Evidence-Based Severity Assessment of Animal Models for Pancreatic Cancer.

Animal models are crucial to preclinical oncological research and drug development. Animal experiments must be performed in accordance with the 3R principles of replacement and reduction, if possible, and refinement where these procedures remain crucial. In addition, European Union legislations demand a continuous refinement approach, as well as pro- and retrospective severity assessment. In this study, an objective databased severity assessment was performed in murine models for pancreatic cancer induced by orthotopic, subcutaneous, or intravenous injection of Panc02 cells. Parameters such as body weight change, distress score, perianal temperature, mouse grimace scale, burrowing, nesting behavior, and the concentration of corticosterone in plasma and its metabolites in feces were monitored during tumor progression. The most important parameters were combined into a score and mapped against a reference data set by the Relative Severity Assessment procedure (RELSA) to obtain the maximum achieved severity for each animal (RELSAmax). This scoring revealed a significantly higher RELSAmax for the orthotopic model than for the subcutaneous and intravenous models. However, compared to animal models such as pancreatitis and bile duct ligation, the pancreatic cancer models are shown to be less severe. Data-based animal welfare assessment proved to be a valuable tool for comparing the severity of differently induced cancer models.

Exploring dose-response variability and relative severity assessment in STZ-induced diabetes male NSG mice.

NSG mice are among the most immunodeficient mouse model being used in various scientific branches. In diabetelogical research diabetic NSG mice are an important asset as a xenotransplantation model for human pancreatic islets or pluripotent stem cell-derived islets. The treatment with the beta cell toxin streptozotocin is the standard procedure for triggering a chemically induced diabetes. Surprisingly, little data has been published about the reproducibility, stress and animal suffering in these NSG mice during diabetes induction. The 3R rules, however, are a constant reminder that existing methods can be further refined to minimize suffering. In this pilot study the dose-response relationship of STZ in male NSG mice was investigated and additionally animal suffering was charted by applying the novel 'Relative Severity Assessment' algorithm. By this we successfully explored an STZ dose that reliably induced diabetes while reduced stress and pain to the animals to a minimum using evidence-based and objective parameters rather than criteria that might be influenced by human bias.

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.

PD-1/CTLA-4 Blockade Leads to Expansion of CD8+PD-1int TILs and Results in Tumor Remission in Experimental Liver Cancer.

Background: Checkpoint inhibitors act on exhausted CD8+ T cells and restore their effector function in chronic infections and cancer. The underlying mechanisms of action appear to differ between different types of cancer and are not yet fully understood. Methods: Here, we established a new orthotopic HCC model to study the effects of checkpoint blockade on exhausted CD8+ tumor-infiltrating lymphocytes (TILs). The tumors expressed endogenous levels of HA, which allowed the study of tumor-specific T cells. Results: The induced tumors developed an immune-resistant TME in which few T cells were found. The few recovered CD8+ TILs were mostly terminally exhausted and expressed high levels of PD-1. PD-1/CTLA-4 blockade resulted in a strong increase in the number of CD8+ TILs expressing intermediate amounts of PD-1, also called progenitor-exhausted CD8+ TILs, while terminally exhausted CD8+ TILs were almost absent in the tumors of treated mice. Although transferred naïve tumor-specific T cells did not expand in the tumors of untreated mice, they expanded strongly after treatment and generated progenitor-exhausted but not terminally exhausted CD8+ TILs. Unexpectedly, progenitor-exhausted CD8+ TILs mediated the antitumor response after treatment with minimal changes in their transcriptional profile. Conclusion: In our model, few doses of checkpoint inhibitors during the priming of transferred CD8+ tumor-specific T cells were sufficient to induce tumor remission. Therefore, PD-1/CTLA-4 blockade has an ameliorative effect on the expansion of recently primed CD8+ T cells while preventing their development into terminally exhausted CD8+ TILs in the TME. This finding could have important implications for future T-cell therapies.

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Results: Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. Discussion: The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

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.

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.

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.

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.

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.

In vivo adenine base editing reverts C282Y and improves iron metabolism in hemochromatosis mice.

Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane. Consequently, the lack of interaction with the transferrin receptors 1 and 2 leads to systemic iron overload. We screened potential gRNAs in a highly precise cell culture assay and applied an AAV8 split-vector expressing the adenine base editor ABE7.10 and our candidate gRNA in 129-Hfetm.1.1Nca mice. Here we show that a single injection of our therapeutic vector leads to a gene correction rate of >10% and improved iron metabolism in the liver. Our study presents a proof-of-concept for a targeted gene correction therapy for one of the most frequent hereditary diseases affecting humans.

Burrowing behaviour of rats: Strain differences and applicability as well-being parameter after intracranial surgery.

In mice, burrowing is considered a species-typical parameter for assessing well-being, while this is less clear in rats. This exploratory study evaluated burrowing behaviour in three rat strains during training and in the direct postoperative phase after complex intracranial surgery in different neuroscience rat models established at Hannover Medical School or Aachen University Hospital. Male Crl:CD (SD; n = 18), BDIX/UlmHanZtm (BDIX; n = 8) and RjHan:WI (Wistar; n = 35) rats were individually trained to burrow gravel out of a tube on four consecutive days. Thereafter, BDIX rats were subjected to intracranial injection of BT4Ca cells and tumour resection (rat glioma model), SD rats to injection of 6-hydroxydopamine (6-OHDA) or vehicle (rat Parkinson's disease model) and Wistar rats to endovascular perforation or sham surgery (rat subarachnoid haemorrhage (SAH) model). Burrowing was retested on the day after surgery. During training, BDIX rats burrowed large amounts (mean of 2370 g on the fourth day), while SD and Wistar rats burrowed less gravel (means of 846 and 520 g, respectively). Burrowing increased significantly during training only in Wistar rats. Complex surgery, that is, tumour resection (BDIX), 6-OHDA injection (SD) and endovascular perforation or sham surgery for SAH (Wistar) significantly reduced burrowing and body weight, while simple stereotactic injection of tumour cells or vehicle did not affect burrowing. Despite the training, burrowing differed between the strains. In the direct postoperative phase, burrowing was reduced after complex surgery, indicating reduced well-being. Reduced burrowing was accompanied with postoperative weight loss, a validated and recognised quantitative measure for severity assessment.

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.

In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution.

Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, "DR4"), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1null/IL2Rγnull (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4+ T cells, and promoted significantly higher development of IgG+ and IgA+ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

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.

Lidocaine and bupivacaine as part of multimodal pain management in a C57BL/6J laparotomy mouse model.

While the use of local anesthesia as part of multimodal pain management is common practice in human and veterinarian surgery, these drugs are not applied routinely in rodent surgery. Several recommendations on the use of local anesthesia exist, but systematic studies on their efficacy and side effects are lacking. In the present study, male and female C57BL/6J mice were subjected to a sham vasectomy or a sham embryo transfer, respectively. We tested whether a mixture of subcutaneously injected Lidocaine and Bupivacaine in combination with systemic Paracetamol applied via drinking water results in superior pain relief when compared to treatment with local anesthesia or Paracetamol alone. We applied a combination of methods to assess behavioral, emotional, and physiological changes indicative of pain. Voluntary Paracetamol intake via drinking water reached the target dosage of 200 mg/kg in most animals. Local anesthesia did not lead to obvious side effects such as irregular wound healing or systemic disorders. No relevant sex differences were detected in our study. Sevoflurane anesthesia and surgery affected physiological and behavioral measurements. Surprisingly, Paracetamol treatment alone significantly increased the Mouse Grimace Scale. Taken together, mice treated with a combination of local anesthesia and systemic analgesia did not show fewer signs of post-surgical pain or improved recovery compared to animals treated with either local anesthesia or Paracetamol.

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.

Inhibition of Na+ /H+ exchanger isoform 3 improves gut fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator-deficient and F508del mutant mice.

BACKGROUND AND PURPOSE: Constipation and intestinal obstructive episodes are major health problems in cystic fibrosis (CF) patients. Three FDA-approved drugs against constipation-prone irritable bowel syndrome were tested for their ability to increase luminal fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator (CFTR) null (cftr-/- ) and F508del mutant (F508delmut/mut ) murine intestine. EXPERIMENTAL APPROACH: Guanylate cyclase C agonist linaclotide, PGE1 analogue lubiprostone and intestine-specific NHE3 inhibitor tenapanor were perfused through a ~3 cm jejunal, proximal or mid-distal colonic segment in anaesthetized cftr-/- , F508delmut/mut and WT mice. Net fluid balance was determined gravimetrically and alkaline output by pH-stat back titration. KEY RESULTS: Basal jejunal fluid absorptive rates were significantly higher and basal HCO3- output was significantly lower in cftr-/- and F508delmut/mut compared to WT mice. In cftr-/- and F508delmut/mut mice, all three drugs significantly inhibited the fluid absorptive rate and increased alkaline output in the jejunum and tenapanor and lubiprostone, but not linaclotide, in the colon. After tenapanor pre-incubation, linaclotide elicited a robust fluid secretory response in WT jejunum, while no further change in absorptive rates was observed in cftr-/- and F508delmut/mut jejunum, suggesting that the increase in gut fluidity and alkalinity by linaclotide in CF gut is mediated via NHE3 inhibition. Lubiprostone also inhibited fluid absorption in cftr-/- and F508delmut/mut jejunum via NHE3 inhibition but had a residual NHE3-independent effect. CONCLUSION AND IMPLICATIONS: Linaclotide, lubiprostone and tenapanor reduced fluid absorption and increased alkaline output in the CF gut. Their application may ameliorate constipation and reduce obstructive episodes in CF patients.

Severity assessment in mice subjected to carbon tetrachloride.

The Directive 2010/63 EU requires classifying burden and severity in all procedures using laboratory animals. This study evaluated the severity of liver fibrosis induction by intraperitoneal carbon tetrachloride (CCl4) injections in mice. 29 male C57BL/6N mice were treated three times per week for 4 weeks with an intraperitoneal injection (50 µl) of either 0.6 ml/kg body weight CCl4-vehicle solution, germ oil (vehicle-control) or handling only. Severity assessment was performed using serum analysis, behavioral tests (open field test, rotarod, burrowing and nesting behavior), fecal corticosterone metabolite (FCM) measurement, and survival. The most significant group differences were noticed in the second week of treatment when the highest AST (1463 ± 1404 vs. 123.8 ± 93 U/L, p < 0.0001) and nesting values were measured. In addition, respective animals showed lower moving distances (4622 ± 1577 vs. 6157 ± 2060 cm, p < 0.01) and velocity in the Open field, identified as main factors in principal component analysis (PCA). Overall, a 50% survival rate was observed within the treatment group, in which the open field performance was a good tracer parameter for survival. In summary, this study demonstrates the feasibility of assessing severity in mice using behavioral tests and highlight the open field test as a possible threshold parameter for risk assessment of mortality.