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Individual differences and a rather long-lasting reproductive cycle, as well as the relatively small number of oocytes that mature during one reproductive cycle makes it difficult to establish a cryopreserved stock of preimplantation embryos of the guineapig (Cavia porcellus) when compared with other laboratory rodents. Only a few data for superovulation protocols that can be used for routine laboratory use in guineapigs are available. However, a huge number of different strains exist for many purposes and the establishment of a frozen repository makes sense. Here, we describe the successful freezing of preimplatation embryos of the strain 2BS with a two-step freezing protocol in a freezing medium containing 1,2- propanediol as cryoprotectant. Human menopausal gonodotrophin induced superovulation in the embryo donors. [ABSTRACT FROM AUTHOR]

Abstract Background Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. Results After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. Conclusion The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

Abstract Background Cell lines represent a key tool in cancer research allowing the generation of neoplasias which resemble initial tumours in in-vivo animal models. The characterisation of early tumour development is of major interest in order to evaluate the efficacy of therapeutic agents. Magnetic resonance imaging (MRI) based in-vivo characterisation allows visualisation and characterisation of tumour development in early stages prior to manual palpation. Contrast agents for MRI such as superparamagnetic iron oxide nanoparticles (SPIOs) and manganese chloride (MnCl2) represent powerful tools for the in-vivo characterisation of early stage tumours. In this experimental study, we labelled prostate cancer cells with MnCl2 or SPIOs in vitro and used 1 T MRI for tracing labelled cells in-vitro and 7 T MRI for tracking in an in-vivo animal model. Methods Labelling of prostate cancer cells CT1258 was established in-vitro with MnCl2 and SPIOs. In-vitro detection of labelled cells in an agar phantom was carried out through 1 T MRI while in-vivo detection was performed using 7 T MRI after subcutaneous (s.c.) injection of labelled cells into NOD-Scid mice (n = 20). The animals were scanned in regular intervals until euthanization. The respective tumour volumes were analysed and corresponding tumour masses were subjected to histologic examination. Results MnCl2in-vitro labelling resulted in no significant metabolic effects on proliferation and cell vitality. In-vitro detection-limit accounted 105 cells for MnCl2 as well as for SPIOs labelling. In-vivo 7 T MRI scans allowed detection of 103 and 104 cells. In-vivo MnCl2 labelled cells were detectable from days 4–16 while SPIO labelling allowed detection until 4 days after s.c. injection. MnCl2 labelled cells were highly tumourigenic in NOD-Scid mice and the tumour volume development was characterised in a time dependent manner. The amount of injected cells correlated with tumour size development and disease progression. Histological analysis of the induced tumour masses demonstrated characteristic morphologies of prostate adenocarcinoma. Conclusions To the best of our knowledge, this is the first study reporting direct in-vitro MnCl2 labelling and 7 T based in-vivo MRI tracing of cancer cells in a model of prostate cancer. MnCl2 labelling was found to be suitable for in-vivo tracing allowing long detection periods. The labelled cells kept their highly tumourigenic potential in-vivo. Tumour volume development was visualised prior to manual palpation allowing tumour characterisation in early stages of the disease.

Abstract Background Neuropeptides may have considerable potential in the treatment of acute and chronic neurological diseases. Encapsulated genetically engineered cells have been suggested as a means for sustained local delivery of such peptides to the brain. In our experiments, we studied human mesenchymal stem cells which were transfected to produce glucagon-like peptide-1 (GLP-1). Methods Cells were packed in a water-permeable mesh bag containing 400 polymeric microcapsules, each containing 3000 cells. The mesh bags were either transplanted into the subdural space, into the brain parenchyma or into the cerebral ventricles of the cat brain. Mesh bags were explanted after two weeks, and cell viability, as well as GLP-1 concentration in the cerebrospinal fluid (CSF), was measured. Results Viability of cells did not significantly differ between the three implantation sites. However, CSF concentration of GLP-1 was significantly elevated only after ventricular transplantation with a maximum concentration of 73 pM (binding constant = 70 pM). Conclusions This study showed that ventricular cell-based delivery of soluble factors has the capability to achieve concentrations in the CSF which may become pharmacologically active. Despite the controversy about the pharmacokinetic limitations of ventricular drug delivery, there might be a niche in this for encapsulated cell biodelivery of soluble, highly biologically-effective neuropeptides of low molecular weight like GLP-1.

Objective: Regenerative medicine is in need of solid, large animal models as a link between rodents and humans to evaluate the functionality, immunogenicity, and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model, genetically close to humans and readily used worldwide in clinical research. Until now, only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Materials and Methods: Therefore, we reprogrammed bone marrow- derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV, SB431542, PD0325901, and ascorbic acid via a novel, excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4, KLF4, SOX2, C-MYC). Results: Endogenous pluripotency markers like OCT3/4, KLF4, SOX2, C-MYC, LIN28, NANOG, and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors, megakaryocytes, adipocytes, chondrocytes, and osteogenic cells. Conclusion: Thus, all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to assess the effectiveness of monocular and bilateral injections of Dexamethasone-21-acetate (Dex-21-Ac) into the murine fornix twice a week as a glucocorticoid-induced ocular hypertension model and investigated potential systemic side effects., Methods: Dex-21-Ac was administered twice weekly in three groups: bilateral injections, monocular injections, and a control group receiving the vehicle solution bilateral. After 21 days, enucleated eyes were examined using immunocytochemistry (ICC), and organ histology was performed., Results: All groups receiving Dex-21-Ac injections had a significant increase in intraocular pressure (IOP). Monocular injections also resulted in a significant increase in IOP in the fellow eye. The Dex-21-Ac-treated groups showed a bilateral increase in IOP of approximately 8 mmHg, accompanied by elevated expression of alpha smooth muscle actin and fibronectin in the anterior chamber angle. There were no significant changes in weight progression. Hepatic steatosis was observed in all Dex-21-Ac-treated animals, and some suffered from residual neuromuscular blockade under fentanyl anesthesia., Conclusion: Bilateral injections of Dex-21-Ac twice a week lead to a significant increase in daytime IOP and fibrotic changes in the trabecular meshwork. Unilateral application has a significant impact on the fellow eye. Local dexamethasone leads to notable systemic effects independent of changes in animal weight. Considering liver damage and associated influence on metabolization, hepatically eliminated injection anesthetics may lead to overdosing and are not recommended. They should be replaced by inhalation anesthesia.

Introduction: Surgical interventions in mice require appropriate pain relief to ensure animal welfare and to avoid influence of pain on research findings. Carprofen is a non-steroidal anti-inflammatory drug commonly used as an analgesic for interventions inducing mild to moderate pain in laboratory rodents. Despite its frequent use, species-specific data on pharmacokinetics (PK), side effects, and potential impact on behavioral pain indicators are limited., Methods: We determined PK and tolerability profiles of carprofen in healthy male and female C57BL/6J mice ( n = 42), administered at highest recommended doses via single subcutaneous (s.c.) injection (20 mg/kg) and oral self-administration (25 mg/kg/24 h) per drinking water (d.w.) for 5 days. Plasma concentrations were measured at various time points after the start of the treatment ( n = 6 per time point), and side effects were evaluated using a modified Irwin test battery, hematology, and histopathology. Additionally, potential interference with cage-side behaviors commonly used for pain assessment, such as the mouse grimace scale, wheel running, burrowing, nesting, and grooming activity, was investigated., Results: Maximum plasma concentrations of 133.4 ± 11.3 μg/ml were reached 1 h after single s.c. injection with an elimination half-life of 8.52 h. Intake from d.w. resulted in a steady state within 24 h after the start of the treatment with plasma levels of around 60 μg/ml over 5 days in both sexes. The medicated water was well-accepted, and increased d.w. intake was observed in the first 24 h after exposure ( p < 0.0001). The Irwin test revealed only minor influence on tested behavior and physiological functions. However, during treatment via d.w., an increase in body temperature ( p < 0.0001) was observed, as well as a reduction in voluntary wheel running activity by 49-70% in male mice. Moreover, grooming behavior was slightly affected. Hematology and histopathology were without pathological findings that could be attributed to carprofen treatment. High-dose carprofen can be considered safe and of favorable PK for both administration routes assessed in healthy C57BL/6J mice of both sexes. Further efficacy evaluation of carprofen as monoanalgesic or component of multimodal post-surgical regimens is clearly encouraged; however, the impact on behavioral markers used for pain assessment should be considered in this context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Glasenapp, Bankstahl, Bähre, Glage and Bankstahl.)

Germ-free (GF) rodents have become a valuable tool for studying the role of intestinal microbes on the host physiology. The major characteristic of GF rodents is an enlarged cecum. The accumulation of mucopolysaccharides, digestion enzymes and water in the intestinal lumen drives this phenotype. Microbial colonization normalizes the cecum size in ex-GF animals. However, whether strain genetics influences the cecal enlargement is unknown. Here we investigated the impact of mouse genetic background on the cecal size in five GF strains frequently used in biomedical research. The cecal weight of GF mice on B6 background (B6J and B6N) represented up to 20% of total body weight. GF NMRI and BALBc mice showed an intermediate phenotype of 5-10%, and those on the C3H background of up to 5%. Reduced cecal size in GF C3H mice correlated with decreased water content, increased expression of water transporters, and reduced production of acidic mucins, but was independent of the level of digestive enzymes in the lumen. In contrast, GF B6J mice with greatly enlarged cecum showed increased water content and a distinct metabolic profile characterized by altered amino acid and bile acid metabolism, and increased acidic mucin production. Together, our results show that genetic background influences the cecal enlargement by regulating the water transport, production of acidic mucins, and metabolic profiles.

The polyamine spermidine is discussed as a caloric restriction mimetic and therapeutic option for obesity and related comorbidities. This study tested oral spermidine supplementation with regard to the systemic, hepatic and pulmonary lipid metabolism under different diet conditions. Male C57BL/6 mice were fed a purified control (CD), high sucrose (HSD) or high fat (HFD) diet with (-S) or without spermidine for 30 weeks. In CD-fed mice, spermidine decreased body and adipose tissue weights and reduced hepatic lipid content. The HSD induced hepatic lipid synthesis and accumulation and hypercholesterolemia. This was not affected by spermidine supplementation, but body weight and blood glucose were lower in HSD-S compared to HSD. HFD-fed mice showed higher body and fat depot weights, prediabetes, hypercholesterolemia and severe liver steatosis, which were not altered by spermidine. Within the liver, spermidine diminished hepatic expression of lipogenic transcription factors SREBF1 and 2 under HSD and HFD and affected the expression of other lipid-related enzymes. In contrast, diet and spermidine exerted only minor effects on pulmonary parameters. Thus, oral spermidine supplementation affects lipid metabolism in a diet-dependent manner, with significant reductions in body fat and weight under physiological nutrition and positive effects on weight and blood glucose under high sucrose intake, but no impact on dietary fat-related parameters.

Mechanisms underlying tumor-promoting inflammatory processes in colitis-associated colorectal cancer (CAC) remain largely elusive. Here, we provide genetic evidence for distinct B cell-mediated immunoregulatory mechanisms that protect from chronic colitis versus CAC. We demonstrate an inherent capacity of interleukin-10 (IL-10)-producing B cells to differentiate into immunoglobulin A (IgA) plasma cells (PCs) upon Toll-like receptor (TLR) activation. Our data show that B cell-derived IL-10 is essential to limit pathogenic T helper type 1 (Th1)/Th17 T cell responses during chronic colitis, while IgA PCs derived from IL-10 + B cells are being implicated in restraining tumorigenesis during CAC. Formation of a tumor-protective intestinal environment was associated with clonal expansion of specific types of colonic IgA PCs and development of an altered microbiota that attenuated CAC. We thus propose that regulatory B cell-mediated immunomodulation entails temporal release of IL-10, which is superseded by the generation of specific IgA affecting the microbial community, thereby controlling chronic inflammation and tumorigenesis in a distinctive but interrelated manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro . After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10 -8 . Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies., Competing Interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (© 2022 The Authors.)

Primary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Ageing provokes a plethora of molecular, cellular and physiological deteriorations, including heart failure, neurodegeneration, metabolic maladaptation, telomere attrition and hair loss. Interestingly, on the molecular level, the capacity to induce autophagy, a cellular recycling and cleaning process, declines with age across a large spectrum of model organisms and is thought to be responsible for a subset of age-induced changes. Here, we show that a 6-month administration of the natural autophagy inducer spermidine in the drinking water to aged mice is sufficient to significantly attenuate distinct age-associated phenotypes. These include modulation of brain glucose metabolism, suppression of distinct cardiac inflammation parameters, decreased number of pathological sights in kidney and liver and decrease of age-induced hair loss. Interestingly, spermidine-mediated age protection was associated with decreased telomere attrition, arguing in favour of a novel cellular mechanism behind the anti-ageing effects of spermidine administration.

The zoonotic intracellular bacterium Chlamydia psittaci causes life-threatening pneumonia in humans. During mouse lung infection, complement factor C3 and the anaphylatoxin C3a augment protection against C. psittaci by a so far unknown mechanism. To clarify how complement contributes to the early, innate and the late, specific immune response and resulting protection, this study addresses the amount of C3, the timing when its presence is required as well as the anaphylatoxin receptor(s) mediating its effects and the complement-dependent migration of dendritic cells. Challenge experiments with C. psittaci on various complement KO mice were combined with transient decomplementation by pharmacological treatment, as well as the analysis of in vivo dendritic cells migration. Our findings reveal that a plasma concentration of C3 close to wildtype levels was required to achieve full protection. The diminished levels of C3 of heterozygote C3 +/- mice permitted already relative effective protection and improved survival as compared to C3 -/- mice, but overall recovery of these animals was delayed. Complement was in particular required during the first days of infection. However, additionally, it seems to support protection at later stages. Migration of CD103 + dendritic cells from the infected lung to the draining lymph node-as prerequisite of antigen presentation-depended on C3 and C3aR and/or C5aR. Our results provide unique mechanistic insight in various aspects of complement-dependent immune responses under almost identical, rather physiological experimental conditions. Our study contributes to an improved understanding of the role of complement, and C3a in particular, in infections by intracellular bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kohn, Lanfermann, Laudeley, Glage, Rheinheimer and Klos.)

Recent advances in complement research have revolutionized our understanding of its role in immune responses. The immunomodulatory features of complement in infections by intracellular pathogens, e.g., viruses, are attracting increasing attention. Thereby, local production and activation of complement by myeloid-derived cells seem to be crucial. We could recently show that C3, a key player of the complement cascade, is required for effective defense against the intracellular bacterium Chlamydia psittaci . Avian zoonotic strains of this pathogen cause life-threatening pneumonia with systemic spread in humans; closely related non-avian strains are responsible for less severe diseases of domestic animals with economic loss. To clarify how far myeloid- and non-myeloid cell-derived complement contributes to immune response and resulting protection against C. psittaci , adoptive bone marrow transfer experiments focusing on C3 were combined with challenge experiments using a non-avian (BSL 2) strain of this intracellular bacterium. Surprisingly, our data prove that for C. psittaci -induced pneumonia in mice, non-myeloid-derived, circulating/systemic C3 has a leading role in protection, in particular on the development of pathogen-specific T- and B- cell responses. In contrast, myeloid-derived and most likely locally produced C3 plays only a minor, mainly fine-tuning role. The work we present here describes authentic, although less pronounced, antigen directed immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kohn, Lanfermann, Laudeley, Glage, Rheinheimer and Klos.)

Signaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25 Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25 Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7 -/- mice. Although palmitoylation of barttin in kidneys of Zdhhc7 -/- animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7 -/- mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension., (© 2020 Gorinski et al.)

Objectives: The aim of the present study was to establish a rodent peri-implantitis model induced by a mixed bacterial infection characterized by bone loss and semi-quantitative graduation of peri-implant inflammation in histological sections., Materials and Methods: Two titanium implants were implanted in Sprague-Dawley rats, bilaterally in each maxilla. After 3 weeks healing, the rats were randomized into three groups according to different treatments over the next 3 months: Antibiotic-Group with oral lavage of antibiotics; Bacteria-Group with oral lavage of Streptococcus oralis and Aggregatibacter actinomycetemcomitans; and Untreated Group with standard housing and no additional treatment. Maxillae were dissected to perform microscopic and histological analysis of bone height and peri-implant tissues., Results: The bone level, measured at one implant site per animal, in the Bacteria-Group (2.60 ± 0.39 mm) was significantly reduced compared to the Antibiotic-Group (2.29 ± 0.32 mm) after 3 months. The differences of bone height in the Bacteria-Group and the Untreated Group (2.46 ± 0.27 mm) did not reach statistical significance. The inflammatory response with respect to the number of inflammatory cells and fibrous tissue compartments of the peri-implant tissues in the Bacteria-Group was significantly increased compared with the Antibiotic-Group (p < .05). S. oralis and A. actinomycetemcomitans DNAs were detected in the Bacteria-Group., Conclusions: This rat model of peri-implantitis used oral bacterial lavage for the induction of an inflammatory host response and bone loss. Additional bacterial treatment enhances the peri-implant phenotype, so that significant differences to a reduced bacterial load similar to the human peri-implantitis disease can be identified., (© 2019 The Authors. Clinical Oral Implants Research published by John Wiley & Sons Ltd.)

Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1 -/- mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-publication and publication bias in animal research is a core topic in current debates on the "reproducibility crisis" and "failure rates in clinical research". To date, however, we lack reliable evidence on the extent of non-publication in animal research. We collected a random and stratified sample (n = 210) from all archived animal study protocols of two major German UMCs (university medical centres) and tracked their results publication. The overall publication rate was 67%. Excluding doctoral theses as results publications, the publication rate decreased to 58%. We did not find substantial differences in publication rates with regard to i) the year of animal study approval, ii) the two UMCs, iii) the animal type (rodents vs. non-rodents), iv) the scope of research (basic vs. preclinical), or v) the discipline of the applicant. Via the most reliable assessment strategy currently available, our study confirms that the non-publication of results from animal studies conducted at UMCs is relatively common. The non-publication of 33% of all animal studies is problematic for the following reasons: A) the primary legitimation of animal research, which is the intended knowledge gain for the wider scientific community, B) the waste of public resources, C) the unnecessary repetition of animal studies, and D) incomplete and potentially biased preclinical evidence for decision making on launching early human trials. Results dissemination should become a professional standard for animal research. Academic institutions and research funders should develop effective policies in this regard., Competing Interests: The authors have declared that no competing interests exist.

Disease activity in Interleukin-10-deficient (Il10 -/- ) mice, a model for IBD, depends on genetic background and microbiome composition. B6.129P2/JZtm-Il10 tm1Cgn (B6-Il10 -/- ) mice are partially resistant to colitis, whereas mice carrying the Cdcs1 C3Bir haplotype on chromosome 3, B6.Cg-Il10 tm1Cgn MMU3(D3Mit11-D3Mit348)/JZtm (BC-R3-Il10 -/- ), are susceptible. This study was performed to clarify Cdcs1 and candidate gene effects on the colitogenic potential of hematopoietic cells using bone marrow (BM) and T-cell transfer models. Acute and chronic graft versus host reaction was excluded by high-density genotyping, in vitro and in vivo approaches. BM-chimeras were created with animals housed in two barriers (I and II) with distinct microbiota composition as identified by sequencing. BM-chimeras of all groups developed comparable moderate-to-severe colitis in Barrier I, however, in Barrier II only recipients of BC-R3-Il10 -/- BM. Subsequent adoptive T cell transfers pointed to a new subcongenic interval within Cdcs1 affecting their colitogenic potential. Transfers excluded Larp7 and Alpk1 but highlighted Ifi44 as potential candidate genes. In this model-system, colitis development after cell transfer heavily depends on microbiome, though Cdcs1 acts mainly independently in hematopoietic cells. A new subcongenic interval, provisionally named Cdcs1.4, modifies colitogenic T cell function. Within this locus, Ifi44 represents an important candidate gene for colitis expression.

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using mb1 -Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using Cd19 -Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in mice.

Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro applications and their in vivo transfection efficiency is still low compared with viral vectors. Here, we show that peptides developed by modular design approaches can spontaneously form compact and monodisperse nanoparticles with poloxamines and nucleic acids via self-assembly. Both messenger RNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles are greatly boosted in vitro and in the lungs of cystic fibrosis mice with negligible toxicity. Peptide-poloxamine nanoparticles containing integrating vectors enable successful in vitro and in vivo long-term restoration of cystic fibrosis transmembrane conductance regulator deficiency with a safe integration profile. Our dataset provides a new framework for designing non-viral gene delivery systems qualified for in vivo genetic modifications.

The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45 + CD11b + CD14 + CD163 + cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.