict unexpected safety events prior to clinical trials and so can be used for this, as well as for efficacy testing. Here, we review several disease areas, and consider how the use of animal models has failed to offer effective new treatments. We also make some suggestions as to how the more human-relevant new approach methodologies might be applied to address this.

Phase-In to Phase-Out-Targeted, Inclusive Strategies Are Needed to Enable Full Replacement of Animal Use in the European Union.

In September 2021, the European Parliament voted overwhelmingly in favour of a resolution to phase out animal use for research, testing, and education, through the adoption of an action plan. Here we explore the opportunity that the action plan could offer in developing a more holistic outlook for fundamental and biomedical research, which accounts for around 70% of all animal use for scientific purposes in the EU. We specifically focus on biomedical research to consider how mapping scientific advances to patient needs, taking into account the ambitious health policies of the EU, would facilitate the development of non-animal strategies to deliver safe and effective medicines, for example. We consider what is needed to help accelerate the move away from animal use, taking account of all stakeholders and setting ambitious but realistic targets for the total replacement of animals. Importantly, we envisage this as a 'phase-in' approach, encouraging the use of human-relevant NAMs, enabling their development and application across research (with applications for toxicology testing). We make recommendations for three pillars of activity, inspired by similar efforts for making the shift to renewable energy and reducing carbon emissions, and point out where investment-both financial and personnel-may be needed.

Patient-Derived Xenograft vs. Organoids: A Preliminary Analysis of Cancer Research Output, Funding and Human Health Impact in 2014-2019.

Cancer remains a major threat to mortality and morbidity globally, despite intense research and generous funding. Patient-derived xenograft (PDX) models-where tumor biopsies are injected into an animal-were developed to improve the predictive capacity of preclinical animal models. However, recent observations have called into question the clinical relevance, and therefore the translational accuracy, of these. Patient-derived organoids (PDO) use patient tumor samples to create in vitro models that maintain aspects of tumor structure and heterogeneity. We undertook a preliminary analysis of the number of breast, colorectal, and lung cancer research studies using PDX or PDO published worldwide between 2014-2019. We looked for evidence of impacts of this research on human health. The number of publications that focused on PDO is gradually increasing over time, but is still very low compared to publications using PDX models. Support for new research projects using PDO is gradually increasing, a promising indicator of a shift towards more human-relevant approaches to understanding human disease. Overall, increases in total funding for these three major cancer types does not appear to be translating to any consequential increase in outputs, defined for this purpose as publications associated with clinical trials. With increasing public discomfort in research using animals and demands for 'alternative' methods, it is timely to consider how to implement non-animal methods more effectively.

Anaerobiosis influences virulence properties of Pseudomonas aeruginosa cystic fibrosis isolates and the interaction with Staphylococcus aureus.

The airways of individuals with cystic fibrosis (CF) are abundantly colonised by Staphylococcus aureus and Pseudomonas aeruginosa. Co-infecting hypoxic regions of static mucus within CF airways, together with decreases in pulmonary function, mucus plugging and oxygen consumption by host neutrophils gives rise to regions of anoxia. This study determined the impact of anaerobiosis upon S. aureus-P. aeruginosa interactions in planktonic co-culture and mixed species biofilms in vitro. Whilst anoxia reduced the ability for P. aeruginosa CF isolates to dominate over S. aureus, this occurred in an isolate dependent manner. Investigations into the underlying mechanisms suggest that the anti-staphylococcal compound facilitating P. aeruginosa dominance under normoxia and anoxia is greater than 3 kDa in size and is heat-stable. Not all interspecies interactions studied were antagonistic, as S. aureus exoproducts were shown to restore and enhance P. aeruginosa motility under normoxia and anoxia in an isolate dependent manner. Collectively, this study suggests changes in oxygen availability within regions of the CF lung is likely to influence interspecies interactions and in turn, potentially influence disease progression.

Parkinson's disease research: adopting a more human perspective to accelerate advances.

Parkinson's disease (PD) affects 1% of the population over 60 years old and, with global increases in the aging population, presents huge economic and societal burdens. The etiology of PD remains unknown; most cases are idiopathic, presumed to result from genetic and environmental risk factors. Despite 200 years since the first description of PD, the mechanisms behind initiation and progression of the characteristic neurodegenerative processes are not known. Here, we review progress and limitations of the multiple PD animal models available and identify advances that could be implemented to better understand pathological processes, improve disease outcome, and reduce dependence on animal models. Lessons learned from reducing animal use in PD research could serve as guideposts for wider biomedical research.

An investigation into E-cigarette cytotoxicity in-vitro using a novel 3D differentiated co-culture model of human airways.

Currently there is a lack of consensus on the possible adverse health effects of E-cigarettes (ECs). Important factors including cell model employed and exposure method determine the physiological relevance of EC studies. The present study aimed to evaluate EC cytotoxicity using a physiologically relevant in-vitro multicellular model of human airways. Human bronchial epithelial cells (CALU-3) and pulmonary fibroblasts (MRC-5) were co-cultured at air-liquid-interface for 11-14 days post which they were exposed to whole cigarette smoke (WCS) or EC vapour (ECV) at standard ISO-3308 regime for 7 m using a bespoke aerosol delivery system. ECV effects were further investigated at higher exposure times (1 h-6 h). Results showed that while WCS significantly reduced cell viability after 7 m, ECV decreased cell viability only at exposure times higher than 3 h. Furthermore, ECV caused elevated IL-6 and IL-8 production despite reduced cell viability. ECV exposure also produced a marked increase in oxidative stress. Finally, WCS but not ECV exposure induced caspase 3/7 activation, suggesting a caspase independent death of ECV exposed cells. Overall, our results indicate that prolonged ECV exposure (≥3 h) has a significant impact on pro-inflammatory mediators' production, oxidative stress and cell viability but not caspase 3/7 activity.

Recommendations toward a human pathway-based approach to disease research.

Failures in the current paradigm for drug development have resulted in soaring research and development costs and reduced numbers of new drug approvals. Over 90% of new drug programs fail, the majority terminated at the level of Phase 2/3 clinical trials, largely because of efficacy failures or unexplained toxicity. A recent workshop brought together members from research institutions, regulatory agencies, industry, academia, and nongovernmental organizations to discuss how existing programs could be better applied to understanding human biology and improving drug discovery. Recommendations include increased emphasis on human relevance, better access and curation of data, and improved interdisciplinary and international collaboration.

A comparative study of electronic cigarette vapor extracts on airway-related cell lines in vitro.

The use of electronic cigarettes (ECs) is rapidly increasing worldwide; however, scientific evidence regarding EC cytotoxicity is limited. The aim of this study was to evaluate the acute cytotoxicity of EC vapor extract (ECE) on airway-related cells in vitro. Cigarette smoke extract (CSE), vapor extract of fifteen brands/flavors of ECs and the extract from the E-vehicle (propylene glycol and glycerin) was collected. Extracts, in concentrations of 100-12.5%, were added to human bronchial epithelial (BEAS-2B, IB3-1 and C38), fibroblast (Wi-38) and macrophage (J774 and THP-1) cell lines. Viability was assessed after 24 h using a standard XTT assay. Viability of <70% of control (no extract) was considered cytotoxic according to UNI EN ISO 10993-5 standards. CSE displayed a concentration-dependent influence on cell viability across all four cell lines with 100% producing the most toxic effect, therefore validating the model and indicating higher cytotoxicity than in ECEs. ECEs did reduce viability although this was not correlated with nicotine content or the E-vehicle. However, several flavors proved cytotoxic, with variation between different brands and cell lines. These data indicate that not all ECs are the same and that use of a particular flavor or brand may have differing effects. The cell line used is also an important factor. More research is crucial to ascertain the health effects of different ECs before they can be accepted as a safe alternative to tobacco cigarettes.

Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations.

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin.

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p<0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p<0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.

Developing accurate models of the human airways.

OBJECTIVES: Particle delivery to the airways is an attractive prospect for many potential therapeutics, including vaccines. Developing strategies for inhalation of particles provides a targeted, controlled and non-invasive delivery route but, as with all novel therapeutics, in vitro and in vivo testing are needed prior to clinical use. Whilst advanced vaccine testing demands the use of animal models to address safety issues, the production of robust in vitro cellular models would take account of the ethical framework known as the 3Rs (Replacement, Reduction and Refinement of animal use), by permitting initial screening of potential candidates prior to animal use. There is thus a need for relevant, realistic in vitro models of the human airways. KEY FINDINGS: Our laboratory has designed and characterised a multi-cellular model of human airways that takes account of the conditions in the airways and recapitulates many salient features, including the epithelial barrier and mucus secretion. SUMMARY: Our human pulmonary models recreate many of the obstacles to successful pulmonary delivery of particles and therefore represent a valid test platform for screening compounds and delivery systems.

The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells.

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material.

Cell exclusion in couette flow: evaluation through flow visualization and mechanical forces.

Cell exclusion is the phenomenon whereby the hematocrit and viscosity of blood decrease in areas of high stress. While this is well known in naturally occurring Poiseuille flow in the human body, it has never previously been shown in Couette flow, which occurs in implantable devices including blood pumps. The high-shear stresses that occur in the gap between the boundaries in Couette flow are known to cause hemolysis in erythrocytes. We propose to mitigate this damage by initiating cell exclusion through the use of a spiral-groove bearing (SGB) that will provide escape routes by which the cells may separate themselves from the plasma and the high stresses in the gap. The force between two bearings (one being the SGB) in Couette flow was measured. Stained erythrocytes, along with silver spheres of similar diameter to erythrocytes, were visualized across a transparent SGB at various gap heights. A reduction in the force across the bearing for human blood, compared with fluids of comparable viscosity, was found. This indicates a reduction in the viscosity of the fluid across the bearing due to a lowered hematocrit because of cell exclusion. The corresponding images clearly show both cells and spheres being excluded from the gap by entering the grooves. This is the first time the phenomenon of cell exclusion has been shown in Couette flow. It not only furthers our understanding of how blood responds to different flows but could also lead to improvements in the future design of medical devices.

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation.

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 µM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

The innate immune system and the clearance of apoptotic cells.

Removal of unwanted, effete, or damaged cells through apoptosis, an active cell death culminating in phagocytic removal of cell corpses, is an important process throughout the immune system in development, control, and homeostasis. For example, neutrophil apoptosis is central to the resolution of acute inflammation, whereas autoreactive and virus-infected cells are similarly deleted. The AC removal process functions not only to remove cell corpses but further, to control inappropriate immune responses so that ACs are removed in an anti-inflammatory manner. Such ″silent″ clearance is mediated by the innate immune system via polarized monocyte/macrophage populations that use a range of PRRs and soluble molecules to promote binding and phagocytosis of ACs. Additionally, attractive signals are released from dying cells to recruit phagocytes to sites of death. Here, we review the molecular mechanisms associated with innate immune removal of and responses to ACs and outline how these may impact on tissue homeostasis and age-associated pathology (e.g., cardiovascular disease). Furthermore, we discuss how an aging innate immune system may contribute to the inflammatory consequences of aging and why the study of an aging immune system may be a useful path to advance characterization of mechanisms mediating effective AC clearance.

Evaluation of disinfecting procedures for aseptic transfer in hospital pharmacy departments.

Current practice in National Health Service (NHS) hospitals employs 70% Industrial Methylated Spirit spray for surface disinfection of components required in Grade A pharmaceutical environments. This study seeks to investigate other agents and procedures that may provide more effective sanitisation. Several methods are available to test the efficacy of disinfectants against vegetative organisms. However, no methods currently available test the efficacy of disinfectants against spores on the hard surfaces encountered in the pharmacy aseptic processing environment. Therefore, a method has been developed to test the efficacy of disinfectants against spores, modified from British Standard 13697 and Association of Analytical Chemists standards. The testing procedure was used to evaluate alternative biocides and disinfection methods for transferring components into hospital pharmacy cleanrooms, and to determine which combinations of biocide and application method have the greatest efficacy against spores of Bacillus subtilis subspecies subtilis 168, Bacillus subtilis American Type Culture Collection (ATCC) 6633, and Bacillus pumilis ATCC 27142. Stainless steel carrier test plates were used to represent the hard surfaces in hospital pharmacy cleanrooms. Plates were inoculated with 10(7)-10(8) colony-forming units per milliliter (CFU/mL) and treated with the various biocide formulations, using different disinfection methods. Sporicidal activity was calculated as log reduction in CFU. Of the biocides tested, 6% hydrogen peroxide and a quaternary ammonium compound/chlorine dioxide combination were most effective compared to a Quat/biguanide, amphoteric surfactant, 70% v/v ethanol in deionised water and isopropyl alcohol in water for injection. Of the different application methods tested, spraying followed by wiping was the most effective, followed closely by wiping alone. Spraying alone was least effective.

Free secretory component from cystic fibrosis sputa displays the cystic fibrosis glycosylation phenotype.

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.

Plasminogen activator inhibitor-1 supports IL-8-mediated neutrophil transendothelial migration by inhibition of the constitutive shedding of endothelial IL-8/heparan sulfate/syndecan-1 complexes.

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.

Safety and use of sputum induction in children with cystic fibrosis.

We assessed the safety and use of induced sputum (IS) in children with cystic fibrosis (CF). Forty-eight children (19 males) with CF, mean age 12.6 (range, 7.3-17.0) years and median forced expired volume in 1 sec (FEV(1)) 48% (range, 14-77%) predicted were recruited. Patients spontaneously expectorated sputum and then performed sputum induction by inhalation of nebulized 7% hypertonic saline. Samples were sent for bacteriological culture, and for measurement of the following inflammatory mediators: interleukin-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase activity. FEV(1) was performed before and after inhalation of hypertonic saline. There was no increase in mediator levels in IS compared to expectorated sputum (ES) samples. Only 3 patients demonstrated significant bronchoconstriction following inhalation of hypertonic saline, by the method used. From the ES samples, Pseudomonas aeruginosa was isolated in 13 patients, Staphylococcus aureus in 7 patients, Stenotrophomonas maltophilia in 1 patient, and both Pseudomonas aeruginosa and Staphylococcus aureus in 5 patients. All these organisms were found in the IS samples. However, in 2 patients whose ES grew no organisms, one patient's IS grew Pseudomonas aeruginosa, and the other patient's IS grew Staphylococcus aureus. In our study, sputum induction was safe, with no proinflammatory effect.