Mouse Model Research Articles

Dayuan Yin alleviates symptoms of HCoV-229E-induced pneumonia and modulates the Ras/Raf1/MEK/ERK pathway

Viral pneumonia is characterized by inflammation in the lungs triggered by respiratory viruses. Dayuan Yin (DYY), a traditional Chinese medicine formula known for treating infectious diseases, is hypothesized to offer therapeutic benefits in treating viral pneumonia, although its specific molecular impacts remain understudied. This study aimed to evaluate the therapeutic effects of DYY in mitigating HCoV-229E virus-induced pneumonia in mice. This study employed an HCoV-229E virus-infected mouse model to investigate the therapeutic potential and underlying molecular mechanisms of DYY on virus-induced pneumonia. The respiratory function and organ indices post-treatment were assessed. Lung tissue and tracheal lesions were evaluated via immunohistochemistry. Spleen immune cell composition was analyzed using flow cytometry. Inflammatory cytokines and viral loads were quantified using hypersensitive multiplex electrochemiluminescence method and PCR analysis, respectively. The expression levels of MAS1, Ras, Raf1, MEK1/2, and ERK1/2 in lung tissues were determined through western blot analysis. DYY significantly improved respiratory function, and reduced organ pathology in infected mice. It effectively decreased viral loads and inflammatory cytokines such as IL-6, IL-1β, and TNF-α in lung tissues. Enhancements in immune response were evidenced by increased CD4/CD8 ratios in the spleen. DYY also notably upregulated MAS1 protein levels and suppressed the activation of the Ras/Raf1/MEK/ERK signaling pathway. DYY enhanced respiratory function and exerted significant antiviral and immunomodulatory effects in mice infected with the HCoV-229E virus, primarily by modulating MAS1 expression and inhibiting the Ras/Raf1/MEK/ERK pathway.Graphical

Hypoxia induced cellular and exosomal RPPH1 promotes breast cancer angiogenesis and metastasis through stabilizing the IGF2BP2/FGFR2 axis.

Metastasis is the major cause of breast cancer mortality, with angiogenesis and tumor-released exosomes playing key roles. However, the communication between breast cancer cells and endothelial cells and its role in tumor metastasis remains unclear. Here, we characterize a long noncoding RNA, RPPH1, which is upregulated in breast cancer tissues and positively associated with poor prognosis. Hypoxia microenvironment upregulates the expression of RPPH1 in breast cancer cells, and promotes its packaging into exosomes through hnRNPA1, leading to the maintenance of stemness and aggressive traits in cancer cells and angiogenesis in endothelial cells. The function of cellular and exosomal RPPH1 was confirmed in the MMTV-PyMT mouse model, in which ASO-RPPH1 therapy effectively inhibited tumor progression and metastasis. Mechanistically, RPPH1 protects IGF2BP2 from ubiquitination-induced degradation, stabilizes N6-methyladenosine (m6A)-modified FGFR2 mRNA, and activates the PI3K/AKT pathway. Our research unveils the role of RPPH1 in breast cancer metastasis and highlights its potential as a therapeutic target.

Identification of Oral Bioavailable Coumarin Derivatives as Potential AR Antagonists Targeting Prostate Cancer.

Androgen receptor (AR) is a crucial driver of prostate cancer (PCa), but acquired resistance to AR antagonists significantly undermines their clinical efficacy. We previously discovered coumarin derivative 1, which is capable of disrupting AR ligand-binding domain dimers, offering the potential for overcoming resistance. However, its poor oral bioavailability limited further development. In this study, comprehensive structure optimizations led to compound 4a (IC50 = 0.051 μM), which exhibited comparable AR antagonistic activity to enzalutamide (IC50 = 0.060 μM) and demonstrated excellent selectivity over other nuclear receptors in vitro. Especially, 4a showed superior efficacy against ARF876L/T877A and ARW741C mutants compared to darolutamide and enzalutamide. Moreover, 4a exhibited favorable pharmacokinetic profiles (F = 66.24%) in vivo and significant tumor growth inhibition in an LNCaP xenograft mouse model upon oral administration. These results highlight the potential of 4a as a promising oral AR antagonist for overcoming drug resistance in PCa.

Analysis of Differential microRNA Expression in the Hippocampus of Scopolamine-Induced Amnesic Mouse Model.

Amnesia is characterized by memory deficits linked to various neurodegenerative pathologies and can be induced by the administration of scopolamine, a cholinergic antagonist. Scopolamine-induced amnesia is a well-studied pharmacological animal model that simulates memory impairment caused by aging, brain illnesses, neuropathologies, and trauma. However, the molecular mechanism of amnesia, more importantly in terms of microRNA (miRNA) regulation, is not well understood. Therefore, this study aimed to analyze miRNA profiles in the hippocampus of both control mice and those treated with scopolamine (amnesic mice). Initially, a short cDNA library was prepared for each sample and then sequenced on the Illumina platform. Among the total differentially expressed miRNAs, 113 were significantly upregulated and 96 were downregulated in the scopolamine group in comparison to the control group. Ten upregulated and ten downregulated miRNAs were validated to confirm the reliability of the sequencing results using qRT-PCR (quantitative real-time PCR). Furthermore, we performed a target prediction analysis intersecting the results from TargetScan, miRDB (miRNA database), and Miranda to analyze the targets of the dysregulated miRNAs. We also conducted a pathway analysis to investigate the molecular, cellular, and biological functions of these targets. miRNA‒target interactions were found to play roles in various signaling pathways during amnesia. These results provide an initial insight for the contribution of miRNAs to scopolamine-induced amnesia, as well as their possible application as markers of disease pathology.

Anti-inflammatory activity of Ziziphus jujuba hydroalcoholic extract in acetic acid-induced ulcerative colitis model.

Ulcerative colitis (UC) is a common gastrointestinal (GI) disorder characterized by chronic inflammation. Current treatments primarily focus on symptom management, but they have inherent limitations. Global attention is increasingly directed towards exploring herbal remedies as complementary approaches. This study aims to investigate the effects of the hydroalcoholic extract of jujuba on an experimental model of ulcerative colitis. In this study, 15 male BALB/c mice were divided into three experimental groups. The first group served as the untreated UC model, acting as the positive control (PC). The second group received treatment with the hydroalcoholic extract of Ziziphus jujuba, while the third group was treated with mesalamine. UC was induced by injecting 100 μL of 4 % acetic acid (AA) intra-rectally several times. Treatment commenced after the onset of symptoms such as diarrhea and bloody stools. The mice were eventually euthanized ethically, and their spleen and intestinal tissues were collected for analysis. Evaluations included the Disease Activity Index (DAI), myeloperoxidase activity (MPO), nitric oxide (NO) levels, cytokine levels (IL-1β, IL-6, TNF-α), and gene expression (iNOS, COX-2, and cytokines). The hydroalcoholic extract of the jujuba plant significantly reduced MPO, NO, the DAI, and the production and expression of inflammatory cytokines, as well as the genes iNOS and COX-2, in the group receiving this extract compared to the positive control group (p<0.05). The study demonstrates that the hydroalcoholic extract of Ziziphus jujuba significantly reduces inflammation markers such as TNF-α, NO, MPO, IL-1β, and IL-6 in a mouse model of ulcerative colitis. Additionally, itdownregulates the expression of pro-inflammatory genes, including iNOS and COX-2. These findings suggest that Z.jujuba extract has potential as an effective anti-inflammatory treatment for managing ulcerative colitis symptoms.

LPS-induced delirium-like behavior and microglial activation in mice correlate with bispectral electroencephalography (BSEEG).

Delirium is a multifactorial medical condition characterized by impairment across various mental functions and is one of the greatest risk factors for prolonged hospitalization, morbidity, and mortality. Research focused on delirium has proven to be challenging due to a lack of objective measures for diagnosing patients, and few laboratory models have been validated. Our recent studies report the efficacy of bispectral electroencephalography (BSEEG) in diagnosing delirium in patients and predicting patient outcomes. We applied BSEEG to validate a lipopolysaccharide (LPS)-induced mouse model of delirium. Moreover, we investigated the relationship between BSEEG score, delirium-like behaviors, and microglia activation in hippocampal dentate gyrus and cortex regions in young and aged mice. There was a significant correlation between BSEEG score and impairment of attention in young mice. Additionally, there was a significant correlation between BSEEG score and microglial activation in hippocampal dentate gyrus and cortex regions in young and aged mice. We have successfully validated the BSEEG method by showing its associations with a level of behavioral change and microglial activation in an LPS-induced mouse model of delirium. In addition, the BSEEG method was able to sensitively capture an LPS-induced delirium-like condition that behavioral tests could not capture because of a hypoactive state.

Kinetics of imidazole propionate from orally delivered histidine in mice and humans

Imidazole Propionate (ImP), a gut-derived metabolite from histidine, affects insulin signaling in mice and is elevated in type 2 diabetes (T2D). However, the source of histidine and the role of the gut microbiota remain unclear. We conducted an intervention study in mice and humans, comparing ImP kinetics in mice on a high-fat diet with varying histidine levels and antibiotics, and assessed ImP levels in healthy and T2D subjects with histidine supplementation. Results show that dietary histidine is metabolized to ImP, with antibiotic-induced gut microbiota suppression reducing ImP levels in mice. In contrast, oral histidine supplementation resulted in increases in circulating ImP levels in humans, whereas antibiotic treatment increased ImP levels, which was associated with a bloom of several bacterial genera that have been associated with ImP production, such as Lactobacilli. Our findings highlight the gut microbiota’s crucial role in regulating ImP and the complexity of translating mouse models to humans.

A dual-purpose humanized mouse model for testing antiviral strategies against both SIV and HIV

Nonhuman primate (NHP) models employing simian/simian-human immunodeficiency viruses (SIV/SHIVs) played a major role in the study of HIV pathogenesis, latency, and cure studies in a preclinical setting. However, it took many years to arrive at the current effective triple drug ARV regimen against SIV due to the genetic differences with that of HIVs. Since new combinations of drugs will be used in the evolving HIV cure studies, a small animal model would be ideal to determine their efficacy against the commonly used SIVs such as SIVmac239 to triage ineffective drugs prior to their application in NHPs. We recently determined that humanized mice (hu-mice) with a transplanted human immune system are permissive to SIVmac strains in addition to HIVs. Based on this novel finding, here we evaluated the utility of this dual-purpose hu-mouse model to test different ART regimens against SIVmac239. Infected mice showing chronic viremia were treated with a combination anti-retroviral treatment (cART) regimen consisting of emtricitabine/elvitegravir/tenofovir disoproxil fumarate (FTC/EVG/TDF). Full viral suppression was seen for several weeks in SIVmac239-infected and treated mice similar to that seen with HIV-1 BaL virus used as a control. However, viral rebound was eventually observed in SIVmac239 infected mice during the treatment period, suggesting viral escape compared to HIV-1 BaL with which viral suppression was fully sustained. Next, a cART regimen consisting of emtricitabine/bictegravir/tenofovir alafenamide fumarate (FTC/BIC/TAF) was similarly evaluated. Our results showed that this ARV regimen was fully effective in rapidly suppressing both SIVmac239 and HIV-1 BaL. Complete viral suppression was maintained until treatment interruption after which viral loads rebounded. These findings highlight the utility of humanized mice for in vivo screening of new combinations of ARV compounds against various SIVs prior to employing them in NHPs. In addition to identifying new effective cART regimens against SIVs, this model would also be amenable to evaluating immunotherapeutic strategies using broadly neutralizing antibodies, LRAs and novel therapeutics in comparative cure studies of SIV and HIV.

Sea Buckthorn Flavonoid Extracted with High Hydrostatic Pressure Alleviated Shrimp Allergy in Mice through the Microbiota and Metabolism.

Sea buckthorn (Hippophaë rhamnoides L.) known as the deciduous shrub has been reported to have effects of antioxidant, anti-inflammatory, and immunomodulatory activities. Tropomyosin (TM) induced a regulatory immune response associated with food allergy. In this study, a mouse model of food allergy sensitized to tropomyosin (TM) was established to assess the antiallergic properties of sea buckthorn flavonoid extract (SBF). SBF alleviated mice's allergic symptoms and exhibited a significant reduction in the levels of IgE and histamine. Meanwhile, SBF repaired the allergic Th2 cell overpolarization generated by TM, via downregulating the IL-4 production and upregulating IFN-γ production to restore the balance of Th1/Th2 cells. Furthermore, the 16S RNA analysis showed that SBF primarily restored the gut microbiota via increasing the abundance in Chitinophilidae and decreasing in Burkholderiaceae, Pneumatobacteriaceae, and Sphingomonadaceae. Gut metabolomes determined by liquid chromatography-mass spectrometry (LC-MS) suggested that TM upregulated PE (14:0/22:1(13Z)) and SBF decreased formimino-l-glutamic acid and urocanic acid levels. According to the KEGG pathway analysis, SBF treatment has been shown to modulate glycerophospholipid and histidine metabolism to improve allergic reactions. SBF holds great promise as a novel potential agent for the treatment of food allergies.

Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.

Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

Bone marrow mesenchymal stem cells overexpressing stromal cell- derived factor 1 aid in bone formation in osteoporotic mice

BackgroundOsteoporosis is characterized by low systemic bone mineral content and destruction of bone microarchitecture. Promoting bone regeneration and reversing its loss by infusion of exogenous bone marrow mesenchymal stem cells (BMSCs) is a potentially effective treatment for osteoporosis. However, their limited migration to target organs reduces the therapeutic effect of the cells. Stromal cell-derived factor 1 (SDF1) is a chemokine that induces targeted cell migration through the SDF1/CXCR4 (C-X-C chemokine receptor 4) axis and can induce migration of exogenous mesenchymal stem cells to sites of high SDF1 concentration. There are no studies on BMSCs overexpressing SDF1 (SDF1-BMSCs) in osteoporotic mice in vivo. We aimed to investigate if the increased SDF1 concentration facilitated cell migration to the bone.MethodsWe used lentivirus to construct BMSCs overexpressing SDF1 or knocking down CXCR4. We verified the proliferation ability of the cells in vitro using Cell Counting Kit-8 (CCK8) and 5-Bromodeoxyuridinc (BrdU), the migration ability of the cells using Transwell, and the osteogenic and lipogenic ability of the cells using osteogenic and lipogenic induction solutions. In in vivo experiments, we induced osteoporosis in 72 female mice by ovariectomy and injected different groups of cells via the tail vein. Femoral tissue samples were collected for a fixed time, and the osteogenic and homing abilities of the cells were verified by MicroCT and tissue section staining.ResultsWe successfully demonstrated that high expression of SDF1 promoted cell proliferation and migration in vitro, without affecting their cell differentiation ability. In an ovariectomized mouse model, SDF1-BMSCs were more likely to be home to the femur than the BMSCs, had a better pro-osteogenic ability, and had higher expression of Wnt-1. Blocking the SDF1/CXCR4 axis reduced the homing of exogenous mesenchymal stem cells (MSCs) to the femur and their osteogenic capacity.ConclusionsSDF1-BMSCs can further promote bone formation by increasing the number of cells homing to the femur in osteoporotic mice. Our study shows that stem cells can promote their proliferation and home to the femur via the SDF1/CXCR4 axis and further help bone formation via Wnt-1 signaling.

The Icelandic mutation (APP-A673T) is protective against amyloid pathology in vivo.

A previous epidemiological study in Northern Europe showed that the A673T mutation (Icelandic mutation) in the amyloid precursor protein gene (APP) can protect against Alzheimer's disease (AD). While the effect of the A673T mutation on APP processing has been investigated primarily in vitro, its in vivo impact has not been evaluated. This is mainly because most existing AD mouse models carry the Swedish mutation. The Swedish and Icelandic mutations are both located near the β-cleavage site, and each mutation is presumed to have the opposite effect on β-cleavage. Therefore, in the AD mouse models with the Swedish mutation, its effects could compete with the effects of the Icelandic mutation. Here, we introduced the A673T mutation into App knock-in mice devoid of the Swedish mutation (AppG-F mice) to avoid potential deleterious effects of the Swedish mutation and generated AppG-F-A673T mice. APP-A673T significantly downregulated β-cleavage and attenuated the production of Aβ and amyloid pathology in the brains of these animals. The Icelandic mutation also reduced neuroinflammation and neuritic alterations. Both sexes were studied. This is the first successful demonstration of the protective effect of the Icelandic mutation on amyloid pathology in vivo. Our findings indicate that specific inhibition of the APP-BACE1 interaction could be a promising therapeutic approach. Alternatively, introduction of the disease-protective mutation such as APP-A673T using in vivo genome editing technology could be a novel treatment for individuals at high risk for AD, such as familial AD gene mutation carriers and APOE ε4 carriers.Significance statement The A673T mutation (Icelandic mutation) in the APP gene can protect against AD. The effect of the A673T mutation on amyloid pathology has not been evaluated in vivo. Utilizing a new AD mouse model that we have recently developed, we show that the APP-A673T attenuates amyloid pathology in vivo. We demonstrate that its protective effects are exerted by inhibiting β-cleavage and reducing the production of Aβ in the brain. Furthermore, we reveal that the Icelandic mutation also reduced neuroinflammation and neuritic alterations. Our findings indicate that specific inhibition of the APP-BACE1 interaction or introduction of protective variants via in vivo genome editing could be a promising therapeutic approach.

RhoA regulates oligodendrocyte differentiation and myelination by orchestrating cortical and membrane tension.

Timely differentiation and myelin formation by oligodendrocytes are essential for the physiological functioning of the central nervous system (CNS). While the Rho GTPase RhoA has been hinted as a negative regulator of myelin sheath formation, the precise in vivo mechanisms have remained elusive. Here we show that RhoA controls the timing and progression of myelination by oligodendrocytes through a fine-tuned balance between cortical tension, membrane tension and cell shape. Using a conditional mouse model, we observe that Rhoa ablation results in the acceleration of myelination driven by hastened differentiation and facilitated through membrane expansion induced by changes in MLCII activity and in F-actin redistribution and turnover within the cell. These findings reveal RhoA as a central molecular integrator of alterations in actin cytoskeleton, actomyosin contractility and membrane tension underlying precise morphogenesis of oligodendrocytes and normal myelination of the CNS.

Cancer immunogenic cell death via pyroptosis with CXCR4-targeted nanotoxins in hepatocellular carcinoma

IntroductionCytotoxic agents have shown limited benefits in hepatocellular carcinoma (HCC), mediated in part by the lack of targeting. As cell-penetrating peptides (CPPs) are capable of delivering various biologically active molecules into cells, including protein, peptides, small chemo-drugs, and nucleic acid with or without targeting, we developed T22-PE24, a CXCR4-targeted self-assembling cytotoxic nanotoxin, to effectively induce HCC pyroptosis.MethodsT22 incorporating enhanced green fluorescent protein (EGFP) or PE24 was purified from DE3 bacterial cells and characterized using transmission electron microscopy, the Zetasizer Nano®, and SEC-HPLC. The internalization effect of T22-EGFP was detected by flow cytometry system (FCS) in CXCR4+/LM3(CXCR4−) HCC cells. The CCK8, lactate dehydrogenase (LDH) release, Western blot, and nude mice HCC models were used to estimate the cell viability of T22-PE24. The complete-immunity HCC tumor-bearing mice model was used to assess the immune response of T22-PE24.ResultsThe round shape under transmission electron microscopy, 49.4 nm hydrodynamic diameter, and −33.33 mV zeta potential indicated that T22-PE24 self-assembled into nanoparticles. T22 incorporating EGFP selectively internalized in CXCR4+ HCC cells and showed no accumulation in CXCR4-knockout HCC cells. The T22-PE24 nanotoxin induced HCC pyroptosis via the caspase-3/GSDME signaling pathway and suppressed tumor growth in the absence of histological alterations in normal organs. Using the complete-immunity HCC tumor-bearing mice model, we found that T22-PE24 nanotoxin effectively induces the global reprogramming of cell components of the immune tumor microenvironment, leading to enhanced antitumor effects compared to those observed in immunodeficient mice.ConclusionOur findings demonstrate the activation of the innate immune response in HCC by inducing pyroptosis with T22-PE24 nanotoxin treatment and support an implementation of this strategy for HCC treatment.

IMPA1-derived inositol maintains stemness in castration-resistant prostate cancer via IMPDH2 activation.

Acquisition of prostate cancer stem cells (PCSCs) manifested during androgen ablation therapy (ABT) contributes to castration-resistant prostate cancer (CRPC). However, little is known about the specific metabolites critically orchestrating this process. Here, we show that IMPA1-derived inositol enriched in PCSCs is a key metabolite crucially maintaining PCSCs for CRPC progression and ABT resistance. Notably, conditional Impa1 knockout in the prostate abrogates the pool and properties of PCSCs to orchestrate CRPC progression and prolong the survival of TRAMP mice. IMPA1-derived inositol serves as a cofactor that directly binds to and activates IMPDH2, which synthesizes guanylate nucleotides for maintaining PCSCs with ARlow/- features leading to CRPC progression and ABT resistance. IMPA1/inositol/IMPDH2 axis is upregulated in human prostate cancer, and its overexpression predicts poor survival outcomes. Genetically and pharmacologically targeting the IMPA1/inositol/IMPDH2 axis abrogates CRPC and overcomes ABT resistance in various CRPC xenografts, patient-derived xenograft (PDX) tumor models, and TRAMP mouse models. Our study identifies IMPDH2 as an inositol sensor whose activation by inositol represents a key mechanism for maintaining PCSCs for CRPC and ABT resistance.

Intestinal human carboxylesterase 2 (CES2) expression rescues drug metabolism and most metabolic syndrome phenotypes in global Ces2 cluster knockout mice.

Carboxylesterase 2 (CES2) is expressed mainly in liver and intestine, but most abundantly in intestine. It hydrolyzes carboxylester, thioester, and amide bonds in many exogenous and endogenous compounds, including lipids. CES2 therefore not only plays an important role in the metabolism of many (pro-)drugs, toxins and pesticides, directly influencing pharmacology and toxicology in humans, but it is also involved in energy homeostasis, affecting lipid and glucose metabolism. In this study we investigated the pharmacological and physiological functions of CES2. We constructed Ces2 cluster knockout mice lacking all eight Ces2 genes (Ces2-/- strain) as well as humanized hepatic or intestinal CES2 transgenic strains in this Ces2-/- background. We showed that oral availability and tissue disposition of capecitabine were drastically increased in Ces2-/- mice, and tissue-specifically decreased by intestinal and hepatic human CES2 (hCES2) activity. The metabolism of the chemotherapeutic agent vinorelbine was strongly reduced in Ces2-/- mice, but only marginally rescued by hCES2 expression. On the other hand, Ces2-/- mice exhibited fatty liver, adipositis, hypercholesterolemia and diminished glucose tolerance and insulin sensitivity, but without body mass changes. Paradoxically, hepatic hCES2 expression rescued these metabolic phenotypes but increased liver size, adipose tissue mass and overall body weight, suggesting a "healthy" obesity phenotype. In contrast, intestinal hCES2 expression efficiently rescued all phenotypes, and even improved some parameters, including body weight, relative to the wild-type baseline values. Our results suggest that the induction of intestinal hCES2 may combat most, if not all, of the adverse effects of metabolic syndrome. These CES2 mouse models will provide powerful preclinical tools to enhance drug development, increase physiological insights, and explore potential solutions for metabolic syndrome-associated disorders.

Cysteine Leukotriene Receptor Antagonist-Montelukast Effects on Diabetic Retinal Microvascular Endothelial Cells Curtail Autophagy.

Diabetic macular edema (DME) is the primary cause of vision impairment in diabetic retinopathy (DR) patients. A previous study has shown the efficacy of montelukast, a cysteinyl leukotriene receptor (CysLTR)1 antagonist, in a diabetic mouse model. This study aims to understand the CysLTR1 signaling in retinal endothelial cells and the impact of montelukast. Primary human retinal microvascular endothelial cells (HRECs) challenged with 20 ng/mL TNF-α and 30mM D-glucose (D-glu) for six to 24 hours served as a model of endothelial activation. HRECs were incubated with L-glucose (L-glu) as an osmotic control. CysLTR1 knockdown and montelukast pretreatment assessed CysLTR1 antagonism. Gene expression, protein expression, and cell-permeable dyes were utilized to measure autophagy and inflammation. Transendothelial electrical resistance (TER) and transendothelial migration of mononuclear leukocytes across HRECs monolayer were measured as a functional assessment of vascular permeability. Endothelial activation induced by hyperglycemia and inflammation increased CysLTR1 expression, triggering autophagy within two to six hours, IL-1β production, loss of junction integrity, decreased TER, and increased leukocyte migration within six to 24 hours. Pretreatment with montelukast effectively alleviated these effects, demonstrating its dependence on CysLTR1. Dysfunctional retinal endothelium initiates a self-reinforcing loop of inflammation, autophagy, and compromised integrity associated with heightened CysLTR1 levels. The antagonistic effect of montelukast against CysLTR1 effectively mitigates these detrimental changes. This study reveals CysLTR1 as a potential therapeutic target in treating DME and offers a novel strategy to mitigate detrimental changes in DR.

IP-10 acts early in CV-A16 infection to induce BBB destruction and promote virus entry into the CNS by increasing TNF-α expression

The mechanisms underlying pathological changes in the central nervous system (CNS) following Coxsackievirus A16 (CV-A16) infection have not yet been elucidated. IFN-γ-inducible protein-10 (IP-10) is often used as a predictive factor to monitor early virus infection. It has also been reported that IP-10 plays a pivotal role in neuroinflammation. In this study, we aimed to explore the role of IP-10 in the neuropathogenesis of CV-A16 infection. We observed that the level of IP-10, as well as the TLR3-TRIF-TRAF3-TBK1-NF-κB and RIG-I/MDA5-MAVS-TRAFS-TBK1-NF-κB pathways, which are the upstream of IP-10, were significantly elevated during the course of CV-A16 infection. This increase was accompanied by an increase in a series of inflammatory cytokines at different time-points during CV-A16 infection. To determine whether IP-10 influences BBB integrity, we examined junctional complexes. Our results revealed that the expression levels of Claudin5, Occludin, ZO-1 and VE-Cadherin were notably decreased in CV-A16-infected HUVECs, but these indicators were restored in CV-A16-infected HUVECs with Eldelumab treatment. Nevertheless, IP-10 is only a chemokine that primarily traffics CXCR3-positive immune cells to inflammatory sites or promotes the production of inflammatory cytokines. Therefore, the interactions between IP-10 and inflammatory cytokines were evaluated. Our data revealed that IP-10 mediated the production of TNF-α, which was also observed to change the junctional complexes. Moreover, in a suckling mouse model, IP-10 and TNF-α treatments exacerbated clinical symptoms, mortality and pathological changes in the brain of CV-A16-infected mice, but Anti-IP-10 and Anti-TNF-α treatments alleviated these changes. Our data also revealed that IP-10 may be detected early in CV-A16 infection, whereas TNF-α was detected late in CV-A16 infection, and the production of TNF-α was also found to be positively correlated with IP-10. In addition, IP-10 and TNF-α were observed to reduce junctional complexes and enhance virus entry into the CNS. Taken together, this study provides the first evidence that CV-A16 activates the IP-10/TNF-α regulatory axis to cause BBB damage and accelerate the formation of neuroinflammation in infected hosts, which not only provides a new understanding of the neuropathogenesis caused by CV-A16, but also offers a promising target for the development of CV-A16 antiviral drugs.

Double transgenic neonatal porcine islets as an alternative source for beta cell replacement therapy

To be clinically efficient, beta cell replacement therapies such as pig islet xenotransplantation must ensure sufficient insulin secretion from grafted islets. While protection from host immune reaction is essential for islet engraftment and their subsequent functioning, intrinsic physiological properties of used cells are also a key factor. We have previously shown that islets with adenoviral-mediated expression of a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP-1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in their beta cells have greatly improved insulin secretory response to glucose stimulation that is otherwise 4 to 10 times lower than human islets. Here, we describe in vitro characterization of the secretory function of pancreatic islets, derived from transgenic pigs expressing the GLP-1M3R cassette under the porcine insulin promoter (InsGLP-1M3R), and their usage to treat insulin-dependent diabetes in an immunodeficient mouse model. Our results show that InsGLP-1M3R islets isolated from neonatal and adult pigs secrete up to 15-fold more insulin in response to glucose stimulation compared to wild-type (WT) islets. They also proved to be more efficient in treating diabetes in a preclinical model as shown by a significantly higher percentage of normoglycemic recipients and higher porcine C-peptide levels up to 9 mo post implantation.

Development and Evaluation of ABI-171, a New Fluoro-Catechin Derivative, for the Treatment of Idiopathic Pulmonary Fibrosis

The persistent challenge of idiopathic pulmonary fibrosis (IPF), characterized by disease progression and high mortality, underscores the urgent need for innovative therapeutic strategies. We have developed a novel small molecule—catechin derivative ABI-171—selectively targeting dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) and proviral integration site for Moloney murine leukemia virus 1 (PIM1) kinases, crucial in the pathogenesis of fibrotic processes. We employed the Bleomycin-induced (intratracheal) mouse model of pulmonary fibrosis (PF) to evaluate the therapeutic efficacy of ABI-171. Mice with induced PF were treated QD with ABI-171, either prophylactically or therapeutically, using oral and intranasal routes. Pirfenidone (100 mg/kg, TID) and Epigallocatechin gallate (EGCG, 100 mg/kg, QD), a natural catechin currently in a Phase 1 clinical trial, were used as reference compounds. ABI-171, administered prophylactically, led to a significant reduction in hydroxyproline levels and fibrotic tissue formation compared to the control group. Treatment with ABI-171 improved body weight, indicating mitigation of disease-related weight loss. Additionally, ABI-171 demonstrated anti-inflammatory activity, reducing lymphocyte and neutrophil infiltration. In the therapeutic setting, ABI-171, administered 7 days post-induction, reduced mortality rates (p = 0.04) compared with the bleomycin and EGCG control groups. ABI-171 also ameliorated the severity of lung injuries assessed by improved Masson’s trichrome scores when administered both orally and intranasally. ABI-171 significantly decreases bleomycin-induced PF and improves survival in mice, showcasing promising therapeutic potential beyond current medications like pirfenidone and EGCG for patients with IPF. Based on these results, further studies with ABI-171 are ongoing in preclinical studies.