Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application.

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Buprenorphine in rats: potent analgesic or trigger for fatal side effects?

With ongoing animal welfare efforts, multimodal analgesia is often recommended to implement in study protocols. Buprenorphine with very potent analgesic effect is a standard opioid for the use in this context in rats. In this study, two rat strains (LEW/NHanZtm, n = 6 and Crl:CD(SD), n = 8) underwent orthopaedic surgery and received carprofen, buprenorphine and a local anaesthetic in a multimodal setup. Crl:CD(SD) rats showed severe side effects in the first 24 h after anaesthesia, predominantly manifesting in pica-behaviour and reaching humane endpoints in two of eight animals, while LEW/NHanZtm rats showed only slight depression in the first postoperative days. In the context of improving animal welfare in experimental studies, buprenorphine is highly recommended not to be used in male Crl:CD(SD) rats and should generally be used very carefully and only if required.

RELSA-A multidimensional procedure for the comparative assessment of well-being and the quantitative determination of severity in experimental procedures.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

Measurement challenges and causes of incomplete results reporting of biomedical animal studies: Results from an interview study.

BACKGROUND: Existing evidence indicates that a significant amount of biomedical research involving animals remains unpublished. At the same time, we lack standards for measuring the extent of results reporting in animal research. Publication rates may vary significantly depending on the level of measurement such as an entire animal study, individual experiments within a study, or the number of animals used. METHODS: Drawing on semi-structured interviews with 18 experts and qualitative content analysis, we investigated challenges and opportunities for the measurement of incomplete reporting of biomedical animal research with specific reference to the German situation. We further investigate causes of incomplete reporting. RESULTS: The in-depth expert interviews revealed several reasons for why incomplete reporting in animal research is difficult to measure at all levels under the current circumstances. While precise quantification based on regulatory approval documentation is feasible at the level of entire studies, measuring incomplete reporting at the more individual experiment and animal levels presents formidable challenges. Expert-interviews further identified six drivers of incomplete reporting of results in animal research. Four of these are well documented in other fields of research: a lack of incentives to report non-positive results, pressures to 'deliver' positive results, perceptions that some data do not add value, and commercial pressures. The fifth driver, reputational concerns, appears to be far more salient in animal research than in human clinical trials. The final driver, socio-political pressures, may be unique to the field. DISCUSSION: Stakeholders in animal research should collaborate to develop a clear conceptualisation of complete reporting in animal research, facilitate valid measurements of the phenomenon, and develop incentives and rewards to overcome the causes for incomplete reporting.

Changes in Intraocular Pressure following Narcosis With Medetomidine, Midazolam, and Fentanyl in Association With Initial Intraocular Pressure in Mice.

PURPOSE: This article describes the development of decreased intraocular pressure (IOP) under general anesthesia with medetomidine, midazolam, and fentanyl in mice with normal and elevated IOP. METHODS: IOP was measured using the iCare Tonolab rebound tonometer. Twelve 3-4 months-old male and female C57BL/6J mice were randomized to a control group with physiological IOP and a high IOP group with experimentally induced ocular hypertension using tarsal injections of dexamethasone-21-acetate. For anesthesia, medetomidine and midazolam were used, subgroups additionally received fentanyl. IOP was measured every 2.5 min for 30 min. RESULTS: Control group differed with 14.89 mmHg (SEM: 0.58) significantly (p = 0.0002) from the high IOP group with initial 20.44 mmHg (SEM: 0.75). All groups showed a significant (p < 0.05) decrease in IOP under general anesthesia. There was no significant difference in IOP development and decrease between the group additionally receiving fentanyl and the group without fentanyl. The decrease in IOP was highly dependent on the initial value, with the high IOP group showing a greater decrease. After 10 min, no significant difference in IOP could be detected between the high IOP and control group. CONCLUSIONS: In mice, general anesthesia with medetomidine and midazolam leads to a declining IOP over time. Adding fentanyl to the anesthesia did not alter these effects. The decline is time-dependent and IOP-dependent.

Voluntary wheel running behaviour as a tool to assess the severity in a mouse pancreatic cancer model.

Laboratory animals frequently undergo routine experimental procedures such as handling, restraining and injections. However, as a known source of stress, these procedures potentially impact study outcome and data quality. In the present study, we, therefore, performed an evidence-based severity assessment of experimental procedures used in a pancreatic cancer model including surgical tumour induction and subsequent chemotherapeutic treatment via repeated intraperitoneal injections. Cancer cell injection into the pancreas was performed during a laparotomy under general anaesthesia. After a four-day recovery phase, mice received either drug treatment (galloflavin and metformin) or the respective vehicle substances via daily intraperitoneal injections. In addition to clinical scoring, an automated home-cage monitoring system was used to assess voluntary wheel running (VWR) behaviour as an indicator of impaired well-being. After surgery, slightly elevated clinical scores and minimal body weight reductions, but significantly decreased VWR behaviour were observed. During therapy, body weight declined in response to chemotherapy, but not after vehicle substance injection, while VWR activity was decreased in both cases. VWR behaviour differed between treatment groups and revealed altered nightly activity patterns. In summary, by monitoring VWR a high impact of repeated injections on the well-being of mice was revealed and substance effects on well-being were distinguishable. However, no differences in tumour growth between treatment groups were observed. This might be due to the severity of the procedures uncovered in this study, as exaggerated stress responses are potentially confounding factors in preclinical studies. Finally, VWR was a more sensitive indicator of impairment than clinical scoring in this model.

Web-based survey among animal researchers on publication practices and incentives for increasing publication rates.

OBJECTIVES: Publication bias, non-publication, and selective reporting of animal studies limit progress toward the 3Rs (Replacement, Reduction, and Refinement) that guide ethical animal testing, waste public resources, and result in redundant research, which collectively undermine the public's trust in scientific reliability. In this study, we aimed to 1) validate findings from a previous follow-up study by our team that examined the publication rates of animal studies from protocol to publication and 2) identify incentives for improving publication rates in animal research. METHODS: The researchers responsible for the animal proposals (n = 210) from our previous study were contacted as participants for a Web-based survey between October 2019 and April 2020. Question types varied between free text questions, answer options based on a 5-point Likert scale and closed yes/no questions. RESULTS: In total, 78 researchers responsible for 101 of 210 animal study proposals participated, yielding a response rate of 48.1%. Results showed that the publication rate increased from 67% in our follow-up study to 70%. According to a 5-point Likert scale (from 1 = "not relevant" to 5 = "extremely relevant"), the most widely accepted suggestions for increasing publication rates were "Publication costs for open access journals are fully covered by funders or universities" (mean 4.02, SD 1.01), "Performance-based allocation of intramural funds for results reporting of animal research not supporting the initial hypothesis (including preprints and repositories)" (mean 3.37, SD 1.05), and "Researchers receive more information from scientific journals that also publish non-significant results" (mean 3.30, SD 1.02). CONCLUSION: While the extent of publication and publication practices have been thoroughly investigated for clinical trials, less data is available for animal research to date. Therefore, the study contributes in complementing the picture of publication practice in animal research. Suggestions from our survey may help improve the publication rates of animal studies.