Adeno-associated virus serotype 2 capsid variants for improved liver-directed gene therapy.

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.

Venetoclax and dexamethasone synergize with inotuzumab ozogamicin-induced DNA damage signaling in B-lineage ALL.

Adult patients with relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a dismal prognosis. To improve pharmacotherapy, we analyzed induction of apoptosis by venetoclax and inotuzumab ozogamicin in terms of cytotoxicity and mode of action. Flow cytometry-based analyses of mitochondrial outer membrane permeabilization (MOMP) and ataxia telangiectasia mutated activation demonstrate rapid induction of MOMP by venetoclax and DNA damage signaling by inotuzumab ozogamicin, respectively. In primary ALL samples and patient-derived xenograft (PDX) models, venetoclax and inotuzumab ozogamicin cooperated and synergized in combination with dexamethasone in vitro in all tested samples of ALL. In murine PDX models, inotuzumab ozogamicin, but not venetoclax, induced complete remission in a dose-dependent manner but constantly failed to achieve relapse-free survival. In contrast, combination therapy with venetoclax, dexamethasone, and inotuzumab ozogamicin induced long-term leukemia-free survival and treatment-free survival in all 3 ALL-PDX models tested. These data demonstrate synergistic and highly efficient pharmacotherapy in preclinical models that qualify for evaluation in clinical trials.

Toward Evidence-Based Severity Assessment in Mouse Models with Repeated Seizures: (II.) Impact of Surgery and Intrahippocampal Kainate.

INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.

Running in the wheel: Defining individual severity levels in mice.

The fine-scale grading of the severity experienced by animals used in research constitutes a key element of the 3Rs (replace, reduce, and refine) principles and a legal requirement in the European Union Directive 2010/63/EU. Particularly, the exact assessment of all signs of pain, suffering, and distress experienced by laboratory animals represents a prerequisite to develop refinement strategies. However, minimal and noninvasive methods for an evidence-based severity assessment are scarce. Therefore, we investigated whether voluntary wheel running (VWR) provides an observer-independent behaviour-centred approach to grade severity experienced by C57BL/6J mice undergoing various treatments. In a mouse model of chemically induced acute colitis, VWR behaviour was directly related to colitis severity, whereas clinical scoring did not sensitively reflect severity but rather indicated marginal signs of compromised welfare. Unsupervised k-means algorithm-based cluster analysis of body weight and VWR data enabled the discrimination of cluster borders and distinct levels of severity. The validity of the cluster analysis was affirmed in a mouse model of acute restraint stress. This method was also applicable to uncover and grade the impact of serial blood sampling on the animal's welfare, underlined by increased histological scores in the colitis model. To reflect the entirety of severity in a multidimensional model, the presented approach may have to be calibrated and validated in other animal models requiring the integration of further parameters. In this experimental set up, however, the automated assessment of an emotional/motivational driven behaviour and subsequent integration of the data into a mathematical model enabled unbiased individual severity grading in laboratory mice, thereby providing an essential contribution to the 3Rs principles.

Toward evidence-based severity assessment in mouse models with repeated seizures: I. Electrical kindling.

OBJECTIVE: Ethical decisions about an allowance for animal experiments need to be based on scientifically sound information about the burden and distress associated with the experimental procedure and models. Thereby, species differences need to be considered for recommendations regarding evidence-based severity assessment and refinement measures. METHODS: A comprehensive analysis of behavioral patterns and corticosterone or its metabolites in serum and feces was completed in kindled mice. The impact of kindling via two different stimulation sites in the amygdala and hippocampus was determined. Data were compared to those from naive and electrode-implanted groups. RESULTS: Amygdala and hippocampus kindled mice exhibited comparable behavioral patterns with increased activity in the open field, reduced anxiety-associated behavior in the elevated-plus maze, and increased anhedonia-associated behavior in the saccharin preference test. In addition, repeated stimulation of the hippocampus caused a reduction in burrowing behavior and an increase in active social interaction. Levels of corticosterone and its metabolites were not altered in serum or feces, respectively. A comparison of mouse data with findings from amygdala kindled rats confirmed pronounced species differences in behavioral patterns associated with the kindling process. SIGNIFICANCE: Taken together the findings suggest a severity classification for the mouse kindling paradigms as moderate regardless of the stimulation site. The outcome of the species comparison provides valuable guidance for species selection for studies exploring behavioral comorbidities. In this context, it is emphasized that the mouse kindling paradigms seem to be well suited for studies exploring the link between ictal events and network alterations on the one hand, and hyperactivity and anhedonia-associated behavior on the other hand. Moreover, the underlying pathophysiological mechanisms and the impact of therapeutic interventions on these behavioral alterations can be studied in these paradigms providing guidance for the clinical management of respective psychiatric comorbidities in patients.

Continuous monitoring of physiological data using the patient vital status fusion score in septic critical care patients.

Accurate and standardized methods for assessing the vital status of patients are crucial for patient care and scientific research. This study introduces the Patient Vital Status (PVS), which quantifies and contextualizes a patient's physical status based on continuous variables such as vital signs and deviations from age-dependent normative values. The vital signs, heart rate, oxygen saturation, respiratory rate, mean arterial blood pressure, and temperature were selected as input to the PVS pipeline. The method was applied to 70 pediatric patients in the intensive care unit (ICU), and its efficacy was evaluated by matching high values with septic events at different time points in patient care. Septic events included systemic inflammatory response syndrome (SIRS) and suspected or proven sepsis. The comparison of maximum PVS values between the presence and absence of a septic event showed significant differences (SIRS/No SIRS: p < 0.0001, η2 = 0.54; Suspected Sepsis/No Suspected Sepsis: p = 0.00047, η2 = 0.43; Proven Sepsis/No Proven Sepsis: p = 0.0055, η2 = 0.34). A further comparison between the most severe PVS in septic patients with the PVS at ICU discharge showed even higher effect sizes (SIRS: p < 0.0001, η2 = 0.8; Suspected Sepsis: p < 0.0001, η2 = 0.8; Proven Sepsis: p = 0.002, η2 = 0.84). The PVS is emerging as a data-driven tool with the potential to assess a patient's vital status in the ICU objectively. Despite real-world data challenges and potential annotation biases, it shows promise for monitoring disease progression and treatment responses. Its adaptability to different disease markers and reliance on age-dependent reference values further broaden its application possibilities. Real-time implementation of PVS in personalized patient monitoring may be a promising way to improve critical care. However, PVS requires further research and external validation to realize its true potential.

Comparing standard screening questionnaires of canine behavior for assessment of cognitive dysfunction.

Background: Canine cognitive dysfunction (CCD) is a common, yet underdiagnosed neurodegenerative disease affecting older dogs. Treatment is most effective when started early, so identifying mild cognitive decline in the earlier stages of the disease is considered important. Hypothesis/objective: To compare the results of three different standard screening questionnaires [Canine Dementia Scale (CADES), Canine Cognitive Assessment Scale (CCAS), and Canine Cognitive Dysfunction Rating Scale (CCDR)] for CCD diagnosis. Trainability, pain sensitivity, and fear were additionally assessed with the Canine Behavioral Assessment and Research Questionnaire (C-BARQ) in order to evaluate associations between the three dementia scales and behavior. Methods: An online survey containing all the mentioned questionnaires was designed for and distributed among owners of elderly dogs. Results: Data from 597 dogs were analyzed. Overall, the scores of the three CCD questionnaires correlated well with each other, especially those of the CADES and CCAS. The CADES was more sensitive in identifying dogs with already mild to moderate cognitive impairment, while the others classified them as still undergoing normal aging. CCD scores increased for all questionnaires with age with spatial orientation being a key feature in CCD development. Trainability assessed with the C-BARQ decreased significantly with severity of CCD signs, while pain sensitivity increased. Fear and anxiety was pronounced in animals with mild but not with severe CCD. These associations based on the C-BARQ were more clearly observable in relation to CADES and CCDR than CCAS. Conclusion/clinical relevance: The choice of screening questionnaire impacts the evaluation of cognitive status and severity of CCD. Thresholds for severity classification differ significantly and may have an impact on reliable assessment. Further longitudinal studies are required to determine which of the questionnaires investigated in this study is best suited for early detection of CCD.

Refinement in Post-Operative Care for Orthopaedic Models: Implementing a Sheep Walking Cast (SWC) for Effective Tibial Fracture Management.

In the healthcare system, lower leg fractures remain relevant, incurring costs related to surgical treatment, hospitalization, and rehabilitation. The duration of treatment may vary depending on the individual case and its severity. Casting as a post-surgical fracture treatment is a common method in human and experimental veterinary medicine. Despite the high importance of sheep in preclinical testing materials for osteosynthesis, there is no standardised cast system ensuring proper stabilisation and functionality of hind limbs during the healing of tibia fractures or defects. Existing treatment approaches for tibial osteosynthesis in laboratory animal science include sling hanging, external fixators, or former Achilles tendon incision. These methods restrict animal movement for 4-6 weeks, limit species-typical behaviour, and impact social interactions. Our pilot study introduces a Standardised Walking Cast (SWC) for sheep, enabling immediate physiological movement post surgery. Seven Rhone sheep (female, 63.5 kg ± 6.45 kg) each with a single tibia defect (6 mm mechanical drilled defect) underwent SWC application for 4 weeks after plate osteosynthesis. The animals bore weight on their operated leg from day one, exhibiting slight lameness (grade 1-2 out of 5). Individual step lengths showed good uniformity (average deviation: 0.89 cm). Group housing successfully started on day three after surgery. Weekly X-rays and cast changes ensured proper placement, depicting the healing process. This study demonstrates the feasibility of using an SWC for up to 72 kg of body weight without sling hanging via ceiling mounting or external fixation techniques. Allowing species-typical movement and social behaviour can significantly improve the physiological behaviour of sheep in experiments, contributing to refinement.

Pathophysiologic Mapping of Chronic Liver Diseases With Longitudinal Multiparametric MRI in Animal Models.

OBJECTIVES: Chronic liver diseases (CLDs) have diverse etiologies. To better classify CLDs, we explored the ability of longitudinal multiparametric MRI (magnetic resonance imaging) in depicting alterations in liver morphology, inflammation, and hepatocyte and macrophage activity in murine high-fat diet (HFD)- and carbon tetrachloride (CCl4)-induced CLD models. MATERIALS AND METHODS: Mice were either untreated, fed an HFD for 24 weeks, or injected with CCl4 for 8 weeks. Longitudinal multiparametric MRI was performed every 4 weeks using a 7 T MRI scanner, including T1/T2 relaxometry, morphological T1/T2-weighted imaging, and fat-selective imaging. Diffusion-weighted imaging was applied to assess fibrotic remodeling and T1-weighted and T2*-weighted dynamic contrast-enhanced MRI and dynamic susceptibility contrast MRI using gadoxetic acid and ferucarbotran to target hepatocytes and the mononuclear phagocyte system, respectively. Imaging data were associated with histopathological and serological analyses. Principal component analysis and clustering were used to reveal underlying disease patterns. RESULTS: The MRI parameters significantly correlated with histologically confirmed steatosis, fibrosis, and liver damage, with varying importance. No single MRI parameter exclusively correlated with 1 pathophysiological feature, underscoring the necessity for using parameter patterns. Clustering revealed early-stage, model-specific patterns. Although the HFD model exhibited pronounced liver fat content and fibrosis, the CCl4 model indicated reduced liver fat content and impaired hepatocyte and macrophage function. In both models, MRI biomarkers of inflammation were elevated. CONCLUSIONS: Multiparametric MRI patterns can be assigned to pathophysiological processes and used for murine CLD classification and progression tracking. These MRI biomarker patterns can directly be explored clinically to improve early CLD detection and differentiation and to refine treatments.

Systematic review and meta-analysis of the effect of fecal microbiota transplantation on behavior in animals.

The bi-directional interaction between gut microbiota and the central nervous system has been coined the gut microbiota-brain axis. Fecal microbiota transplantation (FMT) is the administration of a solution of fecal matter from a donor into the intestinal tract of a recipient. Preclinical FMT experiments are essential to prove causality in the context of the gut microbiota-brain axis. In this systematic review, we assess the body of evidence related to the ability of FMT to modulate an animal's behavior. Accordingly, we provide a detailed summary of the use of FMT in behavior-related animal studies, an extensive risk of bias analysis, and a meta-analysis of the overall effect of FMT on behavioral outcome measures in 64 studies, representing 4889 animals. The resulting meta-analysis revealed FMT was effective at changing animal behavior, thereby substantiating evidence for the gut microbiota-brain axis. However, our study also highlights an urgent need for methodological safeguards within this research field to reduce the risk of bias and improve the internal validity of future studies.

Urinary neurotransmitter analysis and canine behavior assessment.

Behavioral problems are highly prevalent in domestic dogs, negatively affecting the quality of life of dogs and their owners. In humans and dogs, neuropsychological or neurobehavioral disorders can be associated with deviations in various neurotransmitter systems. Previous evidence has revealed correlations between urinary neurotransmitters and various behavioral disorders; however, a causal relationship has not yet been conclusively demonstrated. Non-invasive urinary neurotransmitter analysis may identify specific biomarkers, which enable a more differentiated assessment of canine behavioral disorders in the future and contribute to more effective neuromodulatory treatment decisions and monitoring. This approach could offer new insights into underlying pathomechanisms of canine neurobehavioral disorders. This study assessed urinary neurotransmitter levels and the descriptive behavior profile of 100 dogs using established rating scales (Canine Behavioral Assessment and Research Questionnaire, Attention Deficit Hyperactivity Disorder Rating Scale, Dog Personality Questionnaire, Canine Cognitive Dysfunction Rating Scale), and explored relationships between these variables. No correlation was found between urinary neurotransmitters and the assessed behavior profiles; however, age-, sex- and neuter-related influences were identified. The lack of correlation could be explained by the many confounding factors influencing both behavior and urinary neurotransmitter excretion, including age, sex and neuter status effects, and methodological issues e.g., low discriminatory power between anxiety and aggression in the descriptive behavior evaluation. Urinary neurotransmitter testing could not be validated as a tool for canine behavior evaluation in this study. However, reliable assessment methods with low susceptibility to human biases could be valuable in the future to support behavioral-phenotype diagnoses.

A pro B cell population forms the apex of the leukemic hierarchy in Hoxa9/Meis1-dependent AML.

Relapse is a major challenge to therapeutic success in acute myeloid leukemia (AML) and can be partly associated with heterogeneous leukemic stem cell (LSC) properties. In the murine Hoxa9/Meis1-dependent (H9M) AML model, LSC potential lies in three defined immunophenotypes, including Lin-cKit+ progenitor cells (Lin-), Gr1+CD11b+cKit+ myeloid cells, and lymphoid cells (Lym+). Previous reports demonstrated their interconversion and distinct drug sensitivities. In contrast, we here show that H9M AML is hierarchically organized. We, therefore, tracked the developmental potential of LSC phenotypes. This unexpectedly revealed a substantial fraction of Lin- LSCs that failed to regenerate Lym+ LSCs, and that harbored reduced leukemogenic potential. However, Lin- LSCs capable of producing Lym+ LSCs as well as Lym+ LSCs triggered rapid disease development suggestive of their high relapse-driving potential. Transcriptional analyses revealed that B lymphoid master regulators, including Sox4 and Bach2, correlated with Lym+ LSC development and presumably aggressive disease. Lentiviral overexpression of Sox4 and Bach2 induced dedifferentiation of H9M cells towards a lineage-negative state in vitro as the first step of lineage conversion. This work suggests that the potency to initiate a partial B lymphoid primed transcriptional program as present in infant AML correlates with aggressive disease and governs the H9M LSC hierarchy.

Establishment of the body condition score for adult female Xenopus laevis.

The assessment of animals' health and nutritional status using a Body Condition Score (BCS) has become a common and reliable tool in lab-animal science. It enables a simple, semi-objective, and non-invasive assessment (palpation of osteal prominences and subcutaneous fat tissue) in routine examination of an animal. In mammals, the BCS classification contains 5 levels: A low score describes a poor nutritional condition (BCS 1-2). A BCS of 3 to 4 is considered optimum, whereas a high score (BCS = 5) is associated with obesity. While BCS are published for most common laboratory mammals, these assessment criteria are not directly applicable to clawed frogs (Xenopus laevis) due to their intracoelomic fat body instead of subcutaneous fat tissue. Therefore, this assessment tool is still missing for Xenopus laevis. The present study aimed to establish a species-specific BCS for clawed frogs in terms of housing refinement in lab-animal facilities. Accordingly, 62 adult female Xenopus laevis were weighed and sized. Further, the body contour was defined, classified, and assigned to BCS groups. A BCS 5 was associated with a mean body weight of 193.3 g (± 27.6 g), whereas a BCS 4 ranged at 163.1 g (±16.0 g). Animals with a BCS = 3 had an average body weight of 114.7 g (±16.7 g). A BCS = 2 was determined in 3 animals (103 g, 110 g, and 111 g). One animal had a BCS = 1 (83 g), equivalent to a humane endpoint. In conclusion, individual examination using the presented visual BCS provides a quick and easy assessment of the nutritional status and overall health of adult female Xenopus laevis. Due to their ectothermic nature and the associated special metabolic situation, it can be assumed that a BCS ≥3 is to be preferred for female Xenopus laevis. In addition, BCS assessment may indicate underlying subclinical health problems that require further diagnostic investigation.

Experimenter familiarization is a crucial prerequisite for assessing behavioral outcomes and reduces stress in mice not only under chronic pain conditions.

Rodent behavior is affected by different environmental conditions. These do not only comprise experimental and housing conditions but also familiarization with the experimenter. However, specific effects on pain-related behavior and chronic pain conditions have not been examined. Therefore, we aimed to investigate the impact of different housing conditions, using individually ventilated and standard open top cages, inverted day-night cycles, and experimenter familiarization on male mice following peripheral neuropathy using the spared nerve injury (SNI) model. Using a multimodal approach, we evaluated evoked pain-related- using von Frey hair filaments, measured gait pattern with the CatWalk system, assessed anxiety- and depression-like behavior with the Elevated plus maze and tail suspension test, measured corticosterone metabolite levels in feces and utilized an integrative approach for relative-severity-assessment. Mechanical sensitivity differed between the cage systems and experimenter familiarization and was affected in both sham and SNI mice. Experimenter familiarization and an inverted day-night cycle reduced mechanical hypersensitivity in SNI and sham mice. SNI mice of the inverted day-night group displayed the slightest pronounced alterations in gait pattern in the Catwalk test. Anxiety-related behavior was only found in SNI mice of experimenter-familiarized mice compared to the sham controls. In addition, familiarization reduced the stress level measured by fecal corticosteroid metabolites caused by the pain and the behavioral tests. Although no environmental condition significantly modulated the severity in SNI mice, it influenced pain-affected phenotypes and is, therefore, crucial for designing and interpreting preclinical pain studies. Moreover, environmental conditions should be considered more in the reporting guidelines, described in more detail, and discussed as a potential influence on pain phenotypes.

Evidence-based comparative severity assessment in young and adult mice.

In animal-based research, welfare assessments are essential for ethical and legal reasons. However, accurate assessment of suffering in laboratory animals is often complicated by the multidimensional character of distress and pain and the associated affective states. The present study aimed to design and validate multidimensional composite measure schemes comprising behavioral and biochemical parameters based on a bioinformatics approach. Published data sets from induced and genetic mouse models of neurological and psychiatric disorders were subjected to a bioinformatics workflow for cross-model analyses. ROC analyses pointed to a model-specific discriminatory power of selected behavioral parameters. Principal component analyses confirmed that the composite measure schemes developed for adult or young mice provided relevant information with the level of group separation reflecting the expected severity levels. Finally, the validity of the composite measure schemes developed for adult and young mice was further confirmed by k-means-based clustering as a basis for severity classification. The classification systems allowed the allocation of individual animals to different severity levels and a direct comparison of animal groups and other models. In conclusion, the bioinformatics approach confirmed the suitability of the composite measure schemes for evidence-based comparative severity assessment in adult and young mice. In particular, we demonstrated that the composite measure schemes provide a basis for an individualized severity classification in control and experimental groups allowing direct comparison of severity levels across different induced or genetic models. An online tool (R package) is provided, allowing the application of the bioinformatics approach to severity assessment data sets regardless of the parameters or models used. This tool can also be used to validate refinement measures.

EVIDENCE FROM FATAL COVID-19 FOR TARGETING THE BRADYKININ METABOLISM-A SINGLE-CENTER COHORT STUDY.

ABSTRACT: Background: Severe progression of COVID-19 to critical illness, with pulmonary failure, multiple organ failure, and death, is driven by systemic inflammatory responses with overproduction of inflammatory cytokines. In the past years, the potential role of bradykinin, leading to inappropriate immune responses in the pathogenesis of COVID-19, has been raised in a so-called bradykinin storm. However, clinical investigations of bradykinin, its metabolite des-Arg 9 -bradykinin, or substance P, are rare or completely lacking during intensive care of COVID-19 patients. A prospective prolonged cohort study was conducted, including 44 COVID-19 patients (09/2020-02/2021, prevalent wildtype SARS-CoV-2) from the intensive care unit. Plasma levels of bradykinin, des-Arg 9 -bradykinin, and substance P were measured daily by ELISA in survivors (n = 21) and nonsurvivors (n = 23) of COVID-19 from admission until discharge or death. Results: We found significantly higher plasma levels of des-Arg 9 -bradykinin in survivors and nonsurvivors of COVID-19 compared with healthy controls. In addition, plasma des-Arg 9 -bradykinin levels were higher ( P < 0.001, effect size = 0.79) in nonsurvivors compared with survivors of COVID-19 and correlated significantly with disease worsening, and clinical parameters of inflammation, like leukocyte count, IL-6 or lactate dehydrogenase, and outcome. Consequently, compared with healthy controls, bradykinin and substance P plasma levels were significantly reduced in survivors and nonsurvivors of COVID-19. Furthermore, plasma substance P levels were significantly reduced ( P < 0.001, effect size = 0.7) in nonsurvivors compared with survivors of COVID-19, whereas plasma bradykinin levels did not significantly differ between survivors and nonsurvivors of COVID-19. Conclusion: Our data demonstrates that des-Arg 9 -bradykinin is significantly elevated in COVID-19 intensive care unit patients and is associated with disease severity, clinical inflammatory parameters, and survival. These results indicate that des-Arg 9 -bradykinin, not bradykinin, is one of the pivotal peptides of concern for the lethal COVID-19 aggravation and outcome. Further investigations are necessary to evaluate whether des-Arg 9 -bradykinin exhibits potent blood biomarker properties in COVID-19 and offer new treatment approaches.

Evidence-based severity assessment of the forced swim test in the rat.

The forced swim test (FST) is a traditional assay, which has been used for more than 40 years to assess antidepressant effects of novel drug candidates. In recent years, a debate about the test has focused on the assumption that the FST is highly aversive and burdening for the animals because of the earlier anthropomorphic interpretation and designation as a "behavioral despair test". The Directive 2010/63/EU and the German Animal Welfare law require a prospective severity classification of the planned experimental procedures. Still, an objective examination of the animals' burden in this test has not been performed yet. To fill this gap, we conducted an evidence-based severity assessment of the forced swim test in rats according to a 'standard protocol' with a water temperature of 25°C. We examined parameters representing the physiological and the affective state, and natural as well as locomotion-associated behaviors in three separate experiments to reflect as many dimensions as possible of the animal's condition in the test. Hypothermia was the only effect observed in all animals exposed to the FST when using this standard protocol. Additional adverse effects on body weight, food consumption, and fecal corticosterone metabolite concentrations occurred in response to administration of the antidepressant imipramine, which is frequently used as positive control when testing for antidepressant effects of new substances. We conclude that this version of the FST itself is less severe for the animals than assumed, and we suggest a severity classification of 'moderate' because of the acute and short-lasting effects of hypothermia. To refine the FST according to the 3Rs, we encourage confirming the predictive validity in warmer water temperatures to allow the rats to maintain physiological body temperature.

Robustness of a multivariate composite score when evaluating distress of animal models for gastrointestinal diseases.

The fundament of an evidence-based severity assessment in laboratory animal science is reliable distress parameters. Many readouts are used to evaluate and determine animal distress and the severity of experimental procedures. Therefore, we analyzed four distinct parameters like the body weight, burrowing behavior, nesting, and distress score in the four gastrointestinal animal models (pancreatic ductal adenocarcinoma (PDA), pancreatitis, CCl4 intoxication, and bile duct ligation (BDL)). Further, we determined the parameters' robustness in various experimental subgroups due to slight variations like drug treatment or telemeter implantations. We used non-parametric bootstrapping to get robust estimates and 95% confidence intervals for the experimental groups. It was found that the performance of the readout parameters is model-dependent and that the distress score is prone to experimental variation. On the other hand, we also found that burrowing and nesting can be more robust than, e.g., the body weight when evaluating PDA. However, the body weight still was highly robust in BDL, pancreatitis, and CCl4 intoxication. To address the complex nature of the multi-dimensional severity space, we used the Relative Severity Assessment (RELSA) procedure to combine multiple distress parameters into a score and mapped the subgroups and models against a defined reference set obtained by telemeter implantation. This approach allowed us to compare the severity of individual animals in the experimental subgroups using the maximum achieved severity (RELSAmax). With this, the following order of severity was found for the animal models: CCl4 < PDA ≈ Pancreatitis < BDL. Furthermore, the robustness of the RELSA procedure and outcome was externally validated with a reference set from another laboratory also obtained from telemeter implantation. Since the RELSA procedure reflects the multi-dimensional severity information and is highly robust in estimating the quantitative severity within and between models, it can be deemed a valuable tool for laboratory animal severity assessment.