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A prevalence of cigarette smoking can cause the accumulation of cadmium (Cd2+) in the lungs, kidneys, and blood. The effects of exposure can cause multiple chronic disease types to emerge in the affected organ systems. The only moderately effective therapeutic option is chelation therapy; the health risks associated with this therapy have caused much criticism. The disease types associated with Cd2+ toxicity have inflammatory components and greatly impact innate immunity. These factors are affected at the cellular level and cause pathways like apoptosis, pyroptosis, and necroptosis. A development in understanding these pathways stipulates that these three pathways act as one complex of pathways, known together as PANoptosis. The inflammatory mechanisms of PANoptosis are particularly interesting in Cd2+ toxicity due to its inflammatory effects. Proteins in the gasdermin family act to release inflammatory cytokines, like interleukin-1β, into the extracellular environment. Cytokines cause inflammatory disease pathologies like fibrosis and cancer. RAW 264.7 monocytes are key in the murine immune system and provide an excellent model to investigate Cd2+ toxicity. Exposure of 0–15 µM CdCl2 was sufficient to increase expression of cleaved gasdermin D (GSDMD) and gasdermin E (GSDME) in this cell type. Cd2+ also exhibits a dose–dependent cytotoxicity in this cell type. [ABSTRACT FROM AUTHOR]

In Europe and North America, the prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing. Most pulmonary NTM infections are caused by the Mycobacterium avium complex (MAC). Sadly, the treatment of pulmonary MAC is suboptimal with failure rates ranging from 37% to 58%. Therefore, there is a need to develop new therapeutics. Developing new immunotherapies and studying their interaction with standard or new drugs requires reliable assays. Four different assays including CFSE-based flow cytometry, in vitro protection assays, IFN-γ ELISPOT, and murine infection models were optimized using a reference strain of MAC (ATCC 700898) to help with the development of immunotherapies for MAC. Expansion of proliferating and IFN-γ producing human T cells is optimal after 7 days of stimulation with MAC at a multiplicity of infection (MOI) of 0.1, achieving a stimulation index of 26.5 ± 11.6 (mean ± SE). The in vitro protection assay for MAC works best by co-culturing T cells expanded for 7 days with MAC (MOI 1)-infected autologous macrophages. Aerosol MAC infection of mice allows measurement of the effects of the BCG vaccine and clarithromycin. IFN-γ ELISPOT assays with live MAC (MOI 3) stimulation of splenocytes from mice immunized with BCG help identify differences between unimmunized mice and mice immunized with BCG. In conclusion, multiple assays are available for use to identify MAC-specific effector T cells, which will help in the development of new therapeutics or vaccines against pulmonary MAC. [ABSTRACT FROM AUTHOR]

Adrenocortical carcinoma (ACC) and pheochromocytoma (PCC) are malignancies originating from distinct layers of the adrenal gland. ACCs arise from the adrenal cortex, are often detected at advanced stages and are associated with poor prognosis. PCCs are mostly benign, arise from the adrenal medulla and have a variable prognosis, with 10% of PCCs resulting in metastasis. Genetic background strongly influences metastasis of PCCs, and no reliable biomarkers that predict metastatic behavior exist to date. Current therapeutic strategies for both ACCs and PCCs are overall limited. Thus, novel preclinical models and drug screening approaches need to be established to aid in the identification of more promising drugs and treatment schemes. In this review, we summarize the currently available human and murine cell lines for both tumor entities. [ABSTRACT FROM AUTHOR]

Hepatic ischemia–reperfusion injury (IRI) is a major cause of postoperative hepatic dysfunction and liver failure involving cellular damage to previously ischemic tissues to which blood flow is restored. The reestablishment of blood flow is essential for salvaging ischemic tissues. The reperfusion itself, however, can paradoxically lead to further cellular damage, which involves a multi-factorial process resulting in extensive tissue damage, which can threaten the function and viability of the liver and other organ systems. The following review outlines multiple models for in-lab analysis of the various hepatic IRI mechanisms, including murine, porcine, cell lines, and machine perfusion models. [ABSTRACT FROM AUTHOR]

Background: Ischemic post-conditioning (IPoC) has been shown to improve outcomes in limited pre-clinical models. As down-time is often unknown, this technique needs to be investigated over a range of scenarios. As this tool limits reperfusion injury, there may be limited benefit or even harm after short arrest and limited ischemia-reperfusion injury. Methods: Eighteen male Wistar rats underwent 7 min of asphyxial arrest. Animals randomized to IPoC received a 20 s pause followed by 20 s of compressions, repeated four times, initiated 40 s into cardiopulmonary resuscitation. If return of spontaneous circulation (ROSC) was achieved, epinephrine was titrated to mean arterial pressure (MAP) of 70 mmHg. Data were analyzed using t-test or Mann–Whitney test. Significance set at p ≤ 0.05. Results: The rate of ROSC was equivalent in both groups, 88%. There was no statistically significant difference in time to ROSC, epinephrine required post ROSC, carotid flow, or peak lactate at any timepoint. There was a significantly elevated MAP with IPoC, 90.7 mmHg (SD 13.9), as compared to standard CPR, 76.7 mmHg (8.5), 2 h after ROSC, p = 0.03. Conclusions: IPoC demonstrated no harm in a model of short arrest using a new arrest etiology for CPR based IPoC intervention in a rat model. [ABSTRACT FROM AUTHOR]

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2. [ABSTRACT FROM AUTHOR]

Normalizing RT-qPCR miRNA datasets that encompass numerous preimplantation embryo stages requires the identification of miRNAs that may be used as stable reference genes. A need has also arisen for the normalization of the accompanying conditioned culture media as extracellular miRNAs may serve as biomarkers of embryo developmental competence. Here, we evaluate the stability of six commonly used miRNA normalization candidates, as well as small nuclear U6, using five different means of evaluation (BestKeeper, NormFinder, geNorm, the comparative Delta Ct method and RefFinder comprehensive analysis) to assess their stability throughout murine preimplantation embryo development from the oocyte to the late blastocyst stages, both in whole embryos and the associated conditioned culture media. In descending order of effectiveness, miR-16, miR-191 and miR-106 were identified as the most stable individual reference miRNAs for developing whole CD1 murine preimplantation embryos, while miR-16, miR-106 and miR-103 were ideal for the conditioned culture media. Notably, the widely used U6 reference was among the least appropriate for normalizing both whole embryo and conditioned media miRNA datasets. Incorporating multiple reference miRNAs into the normalization basis via a geometric mean was deemed beneficial, and combinations of each set of stable miRNAs are further recommended, pending validation on a per experiment basis. [ABSTRACT FROM AUTHOR]

Reperfusion after ischemia causes additional cellular damage, known as reperfusion injury, for which there is still no effective remedy. Poloxamer (P)188, a tri-block copolymer-based cell membrane stabilizer (CCMS), has been shown to provide protection against hypoxia/reoxygenation (HR) injury in various models by reducing membrane leakage and apoptosis and improving mitochondrial function. Interestingly, substituting one of its hydrophilic poly-ethylene oxide (PEO) blocks with a (t)ert-butyl terminus added to the hydrophobic poly-propylene oxide (PPO) block yields a di-block compound (PEO-PPOt) that interacts better with the cell membrane lipid bi-layer and exhibits greater cellular protection than the gold standard tri-block P188 (PEO75-PPO30-PEO75). For this study, we custom-made three different new di-blocks (PEO113-PPO10t, PEO226-PPO18t and PEO113-PPO20t) to systemically examine the effects of the length of each polymer block on cellular protection in comparison to P188. Cellular protection was assessed by cell viability, lactate dehydrogenase release, and uptake of FM1-43 in mouse artery endothelial cells (ECs) following HR injury. We found that di-block CCMS were able to provide the same or better EC protection than P188. Our study provides the first direct evidence that custom-made di-block CCMS can be superior to P188 in improving EC membrane protection, raising their potential in treating cardiac reperfusion injury. [ABSTRACT FROM AUTHOR]

Teeth with different chemical compositions can show vastly different physical properties, so knowledge of elemental composition is required to use animal teeth as substitutes for human teeth in research. In vitro, energy dispersive X-ray spectroscopy (EDX), improved by calibration standards and Si3N4-window material, enables determining local elemental compositions of inorganic and organic compounds without sample destruction. Six human molars, bovine incisors, murine incisors, and murine molars were analyzed. EDX-field scans and line scans were analyzed for elements Ca, P, O, C, N, F, Na, Mg, Fe, Cl, and S. Furthermore, Ca/P- and Ca/N-Ratios were calculated. The presence of iron in murine incisor enamel was investigated using additional wavelength dispersive X-ray spectroscopy measurements (WDX) near the enamel surface. Bovine and human enamel and dentin revealed close similarities regarding elemental composition. The median (25–75% percentiles) of At%Ca was 21.1 (20.8–21.3) in human enamel, 21.0 (20.7–21.2) in bovine enamel, and in murine enamel, 18.3 (17.85–18.88) for molars and 18.35 (18.00–18.75) for incisors. In dentin, murine teeth revealed a higher At%Ca compared to human and bovine teeth. Significant differences between human and bovine teeth were found for nitrogen in dentin, with a median of 4.5 (3.3–5) At%N for human dentin and 2.7 (2.3–3.2) At%N for bovine dentin. The Ca/P-Ratio was the highest in human and bovine enamel, which did not differ significantly. Enamel from murine molars had a higher Ca/P-Ratio than murine incisors and the highest Ca/P-Ratio in dentin was observed for human teeth and murine molars (1.49). WDX revealed iron in the outer 10 µm of pre- and post-eruptive enamel of murine incisors. Pre- and post-eruptive enamel on murine incisors only differed significantly in At%Ca (p = 0.041) and At%P (p = 0.026) with both At% higher in the pre-eruptive enamel. Murine teeth differ significantly from human and bovine teeth in terms of the elemental composition of enamel and dentin. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinomas (OSCC) remain a major healthcare burden in Asian countries. In Pakistan alone, it is the most common cancer in males and second only to breast cancer in females. Alarmingly, treatment options for OSCC remain limited. With this context, investigations made to explore the inflammatory milieu of OSCC become highly relevant, with the hope of practicing immunotherapeutic approaches to address this highly prevalent tumor. We investigated the newly identified innate lymphoid cells (ILCs) and associated cytokines in well-defined human oral squamous cell carcinoma (OSCC) as well as in a 7,12-dimethylbenz[a]anthracene (DMBA)-induced murine model of OSCC using flow cytometry and quantitative real-time polymerase chain reaction (qPCR). We further went on to explore molecular circuitry involved in OSCC by developing a murine model of OSCC and using an α-Thy1 antibody to inhibit ILCs. Amongst the ILCs that we found in human OSCC, ILC3 (23%) was the most abundant, followed by ILC2 (17%) and ILC1 (1%). Mice were divided into four groups: DMBA (n = 33), DMBA+antibody (Ab) (n = 30), acetone (n = 5), and control (n = 5). In murine OSCC tissues, ILC1 and ILC3 were down-infiltrated, while ILC2 remained unchanged compared to controls. Interestingly, compared to the controls (DMBA group), mice treated with the α-Thy1 antibody showed fewer numbers of large tumors, and a larger percentage of these mice were tumor-free at this study's end point. We present novel data on the differential expansion/downsizing of ILCs in OSCC, which provides a pivotal basis to dive deeper into molecular circuitry and the OSCC tumor niche to devise novel diagnostic, therapeutic, and prognostic strategies to prevent/treat oral cancers. [ABSTRACT FROM AUTHOR]

Simple Summary: We developed a new labeled mouse cell line to study ovarian cancer. The STOSE mouse cell line was engineered to express a luciferase label to enable real-time tumor monitoring by imaging. We characterized the growth of the cell line in mice and identified the immune cells within the formed tumors. We treated the mice with immunotherapy, which had no effect on tumor growth. Overall, the STOSE.M1 luc model closely resembles ovarian cancer in humans and will further aid in our understanding and treatment of this disease. Despite advances in surgery and targeted therapies, the prognosis for women with high-grade serous ovarian cancer remains poor. Moreover, unlike other cancers, immunotherapy has minimally impacted outcomes in patients with ovarian cancer. Progress in this regard has been hindered by the lack of relevant syngeneic ovarian cancer models to study tumor immunity and evaluate immunotherapies. To address this problem, we developed a luciferase labeled murine model of high-grade serous ovarian cancer, STOSE.M1 luc. We defined its growth characteristics, immune cell repertoire, and response to anti PD-L1 immunotherapy. As with human ovarian cancer, we demonstrated that this model is poorly sensitive to immune checkpoint modulators. By developing the STOSE.M1 luc model, it will be possible to probe the mechanisms underlying resistance to immunotherapies and evaluate new therapeutic approaches to treat ovarian cancer. [ABSTRACT FROM AUTHOR]

The Brassicaceae family constitutes some of the most well-studied natural products in the world, due to their anti-inflammatory, anti-oxidative, and pro-regenerative properties as well as their ubiquitous distribution across the world. To evaluate the potential efficacy of the Brassicaceae family in the treatment of inflammatory skin disorders and wounds, based on preclinical evidence from in vivo and in vitro studies. This systematic review was performed according to the PRISMA guidelines, using a structured search on the PubMed-Medline, Scopus, and Web of Science platforms. The studies included were those that used murine models and in vitro studies to investigate the effect of Brassicaceae on skin disorders. Bias analysis and methodological quality assessments were examined through SYRCLE's RoB tool. Brassicaceae have shown positive impacts on inflammatory regulation of the skin, accelerating the wound healing process, and inhibiting the development of edema. The studies showed that the Brassicaceae family has antioxidant activity and effects on the modulation of cyclooxygenase 2 and the nuclear factor kappa β (NFκβ) pathway. The secondary metabolites present in Brassicas are polyphenols (68.75%; n = 11), terpenes/carotenoids (31.25%; n = 5), and glycosylates (25%; n = 4), which are responsible for their anti-inflammatory, healing, and antioxidant effects. In addition, the current evidence is reliable because the bias analysis showed a low risk of bias. Our review indicates that compounds derived from Brassicaceae present exceptional potential to treat inflammatory skin diseases and accelerate cutaneous wound healing. We hope that our critical analysis can help to expedite clinical research and to reduce methodological bias, thereby improving the quality of evidence in future research. The registration number on the Prospero platform is CRD42021262953. [ABSTRACT FROM AUTHOR]

Purpose: Combined radiotherapy (RT) and immune checkpoint-inhibitor (ICI) therapy can act synergistically to enhance tumor response beyond what either treatment can achieve alone. Alongside the revolutionary impact of ICIs on cancer therapy, life-threatening potential side effects, such as checkpoint-inhibitor-induced (CIP) pneumonitis, remain underreported and unpredictable. In this preclinical study, we hypothesized that routinely collected data such as imaging, blood counts, and blood cytokine levels can be utilized to build a model that predicts lung inflammation associated with combined RT/ICI therapy. Materials and Methods: This proof-of-concept investigational work was performed on Lewis lung carcinoma in a syngeneic murine model. Nineteen mice were used, four as untreated controls and the rest subjected to RT/ICI therapy. Tumors were implanted subcutaneously in both flanks and upon reaching volumes of ~200 mm3 the animals were imaged with both CT and MRI and blood was collected. Quantitative radiomics features were extracted from imaging of both lungs. The animals then received RT to the right flank tumor only with a regimen of three 8 Gy fractions (one fraction per day over 3 days) with PD-1 inhibitor administration delivered intraperitoneally after each daily RT fraction. Tumor volume evolution was followed until tumors reached the maximum size allowed by the Institutional Animal Care and Use Committee (IACUC). The animals were sacrificed, and lung tissues harvested for immunohistochemistry evaluation. Tissue biomarkers of lung inflammation (CD45) were tallied, and binary logistic regression analyses were performed to create models predictive of lung inflammation, incorporating pretreatment CT/MRI radiomics, blood counts, and blood cytokines. Results: The treated animal cohort was dichotomized by the median value of CD45 infiltration in the lungs. Four pretreatment radiomics features (3 CT features and 1 MRI feature) together with pre-treatment neutrophil-to-lymphocyte (NLR) ratio and pre-treatment granulocyte-macrophage colony-stimulating factor (GM-CSF) level correlated with dichotomized CD45 infiltration. Predictive models were created by combining radiomics with NLR and GM-CSF. Receiver operating characteristic (ROC) analyses of two-fold internal cross-validation indicated that the predictive model incorporating MR radiomics had an average area under the curve (AUC) of 0.834, while the model incorporating CT radiomics had an AUC of 0.787. Conclusions: Model building using quantitative imaging data, blood counts, and blood cytokines resulted in lung inflammation prediction models justifying the study hypothesis. The models yielded very-good-to-excellent AUCs of more than 0.78 on internal cross-validation analyses. [ABSTRACT FROM AUTHOR]

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction. [ABSTRACT FROM AUTHOR]

There is increased concern that the quality, generalizability and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides. The only difference detected was that when DKA mice were infected with M. circinelloides, female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides, in both mouse sexes. While little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response. [ABSTRACT FROM AUTHOR]

Featured Application: The presented work details the use of machine learning to left ventricle boundary prediction in high-frequency four-dimensional ultrasound data for rapid and reliable characterization of murine heart function. Automatic boundary detection of 4D ultrasound (4DUS) cardiac data is a promising yet challenging application at the intersection of machine learning and medicine. Using recently developed murine 4DUS cardiac imaging data, we demonstrate here a set of three machine learning models that predict left ventricular wall kinematics along both the endo- and epi-cardial boundaries. Each model is fundamentally built on three key features: (1) the projection of raw US data to a lower dimensional subspace, (2) a smoothing spline basis across time, and (3) a strategic parameterization of the left ventricular boundaries. Model 1 is constructed such that boundary predictions are based on individual short-axis images, regardless of their relative position in the ventricle. Model 2 simultaneously incorporates parallel short-axis image data into their predictions. Model 3 builds on the multi-slice approach of model 2, but assists predictions with a single ground-truth position at end-diastole. To assess the performance of each model, Monte Carlo cross validation was used to assess the performance of each model on unseen data. For predicting the radial distance of the endocardium, models 1, 2, and 3 yielded average R2 values of 0.41, 0.49, and 0.71, respectively. Monte Carlo simulations of the endocardial wall showed significantly closer predictions when using model 2 versus model 1 at a rate of 48.67%, and using model 3 versus model 2 at a rate of 83.50%. These finding suggest that a machine learning approach where multi-slice data are simultaneously used as input and predictions are aided by a single user input yields the most robust performance. Subsequently, we explore the how metrics of cardiac kinematics compare between ground-truth contours and predicted boundaries. We observed negligible deviations from ground-truth when using predicted boundaries alone, except in the case of early diastolic strain rate, providing confidence for the use of such machine learning models for rapid and reliable assessments of murine cardiac function. To our knowledge, this is the first application of machine learning to murine left ventricular 4DUS data. Future work will be needed to strengthen both model performance and applicability to different cardiac disease models. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-18 expression in synovial tissue correlates with the severity of joint inflammation and the levels of pro-inflammatory cytokines. However, the role of the IL-18/IL-18 receptor-alpha (Rα) signaling pathway in autoimmune arthritis is unknown. Wild-type (WT) and IL-18Rα knockout (KO) mice were immunized with bovine type II collagen before the onset of arthritis induced by lipopolysaccharide injection. Disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum inflammatory cytokine and anticollagen antibody levels were quantified by an enzyme-linked immunosorbent assay. Joint cytokine and matrix metalloproteinases-3 levels were determined by a quantitative polymerase chain reaction. Splenic suppressors of cytokine signaling (SOCS) were determined by Western blot analysis as indices of systemic immunoresponse. IL-18Rα KO mice showed lower arthritis and histological scores in bone erosion and synovitis due to reductions in the infiltration of CD4+ T cells and F4/80+ cells and decreased serum IL-6, -18, TNF, and IFN-γ levels. The mRNA expression and protein levels of SOCS3 were significantly increased in the IL-18Rα KO mice. By an up-regulation of SOCS, pro-inflammatory cytokines were decreased through the IL-18/IL-18Rα signaling pathway. These results suggest that inhibitors of the IL-18/IL-18Rα signaling pathway could become new therapeutic agents for rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak−/− mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak−/− and zak+/+ bone marrow–derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak−/− mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak−/− mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak−/− strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak−/− mice have decreased duodenal damage following in vivo ricin challenge. zak−/− mice demonstrate a distinct ribotoxic stress–associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling. [ABSTRACT FROM AUTHOR]

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and "non-self"-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed "non-self" proteins, and potentially improve treatment efficacy. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastoma (GBM) remains the most aggressive brain tumor. Treatment typically includes surgery and radio/chemotherapy, but in spite of intensive treatment, virtually all tumors recur within the time-frame of months with insufficient and unsuccessful second line options. This clinical reality is in contrast to preclinical animal experiments, which often show successful outcomes of novel immunotherapeutic approaches. This discrepancy is largely explained by the small number of animal models and their limited capacity to mimic the complexity of the human disease. Moreover, new treatment options are typically administered as single treatments in animal models, whereas patients receive them in combination with standard-of-care. In this review, we provide an overview of the existing mouse models for GBM research and how each of them mimic (parts of) the human disease spectrum. As such we provide an overview of the advantages and limitations of the currently available options for in vivo drug testing for GBM. Glioblastoma (GBM) is the most aggressive intrinsic brain tumor in adults. Despite maximal therapy consisting of surgery and radio/chemotherapy, GBM remains largely incurable with a median survival of less than 15 months. GBM has a strong immunosuppressive nature with a multitude of tumor and microenvironment (TME) derived factors that prohibit an effective immune response. To date, all clinical trials failed to provide lasting clinical efficacy, despite the relatively high success rates of preclinical studies to show effectivity of immunotherapy. Various factors may explain this discrepancy, including the inability of a single mouse model to fully recapitulate the complexity and heterogeneity of GBM. It is therefore critical to understand the features and limitations of each model, which should probably be combined to grab the full spectrum of the disease. In this review, we summarize the available knowledge concerning immune composition, stem cell characteristics and response to standard-of-care and immunotherapeutics for the most commonly available immunocompetent mouse models of GBM. [ABSTRACT FROM AUTHOR]

Ricin, produced from the castor beans of Ricinus communis, is a cytotoxin that exerts its action by inactivating ribosomes and causing cell death. Accidental (e.g., ingestion of castor beans) and/or intentional (e.g., suicide) exposure to ricin through the oral route is an area of concern from a public health perspective and no current licensed medical interventions exist to protect from the action of the toxin. Therefore, we examined the oral toxicity of ricin in Balb/C mice and developed a robust food deprivation model of ricin oral intoxication that has enabled the assessment of potential antitoxin treatments. A lethal oral dose was identified and mice were found to succumb to the toxin within 48 h of exposure. We then examined whether a despeciated ovine F(ab′)2 antibody fragment, that had previously been demonstrated to protect mice from exposure to aerosolised ricin, could also protect against oral intoxication. Mice were challenged orally with an LD99 of ricin, and 89 and 44% of mice exposed to this otherwise lethal exposure survived after receiving either the parent anti-ricin IgG or F(ab′)2, respectively. Combined with our previous work, these results further highlight the benefit of ovine-derived polyclonal antibody antitoxin in providing post-exposure protection against ricin intoxication. [ABSTRACT FROM AUTHOR]

Simple Summary: Open orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these limitations, this study aimed to evaluate the establishment of an endoscopy-guided minimally invasive model without laparotomy. Different concentrations of the murine CRC cell lines CT26 and MC38 were endoscopically injected into the colorectal wall of BALB/C and C57BL/6J mice, respectively. Consistent tumor growth with the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. Analysis of the learning curve demonstrated that this model is easy to learn and quick to establish. It enables intra-individual follow-up endoscopies, and features tumors to study mechanisms of metastasis and the interaction with the immune system. The application of specific cell lines and concentrations enables a controlled local tumor growth and metastatic formation within short observation periods. Open orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these drawbacks, this study aimed to evaluate the establishment of a minimally invasive model using murine colonoscopy. CT26 and MC38 CRC cells of different concentrations were injected into BALB/C and C57BL/6J mice, respectively. Follow-up endoscopies were performed to assign an endoscopic score to tumor growth. Gross autopsy, histologic and immuno-histochemical evaluation, and immune scoring were performed. To describe the learning curve of the procedures, a performance score was given. Local tumor growth with colorectal wall infiltration, luminal ulceration, the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. The tumors showed cytoplasmic immuno-staining for CK20. Compared to the MC38/C57BL/6J model, tumorigenicity and immunogenicity of the CT26/BALB/C model were higher. Tumor volume correlated with the endoscopic score. This endoscopy-guided orthotopic mouse model is easy to learn and quick to establish. It features early metastasis and enables the study of interactions with the immune system. When specific cell concentrations and cell lines are applied, controlled local tumor growth and metastasis can be achieved within short observation periods. [ABSTRACT FROM AUTHOR]

Simple Summary: The aim of this study was to evaluate the effect of the implementation of an environmental enrichment (EE) plan on reproductive performance in mice, applied during one year period in Swiss Webster breeders and their female offspring used as recipients for embryo transfer. Birth rate, litter size, pups' weight at weaning, interlitter interval and time to first litter were compared against a control group that did not receive EE. In mice breeders, EE significantly increased pups' weight compared to control group (p < 0.01), without changing any other reproductive parameters. In the offspring females used as recipients, EE did not modify reproductive parameters. Our results demonstrate that this enrichment plan, which is usually applied in mouse facilities for animal well-being, enhances pup's weight at weaning, with no negative effects on the reproductive performance of Swiss Webster mice. The aim of this study was to evaluate the effect of an environmental enrichment (EE) plan on the reproductive performance of Swiss Webster mice and their female offspring used as recipients for embryo transfer. A total of 54 breeder mice and 60 F1 females, used as foster mothers, were allocated in two experimental groups to receive or not receive EE for physical well-being. Reproductive outcomes of the Swiss trios such as birth rate and pup number, litter size, pups' weight at weaning, interlitter interval and time to first litter were analyzed. Environmental enrichment significantly increased pups weight from breeding trios compared to the control group (14.4 ± 0.1 vs. 13.8g ± 0.1, EE vs. control, respectively; p < 0.01). Other parameters did not differ between both groups. Reproductive parameters of female offspring used as recipients for embryo transfer did not differ among groups subjected or not to EE. These data demonstrate that the EE protocol applied in Swiss Webster breeder mice positively enhanced pups weight, and did not interfere with other reproductive outcomes. In conclusion, this study supports the implementation of EE plans usually applied for animal welfare in mouse facilities with slight improvement in reproductive performance. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most aggressive intrinsic brain tumor in adults. Despite maximal therapy consisting of surgery and radio/chemotherapy, GBM remains largely incurable with a median survival of less than 15 months. GBM has a strong immunosuppressive nature with a multitude of tumor and microenvironment (TME) derived factors that prohibit an effective immune response. To date, all clinical trials failed to provide lasting clinical efficacy, despite the relatively high success rates of preclinical studies to show effectivity of immunotherapy. Various factors may explain this discrepancy, including the inability of a single mouse model to fully recapitulate the complexity and heterogeneity of GBM. It is therefore critical to understand the features and limitations of each model, which should probably be combined to grab the full spectrum of the disease. In this review, we summarize the available knowledge concerning immune composition, stem cell characteristics and response to standard-of-care and immunotherapeutics for the most commonly available immunocompetent mouse models of GBM.

Interleukin (IL)-18 expression in synovial tissue correlates with the severity of joint inflammation and the levels of pro-inflammatory cytokines. However, the role of the IL-18/IL-18 receptor-alpha (Rα) signaling pathway in autoimmune arthritis is unknown. Wild-type (WT) and IL-18Rα knockout (KO) mice were immunized with bovine type II collagen before the onset of arthritis induced by lipopolysaccharide injection. Disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum inflammatory cytokine and anticollagen antibody levels were quantified by an enzyme-linked immunosorbent assay. Joint cytokine and matrix metalloproteinases-3 levels were determined by a quantitative polymerase chain reaction. Splenic suppressors of cytokine signaling (SOCS) were determined by Western blot analysis as indices of systemic immunoresponse. IL-18Rα KO mice showed lower arthritis and histological scores in bone erosion and synovitis due to reductions in the infiltration of CD4+ T cells and F4/80+ cells and decreased serum IL-6, -18, TNF, and IFN-γ levels. The mRNA expression and protein levels of SOCS3 were significantly increased in the IL-18Rα KO mice. By an up-regulation of SOCS, pro-inflammatory cytokines were decreased through the IL-18/IL-18Rα signaling pathway. These results suggest that inhibitors of the IL-18/IL-18Rα signaling pathway could become new therapeutic agents for rheumatoid arthritis., Competing Interests: Y.N. was received and this work was partially supported from Eli Lilly Japan; Daiichi Sankyo Co., Ltd.; Takeda Pharmaceutical Co., Ltd. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Acute pancreatitis (AP) is a severe disease associated with high morbidity and mortality. Clinical studies can provide some data concerning the etiology, pathophysiology, and outcomes of this disease. However, the study of early events and new targeted therapies cannot be performed on humans due to ethical reasons. Experimental murine models can be used in the understanding of the pancreatic inflammation, because they are able to closely mimic the main features of human AP, namely their histologic glandular changes and distant organ failure. These models continue to be important research tools for the reproduction of the etiological, environmental, and genetic factors associated with the pathogenesis of this inflammatory pathology and the exploration of novel therapeutic options. This review provides an overview of several murine models of AP. Furthermore, special focus is made on the most frequently carried out models, the protocols used, and their advantages and limitations. Finally, examples are provided of the use of these models to improve knowledge of the mechanisms involved in the pathogenesis, identify new biomarkers of severity, and develop new targeted therapies. [ABSTRACT FROM AUTHOR]